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Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Author's personal copy Advanced Drug Delivery Reviews 64 (2012) 1508–1521 Contents lists available at SciVerse ScienceDirect Advanced Drug Delivery Reviews journal homepage: www.elsevier.com/locate/addr RNAi-based nanomedicines for targeted personalized therapy☆ Ala Daka, Dan Peer ⁎ Laboratory of Nanomedicine, Department of Cell Research and Immunology, George S. Wise Faculty of Life Science, Israel Center for Nanoscience and Nanotechnology, Tel Aviv University, Tel Aviv, 69978, Israel a r t i c l e i n f o Article history: Received 5 April 2012 Accepted 13 August 2012 Available online 5 September 2012 Keywords: Short interfering RNA (siRNA) In vivo Systemic delivery Nanoparticles Clinical trials Personalized medicine a b s t r a c t RNA interference (RNAi) has just made it through the pipeline to clinical trials. However, in order for RNAi to serve as an ideal personalized therapeutics and be clinically approved—safe, specific, and potent strategies must be devised for efficient delivery of RNAi payloads to specific cell types, which despite the immense potential, remains a challenge. Through evaluating the recent reported studies in this field, we introduce the progress in designing targeted nano-scaled strategies that are anticipated to overcome the delivery drawbacks and along with the exciting “omics” discipline to personalize RNAi-based therapeutics. © 2012 Elsevier B.V. All rights reserved. Contents 1. 2. 3. 4. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . RNAi: A powerful approach for personalized medicine . . . . . . . . . . . . . 2.1. RNAi: An advantageous strategy over conventional therapeutics . . . . . 2.2. Personalizing RNAi-based medicine . . . . . . . . . . . . . . . . . . 2.2.1. RNAi-based screening . . . . . . . . . . . . . . . . . . . . 2.2.2. The design of specific siRNAs . . . . . . . . . . . . . . . . . Strategies for in vivo delivery of RNAi molecules . . . . . . . . . . . . . . . . 3.1. Local delivery of RNAi molecules . . . . . . . . . . . . . . . . . . . . 3.2. Systemic delivery of RNAi molecules . . . . . . . . . . . . . . . . . . 3.2.1. Delivery challenges: Rapid clearance of RNAi molecules . . . . . 3.2.2. Delivery challenges: Stability of the RNAi molecules . . . . . . 3.2.3. Off-target effects of induced RNAi: Immunostimulatory effects . 3.2.4. Off-target effects of induced RNAi: miRNA-like effects . . . . . 3.2.5. Off-target effects of induced RNAi: Saturation of the RNAi machinery Systemic NP-mediated delivery of RNAi molecules . . . . . . . . . . . . . . . 4.1. Passive delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.1. Lipid-based nanoparticles (LNPs) . . . . . . . . . . . . . . . 4.1.2. Cationic LNPs . . . . . . . . . . . . . . . . . . . . . . . . 4.1.3. Lipidoids . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.4. Polyplexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1509 1509 1509 1510 1510 1510 1510 1510 1510 1510 1511 1511 1511 1511 1512 1512 1512 1512 1513 1513 Abbreviations: mAb, monoclonal antibody; ApoB, apolipoprotein B; BD, biodistribution; CME, clathrin-mediated endocytosis; CNT, carbon-nanotubes; DC-6-14, O,O′ditetradecanoyl-N-(α-trimethylammonioacetyl) diethanolamine chloride; DOPE, dioleoylphosphatidylethanolamine; DOTAP, N-[1-(2,3-dioleoyloxy)-propyl]-N,N,N-trimethylammonium methylsulfate; DOTMA, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride; dsRNA, double stranded ribonucleic acid; EPR, enhanced permeability and retention; I.V., intravenous; miRNA, micro ribonucleic acid; MPS, mononuclear phagocytic system; MW, molecular weight; NP, nanoparticle; nt, nucleotide; PEG, polyethylenglycol; PEI, Polyethyleneimine; PC, phosphatidylcholine; P.I., post injection; PK, pharmacokinetics; RES, reticuloendothelial system; RISC, RNA-induced silencing complex; RNA, ribonucleic acid; shRNA, short hairpin ribonucleic acid; SNALP, stable lipid-nucleic acid particle; T1/2, half time; TLR, Toll-like receptor. ☆ This review is part of the Advanced Drug Delivery Reviews theme issue on “Personalized nanomedicine.” ⁎ Corresponding author at: Center for Nanoscience and Nanotechnology, Tel Aviv University, Tel Aviv, 69978, Israel. Tel.: +972 3640 7925; fax: +972 3640 5926. E-mail address: [email protected] (D. Peer). 0169-409X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.addr.2012.08.014 Author's personal copy A. Daka, D. Peer / Advanced Drug Delivery Reviews 64 (2012) 1508–1521 4.1.5. Carbon-nanotubes (CNTS) . . . . . Active, target-specific RNAi delivery . . . . . 4.2.1. Peptide-based targeting moieties . . 4.2.2. Aptamer/siRNA chimeras . . . . . . 4.2.3. Other small molecules-siRNA NPs . . 5. Intracellular delivery of NP-associated RNAi molecules 5.1. Delivery to the cell membrane . . . . . . . 5.2. Endosomal escape . . . . . . . . . . . . . 6. NP-mediated RNAi: Clinical studies . . . . . . . . . 7. Conclusions . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . 4.2. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Introduction RNAi is a specific regulatory mechanism of most eukaryotic cells, in which small double-stranded ribonucleic acids (dsRNAs) efficiently control gene expression in a complementarity-dependent manner. This natural mechanism, which involves diverse families of small non-coding RNA regulators, is presumed to protect against pathogenic infections as well as regulate various biological pathways [1]. Among the endogenous regulators, microRNAs are of great importance in controlling numerous physiological processes. A long hairpin-structured dsRNA (pre-miRNA) is transported out of the nucleus to the cytoplasm, where it is cleaved by RNase III endonuclease DICER to generate a mature miRNA. The double-stranded miRNA, which is 21–25 nt long with 5′-phosphorylated ends and 2-nt 3′ overhangs, incorporates into a ribonucleoprotein complex, the RNA-induced silencing complex (RISC) that unwinds the RNA duplex and discards the sense strand. The antisense strand (guide strand) then guides RISC to its target mRNA. The miRNA guide strand most often pairs partially with the mRNA transcript and triggers its translation repression or leads to its degradation [2,3]. Utilizing this native mechanism for therapeutic and diagnostic purposes can be achieved by exogenous delivery of synthetic RNAi molecules. However, due to their net negative charge and relatively large size compared to vasculature effective pore size, RNAi molecules are less likely to readily cross biological barriers; thus, efficient systemic delivery necessitates the use of physical protection (i.e. carriers/reagents) and chemical modifications that combined protect RNAi molecules until reaching their site of action and facilitate cell penetration yet maintaining their functionality. One approach to exogenously trigger RNAi in cells is the use of short-hairpin RNA (shRNA) or miRNA mimics. Most commonly, these strategies are applied by introducing DNA [4] or stem-and-loop containing RNAs into the nucleus in a viral vector-based manner where shRNAs are synthesized and processed via the miRNA pathway [5]. Although shRNA method is considered to be an efficient way of inducing RNAi, mainly because of the induction of prolonged and stable gene-silencing that is favored in the case of chronic diseases [6,7], the toxic effects that have been reported in animals models and patients treated with shRNAs [5,8–10] limit its clinical use. These effects are apparently due to: 1) competition with and overload of endogenous RNAi machinery. 2) Imperfect complementarity of shRNA with its target mRNA that may lead to nonspecific silencing and off-target effects. 3) Stimulation of the immune system. 4) Insertional mutagenesisinduced tumorigenicity possibly enhanced upon viral delivery, and, 5) undesirable long-term expression especially in non-chronic disease models [10]. Furthermore, shRNA-based RNAi induction necessitates intranucleus localization; an additional hurdle that must be mitigated. Consequently, non-viral approaches provide an alternative way to induce gene silencing. Conjugating or incorporating RNAi molecules to or into nanoparticles (NPs), are the major non-viral nano-scaled delivery vehicles that have been recently developed, and been approved to overcome major viral vector drawbacks. Nanobiotechnology offers a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1509 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1514 1515 1515 1516 1516 1516 1517 1517 1517 1518 1519 1519 vast number of nanomaterials enabling the development of a variety of novel systems (such as liposomes, nano/micro-spheres, nanotubes etc.) feasible for biological and medical applications [11,12]. In this review, we present the major impediments for translating RNAi from a genomic tool to clinical practice, review the recent progress in designing targeted nano-scale delivery systems to tackle these impediments and highlight the necessity of “omics” technology that anticipates revolutionizing and expediting the development of the area of personalized diagnostics, disease management and therapeutics. 2. RNAi: A powerful approach for personalized medicine Introducing RNAi in medicine is achieved by designing specific RNAi regulators such as miRNA mimics and small interfering RNAs (siRNAs). Antagomirs are novel synthetic 21–23 nt single stranded miRNA analogs that have been recently developed for specific miRNA silencing. Since miRNAs are involved in many biological and pathological processes, and given that Antagomir-mediated gene-regulation is differential and selective as it induces silencing of specific miRNA targets derived from the same transcript [13], it is therefore rational to utilize it for therapeutic intervention. However, because most of the RNAi-related studies and NPs developing were conducted with siRNAs rather than miRNA-based molecules, the focus of this review is mainly on siRNA-carrying systems. siRNA is a chemically synthesized RNA-duplex, which is 19–23 nt in length. siRNAs are designed so they have 2-nt 3′ overhang, similar to endogenous miRNAs, allowing DICER to recognize and further process them. However, while miRNA targets partially complements mRNA, siRNAs trigger a sequence-specific mRNA cleavage of perfectly complementary targets [14,15]. This feature of specific gene-regulation combined with the high potency and safety compared to conventional medicines or other classical anti-sense and gene silencing methods (such as antisense oligonucleotides and shRNAs [8,16]) make it possible to utilize siRNA as a safer platform for therapy in personalized medicine. 2.1. RNAi: An advantageous strategy over conventional therapeutics RNAi-induced gene silencing mirrors the inhibitory effects of conventional pharmaceuticals, such as protein-based drugs (e.g. antibodies and vaccines) and small molecules. The inhibitory effects of these drugs is mainly achieved by blocking their targets function, however some disease-related molecules, primarily proteins, do not have enzymatic function or have a conformation that is not accessible to conventional drugs or small molecules compounds, hence are considered as “non-druggable” targets. Comparable "non-druggable" molecules have been successfully targeted by RNAi approach in vivo [17–21] demonstrating an exclusively allele-specific gene silencing [22,23]. Additionally, blocking target's gene expression rather than its activity is more efficient, as single mRNA molecule translates multiple copies of protein. Another advantage of RNAi over conventional pharmaceuticals is the ease of synthesis and production of potent regulators. Opposing to protein-based drugs, RNAi synthesis does not necessitate cellular expression systems Author's personal copy 1510 A. Daka, D. Peer / Advanced Drug Delivery Reviews 64 (2012) 1508–1521 or refolding steps. As for small molecules-based drugs, the major disadvantage is the slow and difficult lead identification and optimization [24], whereas the identification of functional and specific RNAi constructs is rapid and involves well-demonstrated strategies and algorithms that facilitate their optimization. However, whilst protein and some small molecules-based drugs do not require intracellular access to obtain functionality, RNAi-based therapeutics necessitate intracellular delivery, an additional challenging task for the development of a new efficient RNAi-based therapeutic. 2.2. Personalizing RNAi-based medicine Given that variation in gene expression patterns may lead to variation in response to a treatment often observed among different patients, personalized medicine aims to use specific therapeutics best suited for an individual considering pharmacogenetic and pharmacogenomic data [25]. The arising “omics” technology field, which beyond genomics, proteomics and metabolomics that contribute to further understanding of the bases of diseases as well as the interaction of the transcriptomes with their environment, leading to identify disease-related genes and molecular determinants; it also refers to toxicogenomics and pharmacogenomics, which provide data on reciprocal interactions of the RNAi-based medicine and the genetic and physiological processes, facilitating the design of novel individualized RNAi constructs accordingly [26]. 2.2.1. RNAi-based screening Genome-scale RNAi analysis is one of the more powerful genomics strategies nowadays. It was first conducted in model organisms (Caenorhabditis elegans and Drosophila). Recently, number of studies utilizing RNAi-based screens was reported in human cells, for example, it was used to identify a number of human host genes affecting influenza A virus replication [27] and west Nile virus infection [28]. In another report, siRNAs were utilized for whole genome lethality study; Tiedemann et al. transfected myeloma human cell lines with siRNA constructs against potential “druggable” targets of myeloma and measured viability. Survival genes were determined as genes that induced cell lethality upon siRNA-related silencing. This application allows identification of important survival genes for the non-curative myeloma cancer [29]. A significant advance in this field was described by Neumann et al. who developed an automated system for siRNA screening by time-lapse fluorescent imaging in live human cells for investigating the chromosome segregation during cell division. This automated platform allows detailed time-resolved and quantitative analysis of phenotype signatures and makes genome-scale RNAi screening applicable in human live cells, overcoming the limitations of end-point RNAi-based screens [30]. 2.2.2. The design of specific siRNAs Depending upon the comprehensive data on novel surface and intracellular determinants and cellular pathways involved in various pathological processes identified by pharmacogenomic and epigenomic analyses, it is now possible to design specific siRNAs. The synthesis of personalized siRNA constructs requires well-established designing and bioinformatic tools that identify a 21–23 nt with 3′ overhang double-stranded siRNA sequence against single target from siRNA libraries. This is followed by small-scale synthesis of siRNAs panel and conducting in vitro assays to determine specificity and efficacy and to eliminate off-target effects and cytotoxicity. Based on the in vitro studies, chemical modifications and refinements of the siRNA construct are applied while preserving or improving its potency, specificity and stability [31,32]. Owing to the differences in RNAi-related responses, off-target events and immune response between in vitro and in vivo systems, it is reasonable to identify numbers of siRNA sequences for in vivo applications, that commonly are the most specific, potent and safe lead constructs. Due to the availability of bioinformatic algorithms enabling the design of single siRNA construct that is cross reactive in rodents and primates, the availability of transgenic and knockdown rodents and the assumption that the physiology of non-human primates is comparable with that of humans, in vivo safety screening conducted in rodents and non-human primates presupposed to be of great importance though not sufficient when progressing to clinical studies [33]. 3. Strategies for in vivo delivery of RNAi molecules The main aim of RNAi applied in vivo is to achieve safe gene-specific silencing via tissue-specific targeting using efficient amounts of RNAi molecules. In vivo delivery of siRNA could be accomplished via local or systemic administration routes, depending on the tissues/organs that are targeted. 3.1. Local delivery of RNAi molecules Various approaches for local delivery of siRNA molecules have been recently applied in animal models. This strategy is based on injection of naked or formulated siRNA in vivo in a localized manner, and mainly includes: intranasal [34–36], intraocular [37,38], intratumoral [39,40], intramuscular [41], and intracerebral [42,43] approaches. Local administration is site-specific, i.e. the injected molecules do not circulate through the body, which offers high bioavailability of the RNAi molecules at the target tissue with low effective dosage, and allows utilizing simple siRNA formulations or even naked molecules. As exemplified in a study by Niu et al., effective gene silencing and tumor suppression upon direct subcutaneous and intraperitoneal injection of siRNA complexed with commercial transfection reagent showed specific gene silencing of human papillomavirus (HBV) 16 E6 oncogene in a cervical cancer model [44]. However, despite the technical ease of the delivery approach, efficiency and selectivity, local administration is applicable for restricted types of tissues, which are technically accessible for invasive delivery. 3.2. Systemic delivery of RNAi molecules In contrast to mucosal and subcutaneous tissues, most of body organs and cells are not accessible to RNAi molecules upon direct administration; accordingly systemic delivery strategies must be devised. The fact that nucleic acids in general are exposed to phagocytosis, nuclease degradation and are membrane-impermeable makes systemic RNAi delivery more complicated and challenging. Due to these hurdles as well as anatomical barriers, RNAi delivery entails significant optimizations prior to administration [45]. The first barrier the delivered RNAi molecule has to overcome before reaching its site of action within the target cell cytoplasm is the circulatory system, where it is imposed to deal with serum proteins including nucleases, kidney filtration and the immune system. Once the RNAi molecule reaches the target cell, further obstacles concerning endocytosis routes and endogenous RNAi pathways have to be tackled. Combined together, those impediments are known as gene silencing off-target effects, i.e. undesirable effects that could alter the siRNA-induced phenotypes and interfere with analyzing the obtained results. Below we introduce the major barriers that must be overcome when RNAi molecules are systemically administered while preserving their function. 3.2.1. Delivery challenges: Rapid clearance of RNAi molecules Naked, unmodified RNAi molecule, faces a number of obstacles during its circulation in the blood stream; the first one is its relatively small size compared to the glomerular effective pore size - as molecules less than 50 kDa (~ 10 nm) are exposed to glomerular filtration in the kidney and excretion into the urine [46,47]. Studies show that systemically administered naked siRNAs preferentially accumulated in the kidney (40 times higher than other organs) and excreted into urine within one hour [48,49]. Even though accumulation of siRNAs Author's personal copy A. Daka, D. Peer / Advanced Drug Delivery Reviews 64 (2012) 1508–1521 in the kidney may be favorable for selective renal targeting and gene silencing [48], recognition of naked siRNAs by the phagocytic system is associated with immunological and cytotoxic stimulation, that could occur right after injection through activation of circulating mononuclear phagocytosis or inside the cytoplasm and endosome compartments of the target cell. 3.2.2. Delivery challenges: Stability of the RNAi molecules Although dsRNAs are more stable compared to other oligonucleotides (ssRNA or DNA), unmodified siRNAs are still exposed to nuclease activity when administered systemically, however, chemical modifications of the delivered siRNA could overcome this degradation. Several researchers have studied the effect of modifications on siRNA function and stability [24,50]. For note, increasing stability of the modified siRNA should be accompanied with tolerance to the RNAi machinery; furthermore, more effective modifications possess the ability to increase silencing activity as well as biodistribution (BD) and pharmacokinetic (PK) properties. Since 2′-OH group of the ribose is not essential for siRNA to enter the RNAi machinery [51], diverse modifications at the 2′-position in both strands are widely used, such modifications are commonly known as 2′-modifications, and mainly referred to 2′-O, 2′-Methyl-nucleoside (2′-O-Me) and 2′-deoxyflouridine (2′-F) modifications. Another modification of the 2′ position of the ribose is the use of locked nucleic acids (LNAs), in which a methylene linkage is generated between the 2′ and 4′ positions [52]. An additional type of chemical modification is the phosphorothioate (P= S) linkage at the 3′ end. PS linkage is generated by replacing one of the nonbridging oxygen atoms on the phosphate between two riboses with a sulfur atom, resulting in exonuclease resistance [24]. These siRNA chemical alterations have been shown to successfully enhance siRNA stability both in vitro and in vivo, and in some cases to enhance potency, reduce interference with endogenous RNAi pathways and to increase serum and thermal stability [51–55], yet, more research in this field is needed as it has been reported that bulkier modification in 2′-position could interfere with RNAi machinery [56]. 3.2.3. Off-target effects of induced RNAi: Immunostimulatory effects dsRNA induces immune response as a defense mechanism against viral infection [1] upon interaction with RNA-binding proteins such as Toll-like Receptors (TLRs) and protein kinase receptor (PKR). This recognition occurs on the cell surface or within the cell (in the cytoplasm or endosome compartments) and leads to innate immunity activation, i.e. production of pro-inflammatory cytokines, such as IL-6 and TNFα, and triggering type I Interferon (IFN) pathway [57]. siRNAs can stimulate the immune system in a sequence-independent manner as in the induction of TLR3 [58] and IFN pathways [59], as well as in a sequence-dependent way as in the case of uridine (U) or guanosine adenine/uridine (GA/GU)‐rich regions recognition via TLR7 and TLR8 [60–62]. Immunostimulatory effects can also be length-dependent. While short siRNAs (12 and 16 nt long) have been reported to poorly stimulate immune cells, longer constructs increased significantly cytokine production—independent of their sequence [61]. It is important to note that immunostimulation by siRNA is cell type-dependent [60,63], for example, though non-immune cells do not express TLRs or express them at low levels, they retain the ability to recognize exogenous RNAs via alternative pathways such as the PKR pathway [64]. Besides optimizing the sequence [65], structure [66,67] or length [61] of the siRNA, chemical modifications can also be applied for mitigating immunogenicity [68]. Of note, immunogenic siRNA sequences could be deliberately applicable in specific treatments such as viral infections or cancer [64], where activation of the immune system is required in order to ameliorate the disease. 1511 3.2.4. Off-target effects of induced RNAi: miRNA-like effects Despite the fact that siRNA-directed mRNA cleavage and the native miRNA-directed translational repression are distinct pathways that have different requirements [69,70]; the two RNAi mechanisms still share common components and entry points, such as the DICER-RISC processing machinery. This suggests that transfected siRNA constructs may function as or interfere with the endogenous miRNAs as demonstrated below. By applying Omics tools via microarray profiling and analyzing gene expression of different transcripts (i.e. gene expression signatures) Jackson et al. have examined different siRNA sequences that target the same gene. The rationale for this tool is that different siRNA sequences against the same gene would generate similar gene silencing signatures only if siRNA-induced gene silencing was selective against the targeted mRNA transcript. Their data showed distinct gene silencing signatures that are siRNA sequence-dependent. Those different signatures belong to on- and off-target mRNA transcripts, that is, specific (targeted) and unintended gene silencing, respectively [71]. The off-target gene silencing occurred even when mRNA transcripts partially hybridized with the siRNA, which suggests the existence of a common feature on those mRNA transcripts inducing that non-specificity. Studies reported that a core region in the 3′-untranslated region (3′-UTR) of such mRNA transcripts shares complementarity with residues in the 5′ of the antisense strand of the siRNA duplex [71–73]. This core region on siRNA is comparable to the “seed region” on miRNAs, which has a key role in mRNA target recognition in the endogenous RNAi pathway [74,75]. Thus, when exogenous siRNA enters and arranges in the RISC complex in such a conformation similar to that of the native miRNA, an “miRNA-like off-target gene silencing” occurs and such non-specific signatures observed by Jackson et al. are obtained [76,77]. It is of great importance to note that these gene silencing signatures are mRNA transcript sequence-specific [78], i.e. the signatures of siRNAs against different mRNA transcripts are not similar, indicating that the off-target effects are not only siRNA sequence-dependent but also target-specific. 3.2.5. Off-target effects of induced RNAi: Saturation of the RNAi machinery Cross-reaction with endogenous RNAi machinery side effects is most likely to occur upon shRNA use rather than siRNA. As above-mentioned, shRNAs are delivered to the nucleus and enter the miRNA pathway at early stages; therefore all components of the miRNA machinery could be saturated by using high doses of exogenous dsRNA, particularly in the case of the sustained expression of shRNA. These cross reactionrelated side effects may inhibit the miRNA pathway and cause cell toxicity. In their study, Grimm et al. [79] revealed that treatment with different shRNA constructs given to mice, led to severe toxicity in 73% of the cases and to 47% lethality due to liver failure. The shRNA-related lethality observed was dose-dependent and not shRNA/target-specific; moreover, it did not require the presence of shRNA target, which hints at competition with and saturation of one or more component of the endogenous miRNA pathway. Those side-effects were eliminated upon lowering the shRNA levels. Even though exogenous siRNA enters the RNAi pathway at a later step compared to shRNA and directly incorporates into the RISC, an evidence for miRNA pathway saturation by siRNA has been recently arisen [34,80], although can be minimized by reducing the delivered siRNA amounts [34,81]. On the other hand, mismatches between the delivered siRNA and the target mRNA could cause gene silencing via miRNA-related pathway, which leads to translational inhibition without altering mRNA levels bringing on competition with the native pathway [82]. Interestingly, it is not the number of mismatches between the siRNA and mRNA sequences but the position of the mismatches, suggesting that structure stability of the siRNA determines whether the siRNA enter the miRNA pathway or not. This indicates that sequence complementarity is a crucial factor though not sufficient. Altogether, while some microarray profiling tests show that siRNA molecules could target mRNA sequences in a specific manner [83], it Author's personal copy 1512 A. Daka, D. Peer / Advanced Drug Delivery Reviews 64 (2012) 1508–1521 is now well established that siRNA silencing entails nonspecific off-target gene silencing [71,78]. These effects could be a concentrationdependent, as lowering the siRNA concentration minimizes these nonspecific effects [34,84] albeit not eliminating them [71]. Better approaches for mitigating nonspecific off-target silencing are: 1) using more than one duplex (i.e. siRNA pool) against the on-target mRNA, in such a way different sequences target distinct and distinguishable regions in the targeted mRNA, which minimize miRNA-like off-target effects as well as significantly reduce interference with endogenous gene silencing mechanisms. 2) Avoiding complementarity between non-targeted mRNAs, especially in the 3′-UTR and seed region [73]. 3) The use of chemical modifications, for instance the use of 2′-O-methyl modification on positions 1 and 2 in the sense strand of 8 different siRNAs was shown to reduce off-target silencing both at the mRNA and protein levels without affecting the on-target mRNA silencing [85]. Collectively, those data highlight the necessity of the development of siRNA off-target prediction algorithms that may optimize the construct of the delivered siRNA. Moreover, a physical approach in overcoming in vivo instability of siRNAs and the other off-target effects is by complexation of the siRNA with a carrier/vector. The design of appropriate siRNA carriers facilitates its delivery and trafficking to and within the cell, and makes it feasible for in vivo application, as these strategies: 1) mask the siRNA molecules from the immune system; 2) exceed the size range for kidney filtration, thus minimize rapid clearance [47]; 3) protect the siRNA molecules from nuclease activity and; 4) facilitate endothelial wall and plasma membrane penetration. Also, targeted delivery strategies increase the local concentration of siRNA at the site of action thus mitigates nonspecific biodistribution and adverse effects accompanied with systemic delivery [50,86]. 4. Systemic NP-mediated delivery of RNAi molecules Challenges for in vivo delivery of RNAi molecules could emerge at any level and are dependent on the route of administration; although local administration is favored over systemic delivery it is not always manageable for clinical application. The requirements for effective and safe delivery of naked RNAi molecules such as avoiding renal clearance, stability in the circulatory system, lack of immunogenicity, biocompatibility, immune tolerance of modifications and functionalizing, the ability to pass through biological barriers and specific targeting seem to hold true also in the case of carrier-formulated RNAi systems designed for clinical use. Furthermore, the carrier itself should meet other criteria [87], for instance it should: have low tendency of aggregation, which could induce toxicity and interfere with effective delivery, be soluble or colloidal in water, have an extended circulating half-life (t1/2) that increases effectiveness, release its payload into the cytoplasm before reaching the lysosome and have a long shelf life [86]. The developing field of nanotechnology offers a wide range of NPs for delivery of RNAi regulators. By definition, for a device to be considered a nano-scaled compound it or its essential component(s) must be man-made and in the range of 1–1000 nm, at least in one dimension [11]. When selecting the most suitable tool for delivery, one should take into account the route of administration as well as properties and structural characteristics of the carrier such as size, shape, charge and other surface characteristics of the NP, as these features affect pharmacokinetics (PK) and biodistribution (BD) profiles of the carrier. Accordingly, well-structured and fine tunable design of the NP would result in developing adequate and appropriate device for RNAi delivery [88]. 4.1. Passive delivery Passive delivery takes advantage of highly selective anatomical and functional features of the targeted tissue to specifically deliver the desired therapeutics. A leading approach of passive delivery is exploiting the enhanced permeability and retention (EPR) effect for solid-tumor targeting. The enhanced and abnormal angiogenesis along with the poor lymphatic drainage characterized with tumorigenicity cause extensive permeability of blood vessels and high retention of macromolecules in tumor tissue, respectively [89,90]. While free and low molecular weight (MW) drugs diffuse freely in and out tumor tissues, macromolecules (> 40 kDa) and NPs between 100 and 200 nm do not easily diffuse back to the blood stream, hence accumulate in the tumor tissue [89,91–94]. EPR phenomenon has also been reported in various human solid tumors as well as in inflammatory tissues [95]. In addition, passive delivery can be influenced by the NP formulation, for instance the passive delivery of naked siRNA molecules and NPs ≤10 nm to the kidney upon systemic administration. On the other hand, particles larger than 100 nm, particularly lipid-based NPs, preferentially accumulate in organs of the reticuloendothelial system (RES) better known now as the mononuclear phagocytic system (MPS) [17,68,96–98] due to high blood flow and fenestrated vasculature in these tissues. Mononuclear phagocytes of the MPS located in the liver (Kupffer cells), lung and spleen take up foreign particles following their opsonization by serum proteins via several interactions. Thus, size, geometry, surface charge and hydrophobicity of the NP - could influence recognition by and accumulation within cells of the MPS. An increase in phagocytic activity is observed upon using highly charged NPs, prominently cationic, as well as hydrophobic particles. In order to avoid opsonization, strategies that mask the NP charge and hydrophobicity may be applied, such as PEGylation, a widely used method for NPs stabilization, referred to insertion of the biocompatible poly-ethylene-glycol (PEG) derivative polymers to the surface of the NP providing natural charge and hydrophilicity, thus minimizing aggregation and opsonization [99–101]. In the next subsections we evaluate NP systems that make use of such physiological mechanisms for passive delivery. These data are summarized in Table 1. 4.1.1. Lipid-based nanoparticles (LNPs) Nano-scaled liposomes are lipid-based particles ~100 nm in diameter that consist of lipid bilayer envelope and hydrophilic aqueous core. Neutral liposomes are characterized with low immunogenicity and toxicity, biocompatibility and ease of preparation, thus they have been investigated extensively [102,103]. Widely used neutral liposomes contain various combinations of phosphatidylcholine (PC, mainly 1,2-Oleoyl-sn-Glycero3-phosphatidylcholine—DOPC), phosphatidylethanolamine (PE, mainly 1,2-Dioleoyl-sn-Glycero-3-phosphatidylethanolamine (DOPE)) and cholesterol [102], while the later two lipids are helper lipids that facilitate endosomal escape of the payload to the cytoplasm and increase stability of the liposome, respectively [104,105]. 65% siRNA encapsulation into DOPC liposomes has been achieved [106], along with significant reduction in protein expression, tumor growth and metastasis in ovarian cancer and human melanoma models in mice [106]. This tumor-targeted delivery was attained due to EPR effect of tumor tissues. 4.1.2. Cationic LNPs Cationic lipid-based NPs that interact with the negative RNAi molecules as well as with the cell membrane, thus stabilizing the NP and enhancing cell uptake, are frequently used in cell-culture assays [102]. However, due to high levels of opsonization by serum proteins, immunogenicity, accumulation near the vasculature and uptake by the liver and spleen, specialized systems such as SNALPs those neutralize the NPs net charge with a “stealth coating” such as PEG [47,99–101,105–107], increase the NP circulation t1/2 and improve its PK would be discussed here. On the other hand, other factors beside the net charge should be considered when designing NP for delivery, e.g. the hydrodynamic diameter; as even PEGylated NPs >100 nm may exhibit higher protein adsorption and thus lower Author's personal copy A. Daka, D. Peer / Advanced Drug Delivery Reviews 64 (2012) 1508–1521 1513 Table 1 RNAi-based NPs for systemic administration. Delivery system NP formulation Targeting mechanism Target tissue References Lipid-based NP (LNPs)/ lipoplexes Cationic LNPs Lipidoids Polyplexes DOPC liposomes EPR Ovarian tumor [106] SNALP Epoxide-based lipidoids JetPEI/LNA-siRNA polyplex MPS MPS Aggregation-induced retention, Mucoadhesion Mucoadhesion EPR MPS Antibody-directed delivery Antibody-directed delivery Antibody-directed delivery Liver Liver Lung, Liver, Kidney [17,98,108] [97,109] [50] Kidney Liver tumors, Inflammatory cells Liver Dendritic cells Gut leukocytes, T lymphocytes and monocytes, Melanoma tumors, activated leukocytes [50] [92,94] [116] [117] [118,119] [120,122] Integrin-targeting peptide AchR-targeting peptide PSMA-targeted aptamer σ receptors-targeted Anisamide Tumor neovasculature AchR-expressing neurons Prostate cancer cells Tumor cells [114,122] [123] [124,125] [126] RBP mediated targeting TfR-mediated targeting NAG-mediated targeting Chol-mediated targeting Hepatic stellate (HS) cells Tumor cells expressing TfR Liver heptocytes Liver and jejunum [127] [128] [129] [13,130] Chitosan/LNA-siRNA polyplex Atelocollagen Carbon nanotubes (CNTs) To17-SWCNT Immuno-liposomes I-tsNP Protein-based targeting Antibody-protamine fusion systems protein RGD NPs RVG-9R NPs Aptamer/NPs RNA A10/siRNA NP Targeted cationic lipid-based PEG–Anisamide–LDP NPs VitA-Lipotrust liposomes CDP NPs Tf-CDP/siRNA DPCs PEGylated NAG-PBAVE DPCs Chol conjugates Chol/Antagomer122, Chol/siRNA circulating t1/2 and permeation into tissues as well as higher MPS clearance [47]. SNALP is a 100–150 nm lipid-based particle that encapsulates siRNA within its layers. It is composed of cationic and fusogenic lipids that facilitate cellular uptake and endosomal escape. The lipid bilayer of SNALP is coated with PEG polymer [68]. Tissue BD assays in rodents show significant delivery of SNALPs to the liver. Several studies report minor liver enzymes release upon SNALP application, otherwise the NPs are well tolerated [17,68]. In the first RNAi-induced gene silencing studies reported with SNALP and conducted in non-human primates, Zimmermann et al. reported significant (>90%), specific and durable dose-dependent gene silencing of apolipoprotein B (ApoB) by single injection of SNALP-formulated siRNA (b 100 nm), which lead to specific dose-dependent reduction in serum cholesterol and LDL. Importantly, i.v. administered SNALP selectively induced gene silencing only in the liver consistent with tissue BD pattern [17]. Similarly, another study showed that SNALP, based on optimized derivative of the ionizable cationic lipid DLinDMA, successfully knocked-down Transthyretin in non-human primates with ED50 of 0.3 mg per kg body (mpk) in specific and durable manner [98]. SNALPs containing pooled siRNA against Zaire Ebola virus (ZEBOV) RNA polymerase were reported to specifically inhibit ZEBOV replication and increase survival in non-human primates [108], establishing a core for RNAi-based bio-defensible tool against viral infection. 4.1.3. Lipidoids Lipidoids are non-glycerol-based cationic lipid particles synthesized by conjugation of amines to acrylate or acrylamide [97]. A study exploiting accumulation of lipidoids in the MPS upon systemic administration was conducted in both non-human primates and rodents and showed potent and specific dose-dependent gene silencing of different genes in lipidoid-formulated siRNA system [97]. In the first multitargeting study in vivo, a pool of five siRNA sequences targeting hepatocytes were formulated with epoxidebased lipidoids (80 nm) and was delivered into mice. The study showed effective simultaneous gene silencing of all the targeted genes upon single injection. The same NP formulation was utilized in the same study for potent silencing of transthyretin (TTR) in non-human primates at 0.03mpk dose [109]. The advantage of this approach is that it provides an opportunity for targeting multifactorial diseases by using a pool of several siRNA constructs. 4.1.4. Polyplexes Polyplexes are polycation-based NPs (100–400 nm) formulated siRNAs. The major advantage of using polycations is their structural flexibility. Widely used polycations include biocompatible natural (e.g. chitosan, Atelocollagen, cyclodextrin) as well as synthetic (e.g. Polyethylenimin (PEI), poly (L-lysine) (PLL)) polymers [110,111] (Fig. 1F, H). Upon the use of polyplex-formulated LNA-siRNA Gao et al. reported an extension in circulatory t1/2 and a reduction in renal clearance compared to naked siRNA. Their work also demonstrated the importance of selecting NP features for efficient targeted drug delivery, as different polyplex formulations affected differently the BD pattern as well as the PK characteristics of the delivered siRNA, a significant aspect that could be utilized for targeted delivery. PEI is a polycation used for RNAi delivery by self-assembly of the polymer with siRNA molecules; however, PEI polyplexes bind nonspecifically to cells and adsorb serum proteins, making their use for siRNA delivery inefficient, hence, conjugating PEG groups to PEI nanoplexes or alternatively fine tuning the ratio of siRNA/PEI, as was well demonstrated in this study, can improve their delivery upon systemic administration. The study conducted in healthy mice shows that i.v. injection of JetPEI/LNA-siRNA polyplexes triggers siRNA accumulation in the lung and to a smaller degree in the kidney and liver. The accumulation in the lungs could be explained by electrostatic interactions between the not-fully neutralized polyplex and negatively serum components, which lead to physical entrapment within the relatively small pulmonary capillaries were the blood flow is slow [50]. Alternatively, this opsonization can trigger engulfment by immune cells in the RES organs. Furthermore, electrostatic interactions and consequently adhesion could happen between the positively charged polyplex and the negatively charged endothelium lining the lung, kidney and liver (mucoadhesion). On the other hand, using chitosan polyplexes for LNA-siRNA delivery in the same system results in substantial accumulation of chitosan polyplex in the kidney reasonably due to mucoadhesion to the lining of the kidney [50], a known feature characteristic with chitosan polymers [112]. Atelocollagen is a safe biomaterial that is widely used for in vivo delivery of RNAi molecules. The polyplex size could be monitored by altering the ratio of Atelocollagen to siRNA, for example a polyplex with 100–300 nm diameter can be obtained by using low concentrations of Atelocollagen (0.05%) [92,94,111]. Such formulation was Author's personal copy 1514 A. Daka, D. Peer / Advanced Drug Delivery Reviews 64 (2012) 1508–1521 Fig. 1. Representative targeted RNAi delivery approaches for in vivo use. RNAi molecule could be complexed with mAb/mAb fragments (A) or other targeting peptides (C) via protamine or 9R-nonamer. Also, it could be direct-conjugated to cholesterol (G) or other small molecules (H), Aptamer (D) and polymers (F). Encapsulation of the RNAi molecule within polymeric NPs (E) or liposomes/immunoliposomes (B) could be an alternative approach. Figure adapted and revised with permission from Ref. [134]. utilized for siRNA delivery to the liver in a murine liver metastasis model of lung cancer. The study showed a durable and dosedependent inhibition and apoptosis of tumor growth along with withdrawal in liver metastasis [94]. Another study has reported specific dose-dependent gene silencing of MCP-1 and reduction in infiltration of immune cells to inflamed tissues, hence inflammatory inhibition in murine ear model of contact hypersensitivity (allergic dermatitis) [92]. Atelocollagen polyplexes were selectively delivered to target sites and taken up by inflamed target cells, rather than taken up by migrating inflammatory cells reasonably due to the EPR effect in inflamed tissue. The difference in BD and PK profiles of the various polyplexes reflects the diversity in net charge, hydrodynamic diameter as well as the shape of the NP, features that could be utilized for more efficient gene silencing. 4.1.5. Carbon-nanotubes (CNTS) Carbon-nanotubes (CNTs) are nonspherical, fibrilous nano-cylinders composed of single (1D single-walled CNTs, SWCNTs) or multiple (multi-walled CNTs, MWCNTs) graphine layer(s) with length ranging from 50 nm to 100 mm and diameter of 1–5 nm or 10–100 nm, respectively. Flexibility of CNT layers facilitates multi-valence binding to cells and conjugating to targeting molecules [113,114]. Proper functionalizing of CNTs by covalent or non-covalent strategies (such as coating with PEG polymers or Tween-20) facilitates their solubility in aqueous solutions and prevents non-specific interactions, thus minimizing toxicity observed in the case of non-functionalized raw particles and increases biocompatibility and circulating t1/2 [113,115]. The shape characteristics of the CNT could significantly affect its BD. Whereas small spherical NPs and globular proteins with hydrodynamic diameter ≤ 6 nm are readily cleared by the kidney [46], length Author's personal copy A. Daka, D. Peer / Advanced Drug Delivery Reviews 64 (2012) 1508–1521 and shape of the NP should be taken into account when it comes to the well-individualized cylindrical CNTs. For instance, functionalized CNTs with diameter 1–30 nm and length of 200–2000 nm may exhibit rapid renal clearance and effective excretion from the body [115], on the other hand, bundles-containing CNTs with same dimensions that are not adequately individualized or undergo aggregation in vivo cannot cross the glomerular filters of the kidney thus accumulate in the MPS organs or in the glomerular capillaries, respectively [114,115]. Despite their advantages, the non-biodegradable characteristic of CNTs makes their use for therapeutic purposes more challengeable and requires further study. In a proof-of-concept study of SWNT-formulated siRNA delivery to tumors in vivo, functionalized positively charged SWCNTs (SWCNTs+) were conjugated to human telomerase reverse transcriptase (hTERT) siRNA. The SWNT-formulated siRNA was injected intratumorally and induced reduction in hTERT mRNA and protein levels leading to inhibition in tumor cells growth in a xenograft mouse model [40]. In a recent study by McCarroll et al. [116], the researchers have developed SWNTs functionalized with lysine-based dendrimers covalently attached to lipid chains (Tol 7), while the positively-charged dendrimer served for siRNA binding, the lipid moiety masks the hydrophilicity of siRNA and facilitates cell binding. This system was utilized for systemic delivery of anti-ApoB siRNA and showed effective reduction in ApoB mRNA and protein levels, which led to reduction in serum cholesterol with no evidence of toxicity or immunogenicity. 4.2. Active, target-specific RNAi delivery Targeting molecules highly expressed on the cells' surface in the disease site of action is an attractive strategy for specific delivery while improving PK and minimizing non-specific delivery to bystander tissues, hence allowing the use of minimal therapeutics doses and reducing off-target effects. Antibodies, natural ligands, ligand mimetics and aptamers can be attached to or complexed with free or NPformulated siRNA constructs (Fig. 1) to enhance stability, induce cellular uptake as well as promote targeted delivery. It is important to note that these targeting molecules must undergo internalization and specific endocytosis in order to obtain gene-silencing [86]. In the introduced studies, the researchers validated targeting moieties' functionality upon conjugating as well as specific binding by conducting in vitro assays before systemic delivery. 4.2.1. Peptide-based targeting moieties When conjugated directly to free siRNA molecules or to NPs, monoclonal antibodies (mAbs) and other proteins composed of targeting peptide and nucleic acid-binding domains could serve as targeting moieties for systemic delivery of RNAi molecules. These moieties are of crucial importance as they induce specific binding to and receptor-induced internalization into the targeted cells. Ligands overexpressed in disease-related tissues, such as integrins and other extra-cellular matrix (ECM) receptors in cancer and inflammation as well as growth factors receptors provide golden targets for those NPs. Immuno-NP is an excellent strategy for specific targeted delivery. However, conjugated mAB is likely to promote nonspecific interactions upon systemic administration (e.g. recognition by immune cells via Fc receptors). Alternatively, engineered Ab fragments, such as Fab and scFv, are safer and favorable in this case, albeit less stable compared to the whole antibody, which lowers the NP t1/2 [86,120]. Zheng et al. succeeded in generating immunoliposomes that selectively target dendritic cells (DCs) [117]. The PEGylated CD40 siRNAcontaining immunoliposomes (siILs) are conjugated to NLDC-145, a DC-specific mAB, and contain the cationic DDAB that facilitates siRNA encapsulation with averaged hydrodynamic diameter of 86 nm. Upon i.v. injection NLDC-145 siILs have shown to accumulate in spleen and liver possibly due to specific targeting of DCs that are abundant in these organs, whereas naked siRNA has accumulated mainly in kidney. When 1515 RNAi efficiency was examined, both naked and NLDC-145 siILencapsulated CD40 siRNAs induce gene silencing in splenic DCs 48 h post injection (p.i.), however only siIL-encapsulated siRNA induced prolonged specific silencing that lasted at least 12 days p.i. This observation can be explained by slow release of stable siRNA from siILs upon spleen accumulation hence increasing its t1/2, underlying another advantageous feature of siRNA complexation over the degradable naked siRNA. By utilizing integrins as excellent receptor targets for RNAi delivery, Peer et al. have reported specific gene silencing of CyclinD1 (CyD1), a pivotal cell regulator, and inhibition of inflammation in an experimental murine model of colitis by developing 100–200 nm integrin–targeted and stabilized NP (I-tsNP) system encapsulating siRNA-protamine complex (Fig. 1B). The positively charged small nucleotide-binding protein protamine neutralizes and condenses the negatively charged siRNA, protects it from degradation and aids in its encapsulation within the NP [118]. Anti- β7 integrin was attached to neutral lipid-based NPs coated with hyaluronan (HA), a biocompatible and non-immunogenic glycosaminoglycan that masks the NP from the immune system and increases its circulating time in vivo [112]. Similarly, LFA-1 I-tsNPs were applied in suppression of HIV infection in a humanized mouse model using siRNA against CCR5, a crucial regulator in early stages of HIV infection. SiCCR5/LFA-1 I-tsNP promoted specific and durable (up to 10 days) gene silencing in lymphocytes and macrophages without inducing IFN response and immunogenicity [119]. Another advantage of such immuno-NP systems is achieved by the synergistic effect obtained upon blocking integrin-mediated leukocyte migration via mAB-integrin interaction. Via fusing Fab antibody fragment (F105) against HIV-1 envelope (gp160) to protamine (F105-P), Song et al. have designed antibodyprotamine fusion protein (Fig. 1A) for systemically delivering a mixture of anti-oncogenic siRNAs into mice grafted with gp160expressing B16 melanoma cells. The F105-P complexed siRNAs selectively targeted gp160-B16 cells and suppressed tumor growth [120]. Based on this strategy, Peer et al. have reported a selective targeting for activated leukocytes using Integrin LFA-1 single chain Fv (scFv)protamine fusion strategy recognizing the conformationally distinct activated form of LFA-1 [121], an observation that holds promise for specific in vivo gene silencing while avoiding delivery to normal bystander cells (mainly leukocytes in this specific case) and mitigating off-target effects in general and global immunosuppression, in particular. Although no toxicity was reported in those studies, the immunogenic response issue should be taken into consideration when using AB for systemic delivery. Other peptide-based agents that have target selectivity whilst exhibit less immunogenicity can be applied in RNAi molecules targeted delivery. Arg–Gly–Asp (RGD) peptides have been utilized for targeting αv integrins expressed on many tumor vasculature. Comparable sequences have been shown to recognize other isoforms of integrins [131]. Attaching an RGD peptide to the PEGylated PEI (RPP) nanoplex facilitated its delivery to tumor tissues. It has been reported that i.v. delivered 70–100 nm RPP nanoplex self-assembled with VEGFR2siRNA selectively targeted tumor vasculature overexpressing integrins and inhibited VEGFR2 expression leading to repression in tumor angiogenesis and decrease in growth rate in a murine tumor model [122]. Although CNT-based targeted delivery is still only in its infancy and yet there is no published study regarding targeted systemic delivery of CNT-formulated siRNA, Liu et al. utilized the stabilized, aqueous suspensions of SWCNTs coated with PEG chains adsorbed to phospholipids (PL-PEG, diameter of 1–5 nm, length of 100–300 nm) that developed by Kam and Dai [132] to conjugate RGD peptide (PL-PEG–RGD) and study their BD by microPET scans 24 h post i.v. administration in a xenograft murine model bearing tumor cells highly expressing αv integrins [114]. SWNT conjugates exhibited slow excretion rate and were mainly taken by the MPS (liver and spleen). The PEGylated Author's personal copy 1516 A. Daka, D. Peer / Advanced Drug Delivery Reviews 64 (2012) 1508–1521 SWNT conjugate with the longest PEG chain (PL-PEG5400) exhibited lower uptake level by the liver consistent with their relatively higher circulating t1/2 compared to PL-PEG2000, probably due to higher hydrophilicity and lower non-specific interactions with serum proteins leading to reduction in opsonization. However, when RGD was conjugated to PL-PEG5400 a significant rapid uptake by tumor tissue was obtained reasonably due to specific and targeted delivery of the PL-PEG–RGD SWCNT conjugates and multivalency effect of binding multiple RGD molecules onto the surface of SWCNT backed up with extending circulation time obtained by PEG-mediated MPS escaping. Within the period of 24 h p.i. no toxicity or immune response were observed [114]. A peptide derived from rabies virus glycoprotein (RVG) was used in transvascular siRNA delivery to the central nervous system (Fig. 1C). RVG binds specifically to Acetylcholine receptor (AchR) which is highly expressed by neuronal cells, and was stabilized by attaching 9 residues of Arginine (9R nonamer) that also enables conjugation to and enhance cellular uptake of siRNA. When anti viral siRNA (siFvE J) was complexed with RVG-9R peptide it has reported to protect against fatal viral encephalitis in mice without triggering immunogenicity [123]. 4.2.2. Aptamer/siRNA chimeras Aptamers are nucleic acid-based molecules that are used for diagnostic application as well as gene expression regulation and gene therapy. The RNA-aptamers/siRNA chimera is recognized and processed by DICER generating 21–23 nt siRNA fragments in a miRNA-like manner, this along with aptamer specific binding to target ligands, small size which allow better tissue internalization and amenability for modifications, make it an appropriate strategy for induction of targeted RNAi delivery in vivo. Additionally, this strategy is considered to be advantageous over protein-based targeted moieties in terms of cost, low immunogenicity and production complexity [31,124]. A10 aptamer binds specifically to PSMA (prostate-specific membrane antigen) on the surface of prostate cancer cells and tumor vascular endothelium, and has been shown to specifically inhibit tumor growth when conjugated to siRNAs against anti-survival genes (Plk1 and Bcl2) overexpressed in most human tumors in a xenograft model of prostate cancer. Interestingly, when A10-NP was examined in another model where the tumors do not express PSMA, no tumor regression was obtained demonstrating the targeting selectivity of this system. This feature is worth note, particularly when triggering apoptosis as in the current case, as it is of great importance not to target healthy cells [124]. In another study, a dual inhibitory function anti-gp120 aptamer/siRNA chimera, in which both aptamer and siRNA portions directed against HIV genes, was used to specifically target cells expressing HIV receptor gp120 leading to repression of HIV replication in tissue culture tests. An attractive approach for RNAi induction that could be utilized for systemic delivery [125] (Fig. 1D). 4.2.3. Other small molecules-siRNA NPs An siRNA-delivery system (120–150 nm) that employs characteristics of both cationic LNPs and polyplexes was developed in the lab of Huang [126]. siRNA constructs were mixed with calf thymus DNA before complexing with protamine followed by coating with cationic liposomes (DOTAP and cholesterol), the liposomes-polycation-DNA (LPD) was then mixed with a PEGylated ligand lipid (PEG–Anisamide– DSPE). Anisamide ligand binds to sigma (σ) receptors highly expressed by tumor cells. In one study conducted in a mouse xenograft tumor model Anisamide-NPs systemically administrated were delivered mainly to the tumor tissue and induced specific gene silencing of the EGFR in addition to cell apoptosis and slowed down tumor progression. Reduced liver uptake was also reported, probably due to relatively reduced MPS delivery upon PEGylation. The calf thymus DNA serves as a carrier that reduced particle size and increased delivery efficiency. Immunostimulatory effects were observed in siRNA-NPs and thought to be related to siRNA-immunostimulatory sequences, as empty NPs (without siRNA) showed minimal immunogenic effects albeit these DNA fragments contained immunogenic CpG motifs [126]. Similarly, Lipotrust liposomes that consist of cationic lipid DC-6-14, cholesterol and DOPE attached to vitamine A were used to encapsulate and deliver anti-gp64 siRNA (VA-lip-siRNA gp46, ~150 nm) to hepatic stellate (HS) cells, which overexpress retinol binding protein (RBP) that takes up vitamine A from the circulation in progressive and lethal model of liver fibrosis in rats. Upon i.v. administration of VA-lip, anti gp-46 siRNA led to collagen inhibition in HS cells and cell apoptosis mitigating liver fibrosis and extending rat survival with no immnuostimulatory effects [127]. Cyclodextrin polycation (CDP) self-assembles with and condenses siRNA molecules to generate ~50–70 nm NPs stabilized by PEGylation. Human transferring (Tf)-CDP-siRNA NPs were used for targeting metastatic cells overexpressing Tf-receptor in murine model of Ewing's family of tumors—EFT (mesenchymal malignancy in bone and soft tissues). These conjugates have shown localization in and uptake into tumor cells that led to transient reduction in tumor growth [128] (Fig. 1E). Whereas Tf-CDP-NPs showed no significant immunostimulatory effects upon i.v. administration into mice, the same system induced modest pro-inflammatory cytokine and IFN productions as well as kidney and liver toxicity in non-human primates. Those undesired side effects can be overcome by injection of multiple lower i.v. doses [133]. siRNA dynamic polyconjugate (DPC) is a PEGylated nanosystem composed of targeted-ligand agent linked by the reversible maleamate linkage to the endosomolytic agent PBAVE (Poly butyl and amino vinyl ethers) in which the siRNA is attached to PBAVE by reversible disulfide bond (Fig. 1F). Attaching the ligand agent N-acetylgalactosamine (NAG) has been reported to selectively target hepatocytes and not the nonparenchymal cells (or the immune Kupffer cells) in the liver, whereas attaching mannose redirected the DPC to nonparenchymal cells away from hepatocytes [129]. NAG DPCs (~10 nm) with anti-apoB or anti-peroxisome proliferator-activated receptor alpha (ppara) siRNAs induced specific and durable (t1/2 between 7 and 10 days) gene silencing selectively in the liver, which led to reduction in serum cholesterol and ApoB levels and serum triglycerides, respectively. Moreover, the NAG-mediated selective targeting to hepatocytes along with DPC's relative small size eliminates Kupffer cells-mediated toxicity in the liver upon systemic administration of siRNA DPCs [129]. Generating cholesterol-siRNA (chol-siRNA) by conjugating cholesterol to 3′ end of the sense strand of siRNA constructs has been shown to improve in vivo PK features (reducing circulation t1/2 and reducing clearance from blood system) compared to unconjugated siRNAs [130] (Fig. 1G). Utilizing this system with anti-apoB siRNA led to specific delivery to the liver and the jejunum and to a reduction in the serum lipoprotein and the cholesterol levels in a transgenic mouse model [130]. Similarly, cholesterol-conjugated antagomir122, against miR-122, a liver-specific miRNA, led to efficient and durable (up to 23 days) silencing of this specific miRNA in mouse liver upon i.v. administration [13]. Antagomir-122 was well-tolerated and regulated the expression of genes involved in cholesterol biosynthesis and reduced cholesterol levels in a specific manner. 5. Intracellular delivery of NP-associated RNAi molecules A crucial requirement of efficient systemic siRNA delivery is the ability to mask their net negative charge that consequently minimizes aggregation and recognition by the immune system leading to an increase in circulating t1/2 and most importantly facilitating binding to target-cell membrane. Beyond selective delivery to the intended cell type in the site of action, sufficient binding strength between the NP and the cell surface along with efficient delivery to the appropriate sub-cellular compartment(s), are also required for targeted gene silencing. Author's personal copy A. Daka, D. Peer / Advanced Drug Delivery Reviews 64 (2012) 1508–1521 5.1. Delivery to the cell membrane Particle internalization into the cytoplasm upon cell-membrane binding occurs by direct delivery through the cell membrane and passive diffusion within the cytoplasm, or by energy-dependent endocytosis (Fig. 2). Because of the high protein concentration and viscosity within the cytoplasm, passive diffusion is slow and restricted to NPs smaller than 30 nm [135]. Besides phagocytosis, that is restricted to phagocytes, endocytosis in mammalian cells occurs through diverse pinocytosis-based mechanisms (e.g. clathrin-dependent and clathrin-independent caveole-mediated pathways). Phagocytosis pathway involves endocytosis of particles larger than 1 μm [136] these dimensions are beyond the scope of this review. Targeted-NPs interact with the plasma membrane via specific cell surface receptors/binding molecules, followed by engulfment in membrane invaginations forming the early endosome, which fuse to more specialized vesicles (e.g. late endosomes, lysosomes, perinuclear vesicles). Finally, the cargo is delivered to distinct intracellular compartments or recycled to the ECM [137]. Clathrin-mediated endocytosis (CME) (Fig. 2.2) is a wellcharacterized internalization pathway in mammals by which the engulfment of the cargo occurs through clathrin-coated pits formation and follows the traditional abovementioned pathway of endocytosis. NPs endocytosed via the CME experience a gradual drop in pH while trafficked from early endosome (pH 5.9–6) to lysosome (pH 4.5–5.5) through the late endosome (pH 5–6) as well as an exposure to nucleases that result in siRNA degradation. In the clathrin-independent Caveolae-mediated endocytosis (Fig. 2.1) however, the engulfed NP is trafficked through caveosome route, which increases its likeliness to escape the drop in pH (intra-caveosome pH is neutral) and avoid nuclease degradation as lysosome trafficking does not occur in the caveolae pathway [138,139]. Whether the NP internalization follows the CME or Caveolaemediated pathway is dependent on the NP physical features. Whereas 200–500 nm NPs are endocytosed mainly via caveolae-mediated pathway, CME pathway is favored in the case of NPs ≤ 200 nm [140]. NP charge and ligand moiety attached to it may also determine the route of endocytosis [141]. Cationic NPs interact with the negatively charged cell membrane leading to internalization via fusion route [142] and localization of the payload into the cell [143]. This promising strategy for direct delivery to the cytoplasm does not override the potential for positive charge-mediated off-target and undesirable effects. Therefore, targeted neutral NPs are more tolerable for systemic delivery, albeit endosomal entrapment must be overcome. 5.2. Endosomal escape In order to achieve efficient gene regulation, the NP must release the siRNA molecules within the cell cytoplasm where they suppose to enter the RISC and initiate gene silencing. Several strategies have been developed for triggering endosomal escape of the cargo following CME before reaching late phases of endocytosis (i.e. trafficking to late endosome and lysosome). The bilayer structure of the biological membrane grants its stability and impermeability of large and hydrophilic molecules. The ability of lipid-based system to promote inverted hexagonal (inverted micelle-like) structure, instead of bilayer or lamellar phase is utilized for destabilizing biological membrane and enhanced intracellular delivery. Fusogenic lipids such as PEs as well as cationic lipids promote lamellar-to-inverted hexagonal transition which enhances fusion with and interruption of the endosome membrane at acidic environments, hence are widely used for drug-delivery approaches [104,137,144]. Considering the cationic lipids, the more the hydrophobic domain of the cationic lipid is unsaturated, the more fusogenic the lipid is [100]. Cationic amino lipids-based NPs that are composed 1517 of ionizable amine headgroups with a pKa of 6–8 were studied for drug and siRNA delivery [98,145]. At pH below their pKa (as in endosomal compartments), the amine headgroups are fully protonated enhancing interaction with the endosomal/lysosomal membranes and lead to membrane rupture and escaping to cytoplasm. On the other hand, partial ionization of the amine headgroups at physiological pH allows for minimized interaction with serum proteins thus reducing cytotoxicity. Ionizable amino lipids are composed of a cholesteryl ether tail and an aliphatic tail. The use of longer aliphatic tail in these lipids or increasing the amino lipid concentration results in a decrease of lamellar to inverted-hexagonal transition temperature. It is worth noting that using amine headgroups with lower pKa values enhances phase transition at higher pH values (i.e. in early endosomes or even at cellular uptake phase) [145]. Endosomal swelling is a non-contact strategy for endosomal escape. When delivered to endosomes, NPs with high buffering capacity prevent acidification of the endosome (by absorbing protons) which lead to passive chloride ions (Cl -) influx into the endosome followed by osmotic swelling and eventually rupture of the endosome membrane allowing cargo release to the cytoplasm. Cationic polymers, in particular tetratable polyamines, such as PEI, which contains highly protonable amino groups have been reported to act as “proton sponge” [146], and enhance nucleotide release to cytoplasm [147]. “Proton sponge” mechanism occurs with other polyplexes and cationic polymers, such as the amphiphilic NPs that Wang et al. have designed. These ~ 130 nm NPs are composed of polymerizable cysteine residues, “proton sponge” and hydrophobic domains. The “proton sponge” domain triggers pH-sensitive membrane rupture in the endosome facilitating NP/siRNA escaping to the cytoplasm. The cysteine residues and hydrophobic domains enhance stable complexation of the NP with the siRNA as well as facilitate siRNA dissociation from the NP via reduction of disulfide bonds by and hydrophobic interaction with cytosol components [148]. Another non-contact strategy for endosomal escape is the use of pH-sensitive lipoplexes/polyplexes. A hydrophilic pH-sensitive PEGylated conjugate (POD) was attached to DOPE liposomes. While the POD conjugate is stable at neutral pH, at acidic condition (below pH 5) the POD is completely degraded. Equivalently, POD/DOPE liposomes have been shown to remain stable at neutral pH for up to 12 h, however, at pH 5.5 aggregation of the liposomes has been observed followed by content release within 30 minutes [149]. In another study, PEGylated PLL polyplex modified with the pH-sensitive endosomolytic Melittin peptide (DMMAn-Mel) increases the efficiency of siRNA delivery [150]. Kam and Dai developed stabilized PL-PEG-coated SWCNTs with PEG-amine groups (PL-PEG–NH2). The terminal NH2 group facilitated the incorporation of siRNA molecules via disulfide bond, which in acid conditions is cleaved by thiol-reducing enzymes and leads to payload release [132]. 6. NP-mediated RNAi: Clinical studies Most of the RNAi-related advanced clinical studies are based on localized delivery of naked RNAi molecules (Table 2), however, systemically-applied RNAi research in non-human primate models [17,97,98,108,133,151] hold promise for bringing it to a clinicallyrelevant stage. Since the first RNAi-related clinical trials that introduced naked siRNA intravitreal to patients with age-related macular degeneration (AMD) and diabetic macular edema (DME) targeting vascular endothelial growth factor (VEGF) pathway, many approaches attempting to deliver naked siRNAs in a local manner were conducted. Unfortunately, many of these approaches were terminated due to safety, uncertain scientific achievements or companies' reorganization issues [152,153]. Since then, more research was conducted in an effort to refine and improve this approach and to adapt it for systemic delivery (Table 2). Author's personal copy 1518 A. Daka, D. Peer / Advanced Drug Delivery Reviews 64 (2012) 1508–1521 Fig. 2. Intracellular delivery pathways. NPs enter the cell membrane and trafficked through the cell cytoplasm via endocytosis pathways (1, 2) or passive delivery (3). Upon endocytosis NPs (1) are released from early endosomes and trafficked within the cytoplasm as in Caveolae-mediated pathway, or (2) trafficked via lysosomal pathway until escaping from lysosome compartments near perinuclear region as in Clathrin-mediated endocytosis (CME) pathway. (3) Particularly small NPs exploit the passive diffusion within the cytoplasm. Figure adapted with permission from Ref. [135]. The first-in-human Phase I clinical trial involving systemic administration of targeted NPs for RNAi molecules delivery to solid tumor, CALAA-01, was conducted by Davis et al. in 2008 [154,155]. CALAA-01 70 nm NP employs the Tf-CDP nanosystem previously described in mice and non-human primates [128,133], with a number of modifications and refinements, in order to formulate the non-modified antiribonucleotide reductase subunit 2 (RRM2) siRNA [154]. Biopsies from melanoma patients that undergo systemic administration of Tf-CDPNPs showed intracellular accumulation of the NPs selectively in tumor tissues and not in adjacent epidermal tissues in a dose-dependent manner, in addition to reduction in both RRM2 mRNA and RRM2 protein levels after several cycles of dosing [155]. Since then, seven candidates for systemic delivery were entered the clinical Phase, six of them are SNALP-based systems. In 2008 SNALP-based tool was formulated with two siRNA constructs against VEGF and kinesin spindle protein (KSP) and administered to patients with liver cancer and transthyretin (TTR)-mediated amyloidosis (ATTR). The ALN-VSP02 therapy was well-tolerated and induced anti-VEGF and anti-tumor effects in most patients and continued to second Phase I trial. In 2009 three candidates entered the clinical Phase, two of which, ALN-TTR01 and TKM-ApoB are SNALP-based therapies targeting the hepatocytes and formulated with antiTransthyretin (TTR) and anti-ApoB siRNAs in ATTR and Hypercholesterolemia patients, respectively. Post single dose, ALN-TTR01 led to significant reduction in amyloid deposits in targeted tissues and in serum TTR protein in a dose-dependent manner. Since patients who received TKM-ApoB exhibited transient reduction in serum cholesterol levels, TKM-ApoB Phase I clinical trial was terminated. Moreover, two of 17 patients exhibited immunostimulatory effects and elevated cytokine production upon i.v. administration of the highest dosage of the drug. In 2010, an improved related NP, TKM-PLK1, was clinically tested in patients with advanced solid cancers and lymphoma that are resistant to conventional therapeutics. This NP targets the pololike kinase 1 (PLK1) in an attempt to inhibit cell-cycle progression and induce apoptosis in tumor tissue. Protein kinase N3 (PKN3), which is an essential downstream component of the PI3K pathway mediating metastasis of tumor cells was targeted in Phase I clinical trial of Atu027 lipoplex in 2009 in patients with advanced colorectal cancer, however, it is important to note that activation of the complement pathway was observed in Atu027 clinical trial, a finding that necessitates further improvement of this delivery system. ALNPCS02 and TKM-EBOLA were recently entered Phase I clinical studies for treatment of hypercholesterolemia and biodefense against Ebola viral infection, respectively [153]. In 2011, two other SNALP-based candidates have entered the clinical phase, TKM-EBOLA [108] and ALN-PCS02 (Table 2). The accumulated clinical data regarding ALN-VSP02 and Atu027 support for further development with the sponsors anticipating to license the two compounds in 2012 [152] , this along with the on-going and speeding up research concerning nanobiotechnology is expected to establish for a revolutionizing and promising core for new nano-based pharmaceutical market (Table 3). 7. Conclusions The potential of RNAi molecules to silence genes in an allelespecific manner along with nanobiotechnology generate the basis for a potent tool for validation and medical interventions in many types of diseases such as cancer, inflammation, allergies and viral infections. Per contra, our poor understanding of the biological Author's personal copy A. Daka, D. Peer / Advanced Drug Delivery Reviews 64 (2012) 1508–1521 1519 Table 2 RNAi-based drug candidates in clinical studies (adapted and revised with permission from Ref. [152]). Candidate name Disease Target Delivery system Phase Year Bevasiranib AMD VEGF Local-intravitreal needle injection. 2004 Cand5 ALN-RSV01 DGFi TD101 AMD, DME RSV infection Acute kidney injury, delayed graft function Pachyonychia congenita Local-intravitreal needle injection. Local-inhalation of unformulated sirRNAs Systemic - naked sirRNA Local-intradermal needle injection (skin) QPI-1007 Chronic nerve atrophy, nonarteritic ischemic optic neuropathy Operable pancreatic ductal adenocarcinoma VEGF Viral RNA p53 Mutant keratin (K6a) Caspase 2 III—was interrupted in 2009 II II II I Local-intravitreal needle injection I 2009 Mutated KRAS Local-drug elution I 2010 Metastatic solid tumors. Liver cancer, cancer with liver involvement Advanced solid tumors RRM2 VEGF, KSP PKN3 I I I 2008 2008 2009 Hypercholesterolemia ATTR Solid cancers and lymphoma Hypercholesterolemia Ebola infection (biodefense) ApoB TTR PLK1 PCSK9 Viral RNA polymerase Systemic-CDP NPs Systemic-SNALP liposomes (hepatocytes) Systemic-AtuPLEX lipoplex (vascular endothelial cells) Systemic-SNALP liposomes (hepatocytes) Systemic-SNALP liposomes (hepatocytes) Systemic-SNALP liposomes (solid tumors) Systemic-SNALP liposomes (hepatocytes) Systemic-SNALP liposomes (hepatocytes and phagocytes) I I I I I 2009 2009 2010 2011 2011 siG12D LODER CALAA-01 ALN-VSP02 Atu027 TKM-ApoB ALN-TTR01 TKM-PLK1 ALN-PCS02 TKM-EBOLA 2004 2005 2007 2008 Abbreviations: ApoB: apolipoprotein B. AMD: age-related macular edema. ATTR: transthyretin (TTR)-mediated amyloidosis. CDP: cyclodextrin polycation. DME: diabetic macular edema. KSP: kinesin spindle protein. NP: nanoparticle. PCSK9: proprotein convertase subtilisin/kexin type 9. PKN3: protein kinase N3. PLK1: polo-like kinase 1. RRM2: ribonucleotide reductase subunit 2. RSV: respiratory syncytial virus. siRNA: small interfering RNA. SNALP: stable nucleic acid lipid particles. VEGF: vascular endothelial growth factor. pathways at the molecular and genomic levels as well as the toxicity and tolerability-related issues concerning synthetic NPs may hamper the clinical approval of highly specific, targeted and individualized RNAi-based medicine. The emerging Omics area on the other hand, provides more adequate and comprehensive understanding of the specific mutant genes in diseases and the biological responses upon NP-formulated RNAi delivery both in vitro and in vivo that would aid in designing safer and more potent delivery strategies. Beyond diagnostics, imaging methodologies and conventional drug delivery systems, Nanobiotechnology field offers other exciting tools for medical and therapeutic applications, such as the tissue engineering discipline. This developing field intends to design biocompatible scaffolds aiming to regenerate tissues as well as resemble the ECM and provide a controlled release of the loaded pharmaceutical agents upon the use of a wide range of biomaterials. Recently, this strategy has been adapted for RNAi molecules delivery mainly in in vitro and ex vivo studies [156]. A sustained and specific RNAi molecules release is achieved upon incorporating NP-associated RNAi molecules into scaffolds within the intended site of action, allowing more efficient, specific and potent gene regulation as well as mitigating several conventional delivery tools- related adverse effects. We believe that the combinatorial and inter-disciplinary approach of RNAi, Nanobiotechnology and Omics era will pave the way for tolerable, targeted and highly effective personalized RNAi-based nanomedicines, revolutionizing research, and turning RNAi to a new therapeutic modality. 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