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Mol Ther. Mar 2012; 20(3): 513–524.
Published online Jan 17, 2012. doi:  10.1038/mt.2011.294
PMCID: PMC3293611

Action and Reaction: The Biological Response to siRNA and Its Delivery Vehicles

Abstract

RNA interference (RNAi)-based therapeutics have significant potential for the treatment of human disease. Safe and effective delivery of RNA to target tissues remains a major barrier to realizing its clinical potential. Several factors can affect the in vivo performance of short interfering RNA (siRNA) delivery formulations, including siRNA sequence, structure, chemical modification, and delivery formulation. This review provides an introduction to the principles underlying the pharmacokinetics and pharmacodynamics of systemically administered siRNA and its delivery formulations, including the factors that lead to its degradation, clearance, and tissue uptake, as well as its potential for immunogenicity, toxicity, and off-target effects within the body.

Introduction

Since the discovery that synthetic short interfering RNAs (siRNAs) could trigger gene silencing in mammalian cells,1 there has been great interest in developing RNA interference (RNAi)-based technologies for both genetic study and therapeutic applications. siRNA molecules must be delivered intracellularly in order to trigger RNAi, but their large, anionic, and hydrophilic structure prevents them from diffusing across cell membranes to reach their site of action. A broad array of delivery materials has been developed to mediate delivery and to improve the overall pharmacokinetics of siRNA when administered systemically (reviewed elsewhere, see ref. 2). While the design of siRNA delivery systems is usually focused on the immediate goal of delivering enough of the injected dose into enough target cells to generate a particular therapeutic effect, a broader awareness of the fate of the entire injected dose and its many other interactions with the body is essential in the development of safe and effective systems. siRNAs and their delivery vehicles can induce immunogenicity, toxicity, and off-target effects in addition to target gene silencing. Various factors can mediate the degradation, clearance, and cellular uptake of siRNA delivery systems, affecting their potency. This review presents a brief summary of important pharmacodynamic and pharmacokinetic considerations in siRNA delivery and intends to introduce the reader to the numerous interactions between the body and siRNA, but is not a comprehensive review of pharmacodynamics and pharmacokinetics data for various delivery systems. For more detailed information on particular topics, the reader is referred to reviews of more limited scope and of greater depth.

The Pharmacodynamics of siRNA Delivery Systems

siRNA delivery systems can elicit both intended effects (e.g., target gene silencing) as well as unintended consequences, including immune stimulation, toxicity, and off-target silencing. The pharmacodynamics of siRNA delivery systems are dependent not only upon the siRNA therapeutic, but also upon the biomaterials included in the delivery vehicle.

RNA structure and gene silencing

The central component of any siRNA formulation is the siRNA itself. The silencing of specific genes through RNAi machinery (Figure 1) is the intended pharmacodynamic effect of siRNA. Over the past few years, a number of design criteria have been developed to improve the specificity and potency of RNA, some of which has been driven by an increased understanding of the biochemistry driving RNAi.3,4

Figure 1
siRNA and miRNA pathways. Exogenous double-stranded RNA (dsRNA) introduced into the cytoplasm is cleaved by Dicer into 21-nt fragments with 2-nt overhangs on the 3′ ends. The antisense strand, or guide strand, is loaded into the RNA-induced silencing ...

When a siRNA molecule is delivered inside of a cell, it can be loaded into the RNA-induced silencing complex (RISC) (Figure 1). Argonaute-2 (Ago-2, Figure 1), a protein within RISC, unwinds the siRNA. The two strands of siRNA are referred to the passenger strand and the guide strand. The passenger strand is cleaved while the activated RISC-guide strand complex seeks out and cleaves messenger RNA (mRNA) that is complementary to the guide strand. The nucleotide sequence of the siRNA affects the efficiency of each of these steps and therefore affects the potency of gene silencing. A number of guidelines have emerged for selection of the most active siRNA sequences, and these principles have been incorporated into various siRNA design algorithms.5 The enhancement of siRNA asymmetry and minimization of secondary structure in mRNA target sites are both important considerations in sequence selection.

When siRNA enters the RNAi pathway (Figure 1), one strand is loaded into the RISC complex to become the guide strand and its complementary strand is cleaved. If the incorrect strand is selected for RISC loading, the strand intended to be the guide strand is cleaved, leading to reduced potency of target gene silencing. Incorrect strand selection can also trigger the silencing of off-target genes complementary to the intended passenger strand. siRNA asymmetry refers to the preference for one strand to be loaded into RISC over the other. Increasing the asymmetry of the duplex helps to ensure that the desired strand is loaded into RISC to direct gene silencing. Strand selection by RISC is determined by the thermodynamic stability of the ends of the duplex: the 5′ end with the lowest hybridization stability is loaded into RISC to become the guide strand.6,7,8 Thermodynamic asymmetry is increased by enriching the A-U base pair content in the 5′ end of the antisense strand. Unlocked nucleic acids (UNA, Figure 2) destabilize duplexes and can enhance asymmetry when incorporated into the 5′ end of the guide strand.9,10,11

Figure 2
Common chemical modifications to the siRNA backbone. The 2′-OH of the ribose ring can be replaced with methoxy (2′-OMe) or fluorine (2′-F) moieties. Locked nucleic acids (LNA) contain a methylene bridge connecting the 2′ ...

Asymmetry can also be enhanced by modification of passenger-strand ends. For siRNAs to be functional, the 5′ end of the antisense strand must have a terminal phosphate.8 5′-OH–terminated siRNAs are rapidly phosphorylated in the cytoplasm,12 but modification of the terminal hydroxyl can prevent phosphorylation and prevent RISC loading.13 Methylation of the 5′ end of the passenger strand is a common strategy to ensure proper strand selection by RISC.13

The presence of delivery materials can affect RISC loading. Delivery materials that complex with siRNA to form nanoparticles must dissociate from siRNA once inside the cell to allow RISC loading. For example, the cationic lipids of many lipoplex delivery systems undergo a phase transition in acidic environments such as endosomes. The conversion from a lamellar phase to hexagonal packing promotes both endosomal release and release of siRNA from the delivery material.14 In other systems, delivery materials are conjugated directly to the ends of siRNA strands. As the 5′ end of the guide strand is essential for RISC loading, conjugation of delivery materials is usually avoided in this position. Delivery materials are usually conjugated to the 3′ or 5′ ends of the passenger strand, though conjugation to the 3′ end of the guide strand can be effective as well.6,15,16

The level of accessibility of the mRNA target site also affects the potency of siRNA.7,17,18 Folding of mRNA can block access to the target site, as activated RISC cannot unfold secondary structures in mRNA.17 Secondary structure prediction algorithms can help to choose mRNA target sites with high accessibility.5,7 Self-folding of the siRNA guide strand can also impede target site recognition, and inverted repeats are often avoided in siRNA sequences.19,20 An example of a siRNA design algorithm that considers RNA secondary structures is the OligoWalk web server, which selects active siRNA sequences based on their free energy changes upon hybridization with target mRNA.5

The internal stability of the length of the duplex can also affect activity. A systematic analysis of a siRNA library determined that the most active sequences had a guanine plus cytosine (GC) content of 36–52%.4 It has been proposed that higher GC content may inhibit RISC loading and release of the cleaved sense strand, while lower GC content may inhibit activity by weakening hybridization between the guide strand and mRNA.3

Several studies of siRNA sequences have confirmed a preference for certain nucleotides in specific positions along siRNA strands.4,21 Several of these positional preferences are consistent with the idea of thermodynamic asymmetry of siRNA ends: A or U is preferred at position 1 of the antisense strand, G or C is preferred at position 19, and AU-richness in positions 1–7 is favored. In addition, A or U is favored at position 10, the site of substrate cleavage by Ago-2.

Immunostimulation

siRNA can be recognized by the innate immune system. In addition to mediating RNAi, siRNA molecules have the potential to induce the innate immune system.22 The interactions between siRNA and the immune system are complex and only an overview is provided here. For a detailed review, see reference23. The process of immunological recognition and response to non-self nucleic acids such as siRNA is governed by the innate immune system. Innate immune responses are characterized by an induction of small signaling molecules called cytokines, including interleukins,24 type I interferons,25 and tumor necrosis factor-α.26

The stimulation of pattern recognition receptors (PRRs) triggers the innate immune response. PRRs recognize distinct pathogenic patterns that are not present on self-cells.27,28 Importantly for siRNA delivery, the innate immune system has evolved to include multiple PRRs that recognize different aspects of RNA structure, providing a redundancy that makes immunostimulation difficult to escape (Figure 3). Two general classes of siRNA-recognizing PRRs include toll-like receptors (TLRs) and cytoplasmic receptors.29

Figure 3
siRNA invokes immunostimulation via pathogen recognition receptors. TLR3 (red) exists on both the cell surface and in subcellular compartments of select cells. TLR7 (orange) and TLR8 (yellow) exist solely in the endosomes and lysosomes of specialized ...

Toll-like receptors are a class of PRRs that recognize structurally conserved regions of foreign pathogens, and each member of the TLR family is responsible for the detection of varying pathogen-associated molecules. Of the 10 human TLRs and 12 murine TLRs that have been discovered to date,30 at least three have relevance to siRNA delivery. These include TLR3, which recognizes double-stranded RNA (dsRNA), and TLR7 and TLR8, which recognize single-stranded RNA (ssRNA). TLR3 responds to dsRNA, which is typically a characteristic of viral replication found in lysed or apoptotic virally infected cells.31 In humans, TLR3 is expressed in endosomes and on the cell surface of selected cell populations. TLRs 7 and 8 are important PRRs that respond to ssRNA in a sequence-specific manner. They are located exclusively in the intracellular vesicles, including endosomes, lysosomes, and the endoplasmic reticulum of plasmacytoid dendritic cells, B cells, and myeloid cells.28 More specifically, TLR7 is located primarily in the endosomes of plasmacytoid dendritic cells and TLR8 in endosomes of myeloid cells.32

In addition to endosomal TLRs, several cytoplasmic receptors mediate immune responses to siRNA (Figure 3). PKR, a protein kinase that responds to dsRNA33 and siRNA,28 can cause the inhibition of protein translation and an interferon response when activated.33 RIG-I, another cytoplasmic PRR expressed in fibroblasts and dendritic cells,34 can provoke a strong interferon response in the presence of various forms of siRNA.28

Several features of siRNA delivery systems influence the nature and degree of immunostimulation. These include the siRNA sequence, the siRNA structure and chemistry, and the materials used to construct the delivery vehicle.22 The sequence of siRNA influences immune stimulation through TLRs 7 and 8, which recognize ssRNA in a sequence-specific manner.35,36 Generally, siRNAs rich in guanosine and uridine-rich motifs tended to provoke more immunostimulatory activity while a decrease in the presence of uridine residues had the opposite effect. The substitution of certain residues within an RNA molecule has been demonstrated to diminish pro-inflammatory response. In particular, substituting guanosine or uridine residues with adenosine resulted in decreased cytokine and interferon activity.36

The chemical structure of the siRNA duplex can also influence the degree of innate immune response. RIG-I, for example, has been shown to bind to ssRNA or dsRNA containing uncapped 5′-triphosphate groups resulting in an interferon-mediated immune response.37,38,39 Uncapped RNA is a sign of viral infection, and subsequently induces inflammatory action. In addition, it has been reported that blunt-ended dsRNA (no overhangs) is also capable of provoking immunostimulatory activity through recognition by RIG-I.28

Nucleotide structure also has a profound effect on innate immune system activation. For example, it has been established that modifying the 2′ group on the ribose ring of the RNA backbone can reduce or even eliminate the innate immune response. Locked nucleic acids (LNA),40 UNA,41 as well as 2′-F, 2′-H (i.e., 2′-deoxy) and 2′-O-methyl modifications (2′-OMe) (Figure 2)15,42 have all been shown inhibit immune activity to some extent. Relative to other types of nucleotide modification, 2′-O-Me modifications successfully inhibit TLR7/8-mediated recognition of siRNA without diminishing RNAi potency.35

siRNA delivery vehicles influence immunostimulation. Although single doses of naked siRNA do not necessarily cause innate immune activation,43 siRNA delivery materials can have a substantial effect on siRNA-mediated immune activity. Delivery vehicles can account for immune responses varying up to two orders of magnitude32,44,45 and can act through a variety of mechanisms. Different delivery materials escort siRNA molecules across cell membranes in unique ways and into differing subcellular compartments, exposing the siRNA to differing numbers and types of PRRs.

For example, cationic materials are frequently employed for siRNA delivery applications, as they readily condense RNA due to electrostatic interactions. In general, cationic and lipid materials are believed to deliver siRNA through the mediation of endosomal uptake into the cell and subsequent endosomal escape into the cytoplasm of the cell.46 When formulated with a cationic delivery material, siRNA is trafficked through several subcellular locations, allowing it to interact with TLRs in the endosomal compartments of immune cells as well as with RIG-I and PKR (Figure 3), which are present in the cytoplasm of most cell types. Therefore, this type of delivery material would expose its cargo to many pattern recognition receptors, rendering the siRNA more prone to innate immune recognition than a delivery material that avoided endosomal and lysosomal delivery routes.

The size, charge, and biodistribution of a delivery vehicle may also alter the intensity and nature of a siRNA-induced immune response. Judge and colleagues found that stable nucleic acid lipid particle-encapsulated and polyethylenimine-complexed siRNA caused a different immune response than siRNA incorporated with polylysine, which forms a larger delivery particle. It has also been shown that the immunostimulatory properties of the lipid-like delivery vehicle ND98-5 (Figure 4) depend significantly on formulation approach.47

Figure 4
Examples of siRNA delivery systems. (a) Stable nucleic acid lipid particles (SNALPs) encapsulate siRNA as cargo. They are formulated with cationic and neutral lipids, cholesterol, and poly(ethylene glycol) (PEG). (b) Cyclodextrin polymers form nanoparticles ...

Off-target gene silencing

Early reports described siRNA-induced silencing as extremely specific: a single mismatch in the target site completely abolished silencing.1 However, it was soon demonstrated that siRNAs with partial sequence homology to off-target mRNAs could affect the expression of many genes.48,49 Off-target silencing both complicates experimental results and can lead to toxicity.50 One cause of off-target silencing is improper strand selection by RISC. Passenger-strand silencing can be avoided by selecting siRNA sequences with high thermodynamic asymmetry or by chemically modifying the sense strand, as described above.

Guide strand off-targeting commonly occurs through complementarity of the siRNA 5′ end to the 3′ untranslated regions (UTRs) of mRNA.22 Positions 1–8 of the 5′ end of the guide strand are referred to as the “seed region,” a sequence that plays a key role in target recognition by endogenous microRNA (miRNAs).51 miRNAs regulate gene expression through incomplete hybridization of the seed region with 3′UTRs, which results in gene suppression by translational inhibition and deadenylation (Figure 1).52,53 As 3′ UTR sequences are shared by many mRNAs, a single miRNA can regulate hundreds of genes.54,55,56,57,58 siRNAs and miRNAs share the same silencing machinery, and siRNA off-targeting is thought to result from siRNAs entering the miRNA pathway (Figure 1).22 These miRNA-like effects can result in downregulation of scores of off-target genes.11,59,60

Though miRNA-like effects are responsible for most off-target gene regulation,3,61 partial complementarity of siRNAs to coding regions of mRNA can also cause unintended silencing. Significant gene silencing has been observed with as little as 11 nucleotides of continuous sequence homology in coding regions.49

To avoid miRNA-like off-targeting, siRNA design algorithms attempt to select sequences with minimal seed-region complementarity to 3′ UTRs.62 However, partial homology with some UTRs is unavoidable: an analysis of the human 3′UTR database revealed that even the most infrequent 7-nucleotide seed sequence still had complementarity with 17 different 3′ UTR sequences.63 Alternatively, chemical modification can be used to minimize miRNA-like effects. Efforts have focused on modifications that destabilize hybridization between the seed sequence and the 3′UTR.

The most common modification for minimizing off-target effects is 2′-OMe in the guide strand seed region, specifically at position 2. In a microarray study, this modification mitigated effects on 80% of off-target transcripts.60 It has been suggested that the bulkiness of the 2′-OMe affects the conformation of the complex formed from Ago, the guide strand, and the target mRNA in a way that inhibits binding to partially complementary targets more than to fully complementary targets. Incorporation of UNA in the seed region, particularly in position 7, also destabilizes siRNA-3′UTR hybridization,41 and was able to reduce the number of off-target transcripts by >90%.9,10 Both methylation and UNA were able to reduce off-targeting without reducing the potency of on-target silencing.9,10,60

While destabilization of the seed-region duplex has been shown to mitigate off-targeting, stabilization of the length of the duplex may improve specificity by other mechanisms. Of the four human Argonaute proteins, Ago-2 is the only one with endonuclease activity required for siRNA-mediated target cleavage.17 In coimmunoprecipitation studies, siRNA duplexes interacted with all four Ago proteins, however, strands of duplexes with increased hybridization stability were only efficiently separated by Ago-2. In the same study, duplexes stabilized by LNA demonstrated both reduced seed-region off-targeting and improved on-target potency.64

Modifications such as methylation, UNA, and LNA can provide the additional benefit of reducing the immunostimulatory effects of siRNA without reducing potency.15,35,41,42 Optimization of siRNA sequence along with combinations of chemical modifications enables the design of siRNAs that potently silence target genes with minimal off-target silencing or immunogenicity.

Saturation of the RNAi machinery

Exogenous siRNAs share components of the RNAi pathway with endogenous miRNAs, and competition for RNAi machinery may inhibit normal gene regulation by miRNAs. Several studies have demonstrated the saturability of the RNAi pathway and the toxic effects that can result from saturation.65,66,67,68,69 RNAi pathway saturation was first observed in experiments using high expression of short hairpin RNAs (shRNAs). ShRNAs are transcribed from virus or plasmid vectors and have a stem-loop structure similar to that of pre-miRNAs that enters the RNAi pathway. In cultured cells, overexpression of shRNAs from plasmid vectors delivered by transfection reagents inhibited miRNA activity by saturating Exportin-5,70 a protein required for transport of shRNAs and miRNA from the nucleus to the cytoplasm. In adult mouse livers, sustained high expression of shRNAs, achieved using viral delivery vectors, resulted in dose-dependent liver injury and fatality that was associated with decreased expression of liver miRNAs.65

Subsequent studies demonstrated that competition for RNAi machinery could also occur downstream from Exportin-5, in the pathway relevant to silencing by synthetic siRNAs. When multiple siRNAs were cotransfected into cells, efficacies of individual sequences were reduced, suggesting competition for downstream silencing machinery.66,71 siRNAs were also observed to affect the activity of endogenous miRNAs and of cotransfected shRNAs.66 Overexpression of Expo-5 only partially relieved this competition, suggesting saturation of downstream components of RISC.66 Competition between transfected siRNAs and endogenous miRNAs was also suggested by an examination of genome-wide transcript levels from a set of 150 published experiments. The analysis found that transfection of siRNA, while silencing genes complementary to siRNA sequences, also led to increased expression of common miRNA targets.69

While Xpo-5 was previously identified as one saturable component of the RNAi pathway, recent overexpression studies with shRNA have implicated the Ago proteins as key factors in the saturation of RNAi. In adult mice, overexpression of Xpo-5 enhanced shRNA efficiency but increased hepatotoxicity. Ago-2/Xpo-5 coexpression enhanced shRNA silencing, reduced hepatotoxicity, and increased RNAi stability.72 Of the four human Argonautes, Ago-2 was identified as the primary determinant of RNAi efficacy, toxicity, and persistence both in cell culture and in mouse liver.72

The harmful effects of RNAi saturation may be avoidable by limiting doses of exogenous RNA.72 Hepatotoxic effects in mice were abrogated when shRNAs were expressed under milder promoters.73 Therapeutic levels of gene silencing in vivo have been observed without perturbing miRNA function. In mice and hamsters, lipid nanoparticle -delivered siRNAs achieved 80% silencing of clinically relevant gene targets without affecting endogenous miRNA activity.73

Delivery material toxicity

Systemic or cellular toxicity can also be caused by delivery reagents themselves. In many of the most efficient in vivo delivery systems, the therapeutic dose of siRNA is accompanied by more than seven times its weight in delivery materials,74,75,76 making vehicle-related toxicity an important consideration. Delivery material toxicity can occur through various mechanisms including immunostimulation (see above), membrane destabilization, and alterations in cellular signaling or gene expression.

One element of the toxicity of lipoplexes and polyplexes is often attributed to their positive charge, which is exacerbated in low pH environment of the endosomes and lysosomes.77 Mice treated with certain cationic lipid nanoparticles showed signs of hepatotoxicity and a systemic interferon type I response, attributed in part to activation of TLR4.78 This effect was not observed in mice treated with neutral or negatively charged particles.78 Cellular toxicity of cationic lipids has been linked to increased production of reactive oxygen species79,80 and subsequent rise in cellular calcium levels,79 which were not observed with neutral or negatively charged lipids. Cholesterol-derived cationic amphiphiles are known to inhibit protein kinase c (PKC),77,81 which can cause cytotoxicity.

Cationic lipid toxicity varies greatly with structure and formulation. Cholesterol-derived lipids with quaternary ammonium head groups can be potent PKC inhibitors and are more toxic than those with tertiary amines.77,81 Introduction of heterocyclic rings into head groups can delocalize charge and in some cases reduce this toxicity.77,82,83 Lipids that contain biodegradable linkages between their polar head groups and hydrophobic tails are also associated with lower toxicity.77,84 Use of ionizable cationic lipids, which are neutral in circulation but become positively charged in the acidic endosomal environment, may reduce nonspecific disruption of cell membranes.85 Formulation of cationic lipids with nucleic acids partially neutralizes their positive charge, and the toxicity profiles of lipids alone can be distinct from those of lipids in formulation with nucleic acid.77,86

Unmodified polyethyleneimine (PEI) has been shown to induce systemic and/or cellular toxicity depending on structure and formulation. Both linear and branched PEI can cause cytotoxicity by damage to cell membranes and by depolarization of mitochondrial membranes, leading to apoptosis.87 PEI formulations with high ratios of delivery material to siRNA can also cause systemic toxicity due to their high positive charges. Particles with high surface charges interact with the negatively charged membranes of blood cells, which can lead to erythrocyte aggregation. Large aggregates can have difficulty passing through capillaries, leading to serious systemic effects.88 Grafting of poly(ethylene glycol) (PEG) to PEI can mitigate the problem of erythrocyte aggregation88 and reduce overall cytotoxicity.88,89 PEG-PEI also is not inert and can affect cellular processes differently than PEI alone. PEG-PEI has been shown to activate apoptotic pathways in a cell line- and concentration-dependent manner.90

Microarray studies have begun to reveal that delivery materials can induce gene expression changes, which can potentially cause toxicity and other off-target effects. Commonly used transfection reagents have been shown to induce their own gene expression signatures even in the absence of siRNA.89,90,91 The cationic lipids oligofectamine and lipofectin altered the expression of dozens of genes involved in various cellular processes including proliferation, differentiation, and apoptosis pathways.91 PEI (25 kDa) and PEI-PEG altered the expression of several genes involved in apoptosis and inflammatory signaling.89,90 In some stably transfected cell lines, signaling triggered by PEG-PEI increased cytomegalovirus promoter activity, causing an increase in reporter gene expression rather than siRNA-mediated knockdown.90 Gene signatures of these materials can change when the material is formulated with nucleic acid92 and can vary greatly for different delivery materials, cell types, and treatment times. Structure of the delivery material affects gene expression signatures as well. When injected intratumorally, branched PEI induced more gene expression changes than linear PEI,92 consistent with the higher reported toxicity of branched PEI.93 An understanding of the alterations in gene expression due to delivery materials is necessary to properly interpret experimental results and to develop delivery formulations with minimal off-target effects.

While delivery materials alone can induce gene expression changes and cause toxicity, delivery materials in formulation with siRNA may cause toxicity and off-target effects by simply introducing siRNA into different cellular environments. Delivering siRNAs into endosomes increases their potential to stimulate endosomal TLRs to elicit an immune response. Delivering siRNAs into the cytoplasm increases their potential to saturate the RNAi machinery or to enter the miRNA pathway, causing off-target silencing. As such, delivery materials alone and in formulation with siRNA (even siRNA sequences that lack targets) can have distinct gene signatures.92

The Pharmacokinetics of siRNA Delivery Systems

Pharmacokinetic considerations determine the ability of delivery systems to reach the cytoplasm of target cells and therefore to induce a therapeutic effect. The fate of intravenously administered siRNA depends heavily upon its structure, chemical modification, and delivery formulation. These factors affect a delivery system's stability in circulation, its tendency to extravasate or exit blood vessels, its ability to enter cells, and its likelihood of being cleared from circulation without being delivered.

Pharmacokinetic parameters such as circulation half-lives, area under the concentration–time curve, elimination rates, and bioavailability are functions of these various physiological processes. For example, a short circulation half-life could be explained by rapid excretion of the formulation in urine, or it may result from the rapid uptake of the formulation by tissues. This section aims to describe the mechanisms and processes that shape pharmacokinetic behavior of siRNA delivery systems. Detailed pharmacologic data for various delivery systems are beyond the scope of this review.

siRNA in circulation

Intravenously administered siRNA is exposed to serum proteins, nucleases, and cells of the innate immune system. The resulting processing and biodistribution of injected siRNA varies with both the structure of the siRNA and the delivery system in which it is carried. Successful siRNA delivery has often required the use of a delivery vehicle because naked, unmodified siRNA is degraded by serum RNAses and cleared from the bloodstream within minutes.42,94,95 In contrast, many delivery formulations, such as lipid and polymer systems, protect siRNA from nucleases by encapsulating it as cargo inside nanoparticles.96,97

The nuclease stability of unformulated siRNA can also be dramatically improved by making chemical modifications to the RNA backbone.98 A common strategy involves modification of the ribose 2′-OH group, as this functional group is critical to the mechanism of many serum RNAses.99,100 Among the most effective backbone modifications for serum stability improvement are the substitution of the ribose 2′ hydroxyl with 2′-fluorine or 2′-methoxy groups (Figure 2). If placed judiciously within the duplex, such modifications can add stability without affecting RNAi activity.15,95 A similar effect is achieved by incorporation of LNA nucleotides, which contain a methylene bridge connecting the 2′ oxygen to the 4′ carbon40,101 (Figure 2). In addition, modifications to the linkages between sugars can be incorporated into the 3′ overhangs of siRNA to confer resistance to exonucleases. The most common of these is the replacement of certain phosphodiester linkages with phosphorothioate linkages (Figure 2).102 Combinations of chemical modifications have been shown to improve siRNA stability in plasma15,103 as well as increase its circulation time.15,42,95 The mechanism by which chemical modification improves circulation time in vivo is unknown. As renal filtration is expected to be slower for large molecules (such as intact siRNA) than for small molecules (such as fragments of degraded siRNA), an increase in serum stability may partly explain the increase in circulation time for chemically-stabilized siRNA.

In addition to enzymes, circulating siRNAs also interact with or bind to various components of the blood, such as red blood cells and serum proteins, that can influence their pharmacokinetics. In some cases, interaction between serum proteins and siRNA can improve delivery outcomes by increasing circulation time and enhancing uptake into target tissues.104,105 For example, the incorporation of cholesterol-conjugated siRNA into serum lipoproteins has been shown to improve its circulation time, reduce renal clearance, and facilitate uptake into hepatocytes via lipoprotein receptors.104 It has also been found that certain ionizable lipid nanoparticles are subject to apolipoprotein E adsorption, which can mediate their uptake by hepatocytes via the low-density lipoprotein receptor.105 In other cases, interaction with serum proteins or red blood cells can be detrimental, leading to formation of aggregates that are readily opsonized or worse. Large aggregates can have difficulty passing through capillaries, which can lead to accumulation in lung capillary beds and even lung hemorrhage.88 More commonly, adsorption of serum proteins, especially components of the complement system, leads to rapid clearance of particles by the cells of the mononuclear phagocyte system.106,107 Phagocytosis of particles is stimulated by the interaction of adsorbed proteins with receptors on phagocytes.106 Adsorption of these proteins can be minimized by functionalizing the surface of particles with hydrophilic polymers such as PEG.106,108,109 PEGylation of siRNA delivery systems is a widely used strategy for minimizing protein adsorption and reducing nonspecific uptake to create long-circulating delivery systems.106,107

Extravasation

Circulating siRNA must leave the bloodstream by crossing the vascular endothelium in order to reach many target tissues. The structure and permeability of the endothelium vary across different tissue types, making some tissues more accessible to macromolecular therapeutics than others.110 In healthy tissues, endothelia are described as continuous or discontinuous and fenestrated or nonfenestrated (Figure 5). In continuous endothelium, cells are connected by tight junctions and adherens junctions that allow the passage of water and small solutes but prevent the passage of molecules larger than 3 nm.110 Macromolecules such as albumin traverse continuous endothelia by transcytosis.111 In fenestrated endothelia, cells contain transcellular channels, or fenestrae, that allow direct passage of water and small solutes across endothelial cells. Fenestrae in continuous endothelia are typically 40–60 nm in diameter112 and are spanned by a thin glycoprotein diaphragm that prevents the passage of macromolecules.110,113 Discontinuous endothelium, found primarily in liver sinusoids, is characterized by large fenestrae (100–200 nm)110,114 that lack a diaphragm. The sinusoidal endothelium also lacks an organized basement membrane110 and therefore provides minimal resistance to the passive transport of macromolecules and nanoparticles from the bloodstream to the liver interstitium. This, along with the well-perfused nature of the liver, may explain why the liver is one of the primary sites of accumulation of many systemically administered siRNA formulations.42,74,85,115,116,117 Though the structure of the liver endothelium allows access to hepatocytes, therapeutics can also be absorbed by resident macrophages in the liver, called Kupffer cells.118 In some cases, accumulation of therapeutic particles the liver is due mainly to phagocytosis by Kupffer cells rather than uptake by hepatocytes.118

Figure 5
Endothelial heterogeneity. Capillary endothelia can be continuous or discontinuous, fenestrated or nonfenestrated. The structure of the endothelium affects the permeability of the vasculature to the passage of siRNA therapeutic formulations. EC, endothelial ...

Another site that can have increased vascular permeability is malignant tissue. In tumors, blood vessels can be heterogeneous in size, spacing, tortuosity, and permeability.112,119,120,121 Tumor endothelia can have large fenestrae,122 unusually thick or thin basement membranes,112 and intercellular gaps that can be as wide as a few microns.123 As a result, tumor endothelia are sometimes more permeable than those of healthy tissues,124,125 allowing delivery systems to extravasate relatively easily. As certain tumors also have poorly functioning lymphatic vessels,126,127 interstitial fluid is not efficiently drained from tumor tissue and nanoparticles tend to accumulate in the interstitium. This accumulation, due mostly to high vascular permeability in addition to poor lymphatic drainage, is known as the enhanced permeation and retention effect, which is exploited by many nanoparticle systems for passive tumor targeting.112,128,129 The enhanced permeation and retention effect aids larger particles (~100 nm) to accumulate in regions of leaky vasculature in tumors; however, smaller particles (<60 nm) diffuse more readily through the interstitium and more deeply penetrate tissues.112,130

Phagocytosis can also play a major role in the clearance of siRNA delivery systems from the blood. As a result, accumulation of siRNA in immune cells is also observed.131 In one study, fluorescence molecular tomography imaging showed that siRNA delivered by lipidoid nanoparticles exited the blood pool primarily into the spleen, bone marrow, and liver, with a blood half-life of 8.1 minutes. siRNA taken up by the liver was excreted by the hepatobiliary tract, while siRNA in the spleen and bone marrow localized to phagocytic cells including neutrophils, macrophages, dendritic cells, and monocytes. This biodistribution pattern was exploited to use siRNA to reduce the accumulation of monocytes in sites of inflammation by silencing of the chemokine receptor CCR2. Knockdown of this target in inflammatory monocytes attenuated the damaging effects caused by inflammation in multiple disease models, including atherosclerotic plaques, myocardial infarction, diabetes, and cancer.131

Cell uptake

Delivery systems that extravasate and diffuse into tissues must cross cell membranes into the cytoplasm to trigger RNAi. Most siRNA delivery formulations traverse cell membranes through membrane-derived transport vesicles in the tightly regulated process of endocytosis. Endocytosis encompasses two broad categories: phagocytosis and pinocytosis. While phagocytosis is only associated with specific cell subtypes, pinocytosis is common to all cells.

Phagocytosis is used by specialized immune cells including macrophages, neutrophils, and monocytes to clear the body of large particles such as pathogens or dead cells.132,133,134 Before involution by phagocytosis, foreign materials are opsonized, which involves their binding to opsonin proteins such as immunoglobulins, complement proteins, and blood serum proteins. Complexation with these proteins marks foreign materials for phagocytosis.133,134,135,136 Rho-family GTPases together with specific cell-surface receptors and signaling cascades determine the mode of phagocytosis.132,137 Macrophages, for example, utilize Fc receptors to recognize antibody-bound targets. Resulting activation of Cdc42 and Rac triggers actin assembly and creates finger-like protrusions that engulf antibody-bound targets.133,134,138 The formation of the resulting vesicle, called a phagosome, stimulates the cell's inflammatory responses and the entrapped target is bombarded with hydrolases, oxygen radicals, and a low pH environment in an attempt to destroy the foreign material.139

Macropinocytosis is a nonspecific endocytic process similar to phagocytosis that occurs in most cells. Rho-GTPase mediates actin-driven membrane protrusions that extend out and collapse over large volumes of extracellular milieu, entrapping particles in large vesicles called macropinosomes.140,141,142 These large vesicles can be up to 1–5 µm in diameter, and their intracellular fate is uncertain, although there is evidence that they shrink and fuse with lysosomal compartments for degradation of entrapped materials.

Clathrin-mediated endocytosis (CME), the most prolific internalization mechanism, occurs constitutively in all tissue types and participates in the uptake of vital nutrients and in intercellular signaling through receptor-dependent and receptor-independent pathways.141,143,144,145,146 Iron-bound transferrin protein, for example, is internalized after association with transferrin receptors initiates invagination of the plasma membrane. Pits that form in the membrane around protein-bound receptors are coated by clathrin, a triskelion-structured protein that assembles into polygonal cages on the cytoplasmic face of the membrane.143,144,146 Receptor-independent CME is able to induce these internalization events simply by nonspecific ionic or hydrophobic interactions with the plasma membrane. Fission of the coated pit from the membrane is mediated by the GTPase dynamin, forming a clathrin-coated vesicle roughly 100–120 nm in size. Internalization occurs on the order of minutes, and resulting vesicles undergo endosomal maturation. Early endosomes have a pH around 6 and later fuse with acid hydrolase-containing prelysosomal vesicles to form late endosomes (pH ~5). The acidic and enzymatic environment of the lysosome is aimed at degradation of the vesicular cargo.147

Caveolae-mediated endocytosis (CvME) is used by cells to internalize serum proteins such as albumin and essential metabolites such as cholesterol and folic acid. CvME involves flask-shaped invaginations, on the order of 50–100 nm, of the plasma membrane called caveolae.143,145,148,149 The dimeric protein caveolin is responsible for the shape and structure of caveolae, whose membranes are enriched with cholesterol and sphingolipids. CvME is a tightly regulated process that is believed to be driven by receptor-ligand interactions, with vesicle formation being mediated by the GTPase dynamin. Caveolin-associated vesicles do not contain any enzymatic mixtures and do not enter a degradative pathway by maturing into lysosomes like CME. The internalization kinetics of CvME are much slower than those of CME, with an uptake half-time on the order of 20 minutes.

Recently, endocytic pathways have been uncovered that do not fall into any of the previously described categories.145,148,149,150 These processes are often referred to as clathrin- and caveolae-independent pathways, and occur at cholesterol-rich microdomains of the plasma membrane that are similar to caveolae. These lipid “rafts” are small structures approximately 40–50 nm in size, and have unique membrane protein compositions that may play a role in vesicle formation. The mechanisms that stimulate this endocytic pathway are still poorly understood, and currently the only way to determine the contribution of this pathway to a cargo's internalization is by elimination of the other pathways.

In general, siRNA delivery particles enter cells via endocytosis. Some systems incorporate targeting ligands designed to stimulate receptor-mediated endocytosis.151 Other systems, such as lipophilic conjugates and some ionizable lipid nanoparticles, exploit endogenous targeting ligands by associating with serum lipoproteins, which are taken up by hepatocytes by receptor-mediated endocytosis.104 Untargeted siRNA lipoplexes are also taken up mostly by endocytosis, but a small number of complexes enter cells through another pathway, possibly by direct fusion of the lipoplex with the cell membrane. Interestingly, this minor pathway was shown to be responsible for functional siRNA delivery in certain systems, as blocking endocytosis did not abolish gene silencing.152

Uptake of siRNA delivery systems by endocytosis leads to sequestration of the delivery system inside intracellular vesicles. Escape from these vesicles may represent a major barrier to triggering RNAi and thereby affect the efficacy of delivery systems. While the mechanisms of vesicular escape of synthetic materials are still poorly understood, polymers with many protonizable groups are thought to induce lysis of endosomes by pH buffering.46,153 Protons are transported into endosomes accompanied by counter ions, and absorption of protons by polymers leads to increased concentrations of counter ions, osmotic swelling, and endosomolysis. Certain ionizable lipid systems undergo a phase transition at low pH, resulting in membrane destabilization that promotes release of material from endosomes.85,154 By converting from a lamellar to an inverted hexagonal phase, these lipids are thought to increase their interactions with the endosomal membrane, leading membrane destabilization and inducing endosomal release. Still other systems employ acid-labile materials155 or modified cell-penetrating peptides.156 Dominska and Dykxhoorn provide a more detailed review of how various delivery systems overcome the problem of endosomal release.157

Elimination

siRNA that is not degraded or endocytosed is removed from circulation by excretion in urine or bile. Renal filtration occurs in the specialized capillaries of the kidney glomerulus (Figure 6). Water and small molecules are able to pass through capillary walls into the urinary space, while macromolecules remain in circulation. The glomerular filtration barrier is composed of three layers: the endothelium, the glomerular basement membrane, and a layer of epithelial cells called podocytes.158 The glomerular endothelium is continuous and populated with abundant 60–100 nm fenestrations.159 Endothelial cells are supported by the glomerular basement membrane, which provides an additional diffusion barrier.160 Podocytes play an important role in the size selectivity of the glomerular filtration barrier.161 Slit diaphragms, composed of proteins that bridge the space between podocytes, create ~8 nm pores162,163,164 that filter waste before excretion in the urine.165,166

Figure 6
The glomerular filtration barrier. The capillaries of the kidney glomerulus participate in the filtration of blood to form urine. The glomerular filtration barrier consists of three layers: endothelial cells, the glomerular basement membrane, and podocytes. ...

The liver, which is also part of the body's clearance system, mediates the secretion of compounds into bile ducts, and are eliminated from the body via the intestine.167,168,169 Hepatocytes secrete bile into hepatic ducts, where it can be emptied directly into the small intestine or stored in the gall bladder. Various studies have shown that naked, unstabilized siRNA is rapidly eliminated from the bloodstream via renal filtration, with serum half-lives of less than 5 minutes.42,94,103 Chemically stabilized siRNAs have been reported to circulate for 30–50 minutes,42,103 and also to undergo renal clearance.42,103 While kidney filtration has often been assumed to be the major route of siRNA elimination, a recent study demonstrated the importance of the hepatobiliary pathway in the clearance of chemically stabilized siRNAs, both naked and in formulation.170

Conclusions

The many interactions between siRNA and the body continue to be elucidated as more delivery systems are studied. In the design of delivery vehicles, it is natural to focus on the aspects of pharmacokinetics and pharmacodynamics that contribute directly to the desired therapeutic effect, yet many other interactions exist that affect the overall potency and tolerability of a formulation. The information contained in this review provides a brief overview of these interactions, which can serve as a guide in design of new systems and in the optimization of existing systems.

Notes

The authors declared no conflict of interest.

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