Advances in siRNA delivery in cancer therapy

Abstract

RNA interference (RNAi)-based therapeutic approaches are under vibrant scrutinisation to seek cancer cure. siRNA suppress expression of the carcinogenic genes by targeting the mRNA expression. However, in vivo systemic siRNA therapy is hampered by the barriers such as poor cellular uptake, instability under physiological conditions, off-target effects and possible immunogenicity. To overcome these challenges, systemic siRNA therapy warrants the development of clinically suitable, safe, and effective drug delivery systems. Herein, we review the barriers, potential siRNA drug delivery systems, and application of siRNA in clinical trials for cancer therapy. Further research is required to harness the full potential of siRNA as a cancer therapeutic.

Keywords:

• Cancer therapy
• gene silencing
• RNA interference
• siRNA
• biological barriers

Introduction

Cancer is a group of diseases that sequences from more than one genetic changes and involving abnormal cell boom with the capability to invade or spread to other parts of the body [1]. Cancer is one of the major targets of RNAi-primarily based therapy, as oncogenes, mutated tumour suppressor genes and numerous different genes contributing to tumour development are potentially important for gene silencing by RNAi. The delivery of nucleic acid therapeutics (e.g. DNA, siRNA, shRNA, and antisense oligonucleotides) to down-regulate mutated genes, and to silence unwanted gene expression; is turning into an exceptionally appealing method to suppressing tumour cell growth and invasion. Optimal combinations of potent anticancer siRNAs and effective delivery systems can also pave the way for the successful clinical application of siRNAs.

The history of RNAi

In the 1990s, a surprising observation on gene-silencing phenomenon was made in Petunias as shown in Figure 1. Plant scientist Richard Jorgensen and colleagues published research on the bizarre effect of introducing extra genes for an enzyme into Petunias [2]. Fire and Mello, were first to report that the double-stranded RNAs (dsRNAs) can trigger gene silencing of complementary messenger RNA sequences in the nematode worm Caenorhabditis elegans [3] and the term “RNA interference” (RNAi) was coined. Elbashir et al. [4] reported that synthetic exogenous siRNA induce sequence specific gene knock down in mammalian cells in vitro [4]. The first observation of sequence specific gene silencing in mice using siRNA was achieved for hepatitis C virus [5]. In 2004, only six years after the discovery of RNAi, the first siRNA-based human therapeutic was developed as treatment for wet age-related macular degeneration, and entered phase I clinical trials [6]. In 2006, Fire and Mello were awarded the Nobel Prize for their discovery of RNAi. Davis et al. [7] reported the first targeted siRNA delivery of nanoparticles in humans via systemic injection. The field of RNAi is moving forward at a remarkable pace as shown in Figure 2. Currently the explosive growth in siRNA is used both for basic research and therapeutic intervention in various diseases including cancer.

Figure 1. Example of petunia plants wherein genes for pigmentation are silenced by RNAi [82].

Figure 2. Timeline of discoveries and milestones in the field of RNAi.

Mechanism of RNA interference

Therapeutic gene silencing by siRNA

RNAi is a fundamental pathway found in many eukaryotes, including animals. As demonstrated in Figure 3, RNAi is triggered by the presence of long double-stranded RNA, which is cleaved into the fragments known as siRNA (21–23 nucleotides long) by the endonuclease dicer. siRNA are loaded onto RNA-induced silencing complex (RISC). RISC contains Argonaute protein (Ago-2) capable of cleaving and removing the passenger strand of the siRNA duplex. The single stranded guide RNA, in association with protein of the RISC complex directs the specificity of the target mRNA recognition through complementary base pairing [8,9]. Argonaute-2 degrades the mRNA complementary to the antisense strand [10], and endonucleolytic cleavage occurs between bases 10 and 11 relative to the 5′ end of the antisense siRNA strand [11–14], thereby causing gene silencing and mRNA degradation.

Figure 3. The mechanism of RNA interference. Long dsRNA is introduced into the cytoplasm, where it is cleaved into siRNA by Dicer. Alternatively, siRNA can be introduced directly into the cell. The siRNA incorporated into RISC assembly, and the sense (passenger) strand is degraded by the protein Argo-2 in the RISC. The remaining antisense strand serves as a guide to recognise the corresponding mRNA. The activated RISC–siRNA complex bind to and degrades the target mRNA, which leads to the silencing of the target gene.

Barriers to siRNA delivery

siRNA hold promise as therapeutic gene silencing, however several barriers still exist in order to achieve effective and controlled in vivo delivery and limit the use of RNAi in the clinic [15]. siRNA formulations for systemic application face a chain of hurdles in vivo before reaching the cytoplasm of the target cell. Post-injection, the siRNA complex must navigate the circulatory system of the body while avoiding kidney filtration, uptake by phagocytes, aggregation with serum proteins and enzymatic degradation by endogenous nucleases [16]. One of the first biological barriers encountered with the aid of administered siRNA is the nuclease activity in plasma and tissues. The major nuclease in plasma is a 3′exonuclease however cleavage of internucleotide bonds can also take place. The reported half-life for unmodified siRNA in serum ranges from several minutes to 1 h [17]. In addition, the kidney plays a key role in siRNA clearance and several studies in animals claimed that the biodistribution of siRNA shows the highest uptake in the kidney. In addition to circulating nuclease degradation and renal clearance, a major barrier to in vivo delivery of siRNA is uptake by the reticuloendothelial system (RES). The RES is composed of phagocytic cells, including circulating monocytes and tissue macrophages, the physiological function of which is to clear foreign pathogens and to remove cellular debris and apoptotic cells [18]. Tissue macrophages are most abundant in the liver and the spleen; tissues that also receive high blood flow and exhibit a fenestrated vasculature. Thus, it is not surprising that these organs accumulate high concentrations of siRNA following systemic administration [19].

Free siRNA, which is a type of anionic and hydrophilic double-stranded small RNA, is not readily taken up by cell. Moreover, the hydrophilicity and negative charge of siRNA molecules prevents them from readily crossing biological membranes. This suggests that siRNA needs to be packaged in vesicles in order to enter cells [20].

The most important challenge that needs to be overcome is the potential for “off-target” effects. A gene that shares a sturdy homology to the target gene has the potential to be inadvertently knocked down, leading to possibly severe unintended side effects. siRNA shows miRNA-like off-target silencing of a large number of unintended transcripts with partial identity to its sequence [21,22]. This mechanism has unpredictable cellular consequences, with important toxic phenotypic effects. Another challenge to siRNA therapy is “immune stimulation”, which is recognition of a siRNA duplex by the innate immune system. Introduction of too much siRNA is known to result in the activation of innate immune responses. The immune system is probably activated via the dsRNA sensor; protein kinase R, inflammatory cytokines and interferon were found to be induced by activation of NF-kB and interferon regulatory factors following the recognition of siRNA by toll-like receptor7 (TLR7), TLR8 and TLR9 [23–25]. To overcome these difficulties, the development of safe and effective in vivo delivery system is essential (Table 1).

Table 1. Problems with naked siRNA for clinical applications.

Problems with Naked siRNA	Reason	Approach
Short half-life	Degradation by serum nucleases Rapid clearance by RES and renal filtration Phagocyte uptake	Chemical modification in siRNA, such as phosphorothioate Chemical modification of the sugar or the bases of oligoribonucleotides for stabilization Nanoparticle carriers
Reduced cellular uptake	Anionic nature Too large to pass membrane	siRNA complexed in a cationic polymeric nanoparticle Cell-penetrating peptide(CPP) mediated internalization
Toxic effect  Saturation of RNAi machinery  Immune response stimulation Off target effect	Reduced accessibility to miRNA TLR activation and type I IFN response Non TLR mediated innate immune response miRNA like off-target silencing	Correct dosing and specific binding to a target cell Chemical modification 2′OH methylation-screening siRNA effect in vitro

Potential systemic siRNA drug delivery system for cancer therapy

The major problem facing siRNA-based therapeutics for cancer and different diseases is delivering siRNA to the target cell population in vivo. The efficacy of siRNA-based drugs in combating cancer requires potent and effective gene silencing in the tumour cells. To achieve efficient delivery of siRNA, an ideal systemic siRNA delivery system should have the following characteristics: (i) be biocompatible, biodegradable and non-immunogenic, (ii) must guard siRNA from serum nucleases during transit through the circulation and on release into endosomes, (iii) avoid rapid hepatic or renal clearances, and (iv) mediate siRNA delivery into target cells while sparing normal tissues. Thus, the design criteria of an in vivo, systemic siRNA delivery system should involve methods for increasing the serum half-life of the siRNA, its distribution to target tissue, its cellular uptake with subsequent intracytoplasmic release without degradation, and avoiding off-target gene silencing activity.

The currently developed siRNA delivery systems for cancer therapy mainly include: (i) chemical modifications of siRNA, (ii) lipid based siRNA delivery system, (iii) polymer based siRNA delivery system, (iv) conjugate siRNA delivery systems, (v) co-delivery of siRNA and anticancer drugs, and (vi) inorganic nanoparticles (quantum dots, carbon nanotubes, and gold nanoparticles as explained in Figure 4).

Figure 4. Various types of siRNA cancer therapeutics: (a) siRNA strand chemical modification, (b) Lipoplexes, (c) stable nucleic acid-lipid particles (SNALPs), (d) polymer-based delivery, (e) dendrimer-siRNA conjugate, (f) conjugate siRNA delivery, (g) aptamer-siRNA chimaeras, (h) quantum dot complexed with siRNA, (i) carbon nanotube-siRNA conjugate, (j) gold nanoparticles.

These modifications help to address the problems in naked siRNA related to (1) serum stability, (2) clearance of large molecular mass material, (3) high toxicity (cytotoxicity), (4) ligand–receptor interaction, (5) vascular permeability to reach cancer tissues, and (6) renal clearance. These benefits are difficult to obtain from a single modification. The siRNA delivery interact with various components, therefore, if these modifications are not designed well, they may also lead to problems. For example, nano-modification may leave high positive charge on the surface of nanoparticles causing unfavourable aggregation with erythrocytes. Another issue with using nanoparticles is the tendency of absorption of serum opsonin proteins, clearing it from the blood and hence barring it from reaching targets. On the other hand, if not well engineered, synthetic or polymer modification may cause issues related to large molecular mass and non-biodegradability leading to toxicity.

Chemical modifications of siRNA for cancer therapy

For anticancer siRNAs to exert their activity in cancer tissues, chemical modifications of naked siRNA have been used to generate nuclease-resistant siRNA to avoid degradation, enhance the stability of siRNA, and improve circulation time as well as tumour uptake in vivo. The performance of siRNA is considerably improved after chemical modification to siRNA strands; the sites include sugar, base, phosphorous acid, strand end or backbone of each sense and antisense strands (Figure 4(a)). A common modification is the replacement of the phosphodiester (PO₄) group with phosphorothioate (PS) at the 3′end [26]. The introduction of PS backbone linkages at the 3′ end of the RNA strands can inhibit enzymatic degradation by reducing siRNAs susceptibility to exonucleases. The introduction of an O-methyl group (2′-O-Me), a fluoro (2′-F) group or a 2-methoxyethyl (2′-O-MOE) group resulted in prolonged half-lives and RNAi activities in cultured cells and plasma [27]_. The modification of siRNA with 2,4-dinitrophenol (DNP) leads to improved nuclease resistance along with an increase in membrane permeability of the modified siRNA [28]. The basic requirement of successful modifications is enhancing siRNA serum stability without negative effects on its gene silencing activity. Indeed boranophosphonate siRNA showed a better resistance to nuclease degradation in spite of the fact that it reduces RNAi activity [29]. The degradation of modified siRNA into non-natural molecules results into reduced RNAi activity and production of toxic metabolites. As a result the delivery systems in which unmodified siRNA will be loaded as a cargo for targeted systemic delivery, have gained prominence.

Lipid-based siRNA delivery system for cancer therapy

Lipid-based systems for delivering anticancer siRNAs embody varied lipid nanoparticles, including liposomes, micelles, emulsions, and solid lipid nanoparticles [20]. Cationic lipid components in the carriers are essential elements for interacting with negatively charged siRNAs, they also play a pivotal role in the delivery efficiencies of siRNAs [30].

Liposomes/lipoplexes

Lipoplexes are one of the most attractive nonviral vectors for plasmid and siRNA delivery [31]. The transfection mechanism of liposomes involves static interactions between negatively charged nucleic acids and cationic lipids as shown in Figure 4(b). Once mixed along, they spontaneously form lipoplexes [32,33]. Cationic lipids (100–300 nm in size) can protect siRNA from enzymatic degradation and increase the circulating half-life and uptake by cells. Cationic lipids such as 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and N-{1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulfate (DOTMA), along with helper lipids such as DOPE, are often used to form cationic liposomes and complex with negatively charged deoxyribonucleic acid and siRNA, resulting in high in vitro transfection efficiency [34]. Commercially available formulations like lipofectamine 2000 are used for in vitro transfection [35]. Cationic liposomes have limited success in vivo, they show dose-dependent toxicity and pulmonary inflammation can arise as a result of reactive oxygen intermediates [36–38]. In a recent study, chemotherapeutics and MCL1-specific siRNA co-delivered using trilysine-derived cationic lipid-based liposomes was found to decrease the expression of MCL1 in the tumour tissues of keratin-forming human epidermal carcinoma (KB) cell-xenografted mice [39]. In one study of anticancer siRNA was co-formulated with a diagnostic agent in cationic liposomes for theranostic purposes [40].

Neutral nanoliposomes

The development of neutral 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) based nanoliposomes (∼mean size 65 nm), which encapsulate siRNA can deliver siRNA in vivo into tumour cells more efficiently than cationic liposomes and naked siRNA, respectively [41]. DOPC-encapsulated siRNA liposomes, which target genes, e.g. EphA2 [42], FAK [43], neuropilin-2 [44] demonstrated significantly inhibited tumour growth in an orthotropic model of ovarian carcinoma and colorectal cancer xenografts in mice.

Stable nucleic acid lipid particles (SNALP)

Stable nucleic acid lipid particles (SNALPs) are a major advance in lipid-based siRNA delivery. The first non-human primate study on siRNA delivery was carried out with SNALPs [45]. SNALPs are microscopic particles approximately 120 nm in diameter, which have been used to deliver siRNAs therapeutically to mammals in vivo. In SNALPs, the siRNA is surrounded by a lipid bilayer containing a mixture of cationic and fusogenic lipids, coated with diffusible polyethylene glycol [46] (Figure 4(c)). The siRNA-lipid complexes showed considerably enhanced cellular internalization and endosomal escape of siRNA. Morrissey et al. suggested that HBV replication was inhibited through the delivery of a siRNA-SNALP complex that targeted HBV RNA. Three daily intravenous injections of 3 mg/kg/day reduced serum HBV levels by at least one order of magnitude, and the effect was specific, dose-dependent and lasted for up to seven days after dosing [47]. Zimmerman et al. demonstrated the ability of SNALPs to enable knockdown of ApoB in the liver of cynomolgus monkeys [48]. Alnylam Pharmaceuticals (Cambridge, MA, USA) has developed the first dual-targeted siRNA drug, SNALP-FORMULATED siRNAs targeting vascular endothelial growth factor (VEGF) and KSP in ALN-VSP02. Tekmira (Barnaby, Canada) started in 2015 a Phase II clinical trial to evaluate the efficacy of siRNA containing SNALP for the treatment of Ebola virus (TKM-100201) [49].

Other lipid-like delivery systems are lipidoid nanoparticles, which are comprised of cholesterol and PEG-modified lipids specific for siRNA delivery [50]. To improve SNALP-mediated delivery, Akinc et al. developed a new chemical method to allow rapid synthesis of a large library of lipidoids and tested their efficacy in siRNA delivery [51]. The 98N₁₂–5 lipidoid-based siRNA formulation, showed 75–90% reduction in ApoB or FVII factor expression in hepatocytes in nonhuman primates and mice [51].

Polymer-based siRNA delivery system for cancer therapy

Cationic polymer-based delivery systems have been investigated for the non-viral delivery of siRNAs. In polymer-based delivery, the siRNA is condensed within different kinds of cationic polymers such as chitosan, cyclodextrin, PEI that form nanoparticles, and the surface of the nanoparticles is decorated with PEG and targeting moieties as shown in Figure 4(d). Examples of cationic polymers used for the delivery of anticancer siRNA are enlisted in Table 2.

Table 2. Examples of siRNA delivery systems for cancer therapeutics.

Delivery system	Carrier	Target genes	Co-treated drugs	Animal models	References
Lipid-based systems	Cationic liposomes	MCL1	Suberoylanilidehydroxamic acid	Human epithelial carcinoma KB-xenografted mice	[39]
	Cationic liposomes	Survivin	Gadolinium-linked lipid, rhodamine-labelled lipid for imaging	Human ovarian cancer OVCAR-3-xenografted mice	[40]
	Neutral nanoliposomes	EphA2	Paclitaxel	Orthotopic ovarian cancer mice	[42]
Polymer-based system	Chitosan-coated polyisohexylcyanoacrylate nanoparticles	RhoA	–	Human breast cancer MDA-MB-231-xenograted mice	[52]
	α-Tocopherololigochitosan nanoparticles	MCL1	–	Human epithelial carcinoma KB-xenografted mice	[53]
	Cyclodextrin-containing polycation nanoparticles	FLI1-EWS	–	Human Ewing’s sarcoma TC71-LUC-xenografted mice	[61]
	PEI polyplexes	HER-2	–	Human ovarian carcinoma SKOV-3-xenografted mice	[54]
	Lipid-linked PEI polyplexes	STAT3	–	Murine melanoma B16-bearing mice	[55]
	Dendrimers	TWIST1	–	Orthotopic tumours	[56]
siRNA conjugates	Aptamer-siRNA chimera	PSMA	–	Xenograft model of prostate cancer	[57]
	EpCAM aptamer-siRNA chimera	PLK1	–	MDA-MB-468 (basal A triple-negative breast cancer/TNBC) nude mice xenografts	[58]

Chitosan

Chitosan is one of the most widely investigated nonviral, naturally derived polymeric gene delivery vectors. It is a cationic polysaccharide composed of β-(1–4)-linked d-glucosamine and N-acetyl-d-glucosamine units. Because of its cationic property, chitosan and its derivatives have been extensively studied for the delivery of plasmid DNA and siRNA in vitro and in vivo [59]. Chitosan-coated polyisohexylcyanoacrylate nanoparticles have been studied for the delivery of RhoA-specific siRNA, efficiently inhibited the growth of aggressive xenografted breast cancer in mice [52]. Self-assembling α-tocopherol oligochitosan nanoparticles have been formulated for the delivery of siRNA against MCL1 [53].

Cyclodextrins

Cyclodextrins are natural polymers, which can form water soluble inclusion complexes with small and large molecules [60]. The cyclodextrin-containing polycation system for the targeted delivery of siRNA was developed [61] . This system consisted of a cyclodextrin – containing polymer, PEG for stability, and human transferrin as the targeting ligand for binding to transferrin receptors, which are often overexpressed on cancer cells. This targeted nanoparticle system, called CALLA-01, was developed for the first siRNA Phase I trial by Calando Pharmaceuticals (Pasadena, CA, USA) [7]. The siRNA in CALLA-01 is designed to inhibit tumour growth via a mechanism to reduce expression of the M2 subunit of ribonucleotide reductase (R2).

Polyethylenimine

Polyethylenimine (PEI), a commonly used synthetic cationic polymer for the delivery of anticancer siRNAs [30]. PEI is available as branched or linear form in various molecular weights. Because of its high cationic charge density, PEI forms small and compact nanoparticles with nucleic acids, and provides silencing of target gene expression after siRNA delivery in vitro and in vivo. Polyplexes of PEI and HER-2 receptor-specific siRNA were shown to produce gene silencing and exert anti-tumour effects in mice [54]. A lipid-linked PEI was reported to improve the siRNA delivery function of PEI [55].

Dendrimers

Dendrimers are synthetic, highly branched macromolecules with three-dimensional nanometric structure (Figure 4(e)). The unique structural properties such as tunable size, accessible terminal functional groups and cargo encapsulation in a nanometer size add to their potential as drug carriers [62]. Cationic dendrimers have proven useful in masking the charge of siRNA long enough for in vivo delivery. Polycationic dendrimers have found applications as non-viral siRNA delivery vectors. In this approach polycationic dendrimers, conjugated with targeted lipid moieties, complex with 2-modified siRNA. Polycationic dendrimers such as poly(amidoamine) (PAMAM) and poly(propylenimine) (PPI) dendrimers have been studied for siRNA delivery in recent years. PAMAM dendrimers have become the most used dendrimer-based carriers for gene delivery. However, PAMAMs were demonstrated to be cytotoxic, predominately related to apoptosis mediated by mitochondrial dysfunction [63]. Surface-modified and cationic PAMAM dendrimers show very low cytotoxicity, even at high concentrations and efficiently penetrated cancer cells in vitro [64]. Finlay et al. [56] targeted the in vivo breast cancer cell metastasis by downregulating TWIST1, a transcription factor activates the EMT, using third generation amphiphilic PAMAM dendrimer YTZ3–15 complexed with TWIST1 siRNA. PPI dendrimers were also used to formulate siRNA nanoparticles, and these nanoparticles showed efficient gene silencing [65].

Conjugate siRNA delivery system for cancer therapy

A common approach for targeted delivery of siRNA to specific cells or tissues is conjugation to deliver materials such as functional peptides, antibodies, aptamers, oleophilic molecules or PEG to improve stability, prolong circulation time, and facilitate cellular uptake of siRNAs on in vivo administration as shown in Figure 4(f) [66].

Lipophile-siRNA conjugates, which were the first conjugate delivery systems to indicate efficacy in vivo, consist of siRNA conjugated to cholesterol [45]. Cholesterol was conjugated to the 3′-terminus of the sense strand of siRNA via a pyrrolidone linkage. To further optimise cholesterol-siRNA, high density lipoprotein was bound, which increased gene silencing efficacy by 8–15 fold in vivo [67]. For enhanced intracellular delivery, siRNA may be conjugated with cell-penetrating peptides or protein transduction domains [68]. Penetratin, transportan and trans-activator of transcription (TAT) protein enable the cellular uptake of hypophilic macromolecules like peptides and nucleic acids [69,70]. However, conjugation of cationic peptides to anionic siRNA may neutralise and reduce the penetrating efficacy of these peptides [68]. In addition, CPP-siRNA conjugates may exhibit cytotoxicity caused by cell membrane perturbation or immunogenicity [71]. Aptamers are oligonucleotide or peptide molecules that bind to specific target molecules. Aptamers are explored for targeted siRNA delivery as an alternative to antibodies because of their chemical versatility, stability, and low immunogenicity [72]. For targeted delivery of siRNA, aptamer-siRNA chimeric (Figure 4(g)) RNAs have been developed [57] and employed to target prostate-specific membrane antigen (PSMA), which is over expressed in prostate cancer cells. The chimeric RNA was demonstrated to bind only PSMA-expressing cells, resulting in depletion of siRNA target proteins and cell death. According to a recent study, gene knockdown by EpCAMaptamer-siRNA chimeras suppresses epithelial breast cancers and their tumour -initiating cells [58].

Co-delivery of siRNA and anticancer drugs

Various types of nanocarriers have been investigated for the co-administration of siRNA and antineoplastic drugs in an effort to enhance anticancer effects by overcoming multidrug resistance or inducing different caspase-mediated cell death pathways. Judiciously designed multifunctional drug/siRNA co-delivery nanocarriers will considerably increase their in vivo tumour accumulation via both passive and active tumour-targeting abilities. Thus, multifunctional nanomedicines offer great promise in overcoming the drawbacks of current treatment modalities, including chemotherapy [73].

Inorganic nanoparticles

A number of inorganic nanoparticles have been emerging as potential siRNA delivery systems devised for therapeutic purposes. They include quantum dots (QDs), carbon nanotubes (CNTs), and gold nanoparticles.

Quantum dots

Quantum dots are semiconducting inorganic crystals with superior photo stability and tunable optical properties for an extensive selection of nonoverlapping colours (Figure 4(h)) [74]. They have bioactivity, and in certain cases, depending on the bulk material used, have no toxicity but a low payload capacity. Quantum dots are a powerful tool in cancer targeting, imaging living animals and investigation of pathophysiology in tumour tissue [75].

Carbon nanotubes

Carbon nanotubes have recently emerged as a replacement choice for cancer treatment, as a carrier for siRNA delivery (Figure 4(i)). CNTs with their nanoneedle structure have been able to independently translocate into cytoplasm without inducing necrobiosis [76,77]. Carbon nanotubes can be divided into single-walled and multiwalled categories. Single walled CNTs functionalised with –CONH–(CH₂)6–NH₃ + Cl⁻ act as siRNA carriers; siRNA is free from the nanotube side-wall to silence the expression of enzyme polymerase. This action inhibits the synthesis of enzyme and prevents cancer cells from getting replicative immortality thus suppressing tumour growth [78,79]. A number of excellent articles have been published that highlight the use of carbon nanotubes for delivery of small molecule drugs and nucleic acids [80,81].

Gold nanoparticles

Gold nanoparticles (Au NPs) (usually 10–20 nm) (Figure 4(j)) are used as a promising siRNA delivery carrier due to their excellent biocompatibility, ease of synthesis, high surface-to-volume ratio, and facile surface functionalization [82]. For the delivery of nucleic acids, gold nanoparticles functionalised with positively charged quaternary ammonium or branched PEI [83], or coated with a cationic lipid bilayer have been reported [83]. The attachment of oligonucleotides to the surface of gold nanoparticles has also been reported. Ding et al. [84] compiled a review on gold nanoparticles for nucleic acid delivery.

Conclusion and future perspectives

Post-transcriptional gene silencing with the aid of RNAi represents a promising new gene therapy approach. An abnormally high number of oncogenes are one of characteristic of cancer. siRNA has a potential to target any cancer-related genes and represents a new class of cancer therapeutics. The engineering of siRNA carriers has gained a lot of interest in recent years, as a result of development of effective delivery systems that can carry siRNA into tumours tissues and the cytoplasm of tumour cells. Several clinical trials of siRNA-based anticancer therapies are summarised in Table 3. The momentous progress in siRNA-based formulation development shall continue to enlighten/apprehend its possible therapeutic application. The ideal nanocarrier system should protect the RNAi therapeutic agent from the circulatory environment and efficiently deliver the therapeutic agent to tumour cells. The future research has to focus on achieving well-organised delivery of siRNA to the desired cells (with no off-target effects), increasing resistance to nuclease, and avoiding immune responses. Great potential exists for nano-siRNA drugs in cancer treatment; further interdisciplinary research is needed in this developing and promising field of nano-siRNA drugs. The siRNA based drug delivery systems hold promise provided their safety and efficacy are adjudged as “satisfactory” by the regulatory agencies.

Table 3. siRNA Cancer therapeutics in clinical trials.

Clinical trial approval	Company	Drug	Vehicle	Target gene	Disease	Delivery route	Phase	Stage	ClinicalTrials.gov Identifier
2008	Calando Pharmaceuticals	CALAA-01	Transferrin receptor-targeted cyclodextrin nanoparticle	RRM2	Solid tumour	IV	I	Terminated	NCT00689065
2009	Alnylam Pharmaceuticals	ALN-VSP02	SNALP	VEGF and KSP	Liver cancer	IV	I	Completed	NCT00882180
2009	Silence Therapeutics GmbH	Atu027	Lipoplex-siRNA	PKN3	Advanced solid tumours	IV	I	Completed	NCT00938574
2010 2012	Silenseed Ltd.	siG12D LODER siG12D LODER + chemotherapy	Miniature biodegradable polymeric matrix	KRASG12D	Pancreatic cancer	EUS biopsy needle	I II	Completed Not yet recruiting	NCT01188785 NCT01676259
2010	Arbutus Biopharma Corporation	TKM 080301	SNALP	PLK-1	Advanced solid tumours	IV	I and II	Completed	NCT01262235
2011	National Cancer Institute (NCI)	TKM 080301	Lipid nanoparticles	PLK-1	Primary or secondary liver cancer	IV	I	Completed	NCT01437007
2012	M.D. Anderson Cancer Centre	siRNA-EphA2-DOPC	Neutral liposome	EphA2	Advanced cancers	IV	I	Recruiting	NCT01591356
2010	National Cancer Institute (NCI)	EZN-2968	Locked nucleic acid	HIF-1	Advanced solid tumours with liver metastases	IV	I	Completed	NCT01120288

RRM2: M2 subunit of ribonucleotide reductase; VEGF: vascular endothelial growth factor; KSP: kinesin spindle protein; PKN3: protein kinase N3; PLK1: polo-like kinase 1; HIF-1: hypoxia-induced factor; KRAS: V-Ki-ras2 Kristen rat sarcoma viral oncogene homolog; EphA2: Ephrin A2 receptor. Cited from http://www.clinicaltrials.gov.

Disclosure statement

The authors report no conflict of interest.