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Advances in siRNA delivery in cancer therapy
Aishwarya Singh, Piyush Trivedi & Narendra Kumar Jain
To cite this article: Aishwarya Singh, Piyush Trivedi & Narendra Kumar Jain (2018) Advances in
siRNA delivery in cancer therapy, Artificial Cells, Nanomedicine, and Biotechnology, 46:2, 274-283,
DOI: 10.1080/21691401.2017.1307210
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Advances in siRNA delivery in cancer therapy
Aishwarya Singh, Piyush Trivedi and Narendra Kumar Jain
School of Pharmaceutical Sciences, Rajiv Gandhi Technical University, Bhopal, Madhya Pradesh, India
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 chal-
lenges, 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.
ARTICLE HISTORY
Received 27 July 2016
Accepted 13 March 2017
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-regu-
late mutated genes, and to silence unwanted gene expres-
sion; is turning into an exceptionally appealing method to
suppressing tumour cell growth and invasion. Optimal combi-
nations of potent anticancer siRNAs and effective delivery sys-
tems 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 phe-
nomenon 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.
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 trig-
gered by the presence of long double-stranded RNA, which is
cleaved into the fragments known as siRNA (21–23 nucleoti-
des 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 endo-
nucleolytic cleavage occurs between bases 10 and 11 relative
to the 50 end of the antisense siRNA strand [11–14], thereby
causing gene silencing and mRNA degradation.
Barriers to siRNA delivery
siRNA hold promise as therapeutic gene silencing, however
several barriers still exist in order to achieve effective and
CONTACT N.K. Jain Emeritus dr.jnarendr@gmail.com School of Pharmaceutical Sciences, Rajiv Gandhi Technical University, Airport Bypass Road, Gandhi
Nagar, Bhopal 462036, India
� 2017 Informa UK Limited, trading as Taylor & Francis Group
ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY, 2018
VOL. 46, NO. 2, 274–283
http://dx.doi.org/10.1080/21691401.2017.1307210
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 fil-
tration, uptake by phagocytes, aggregation with serum pro-
teins 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 30exonuclease
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 kid-
ney 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 nucle-
ase degradation and renal clearance, a major barrier to in
vivo delivery of siRNA is uptake by the reticuloendothelial sys-
tem (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 fenes-
trated 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 knockeddown, 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
Figure 2. Timeline of discoveries and milestones in the field of RNAi.
Figure 1. Example of petunia plants wherein genes for pigmentation are silenced by RNAi [82].
ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY 275
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 acti-
vated via the dsRNA sensor; protein kinase R, inflammatory
cytokines and interferon were found to be induced by acti-
vation 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 devel-
opment of safe and effective in vivo delivery system is
essential (Table 1).
Potential systemic siRNA drug delivery system for
cancer therapy
The major problem facing siRNA-based therapeutics for can-
cer 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 silenc-
ing in the tumour cells. To achieve efficient delivery of siRNA,
an ideal systemic siRNA delivery system should have the fol-
lowing 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
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.
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
20OH methylation-screening siRNA effect in vitro
276 A. SINGH ET AL.
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 intracyto-
plasmic 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) inor-
ganic nanoparticles (quantum dots, carbon nanotubes, and
gold nanoparticles as explained in Figure 4).
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-biodegrad-
ability 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 replace-
ment of the phosphodiester (PO4) group with phosphoro-
thioate (PS) at the 30end [26]. The introduction of PS
backbone linkages at the 30 end of the RNA strands can
inhibit enzymatic degradation by reducing siRNAs susceptibil-
ity to exonucleases. The introduction of an O-methyl group
(20-O-Me), a fluoro (20-F) group or a 2-methoxyethyl (20-O-
MOE) group resulted in prolonged half-lives and RNAi activ-
ities in cultured cells and plasma [27]. The modification of
siRNA with 2,4-dinitrophenol (DNP) leads to improved
Figure 4. Various types of siRNA cancer therapeutics: (a) siRNA strand chemical modification, (b) Lipoplexes, (c) stable nucleic acid-lipid particles (SNALPs), (d) poly-
mer-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.
ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY 277
nuclease resistance along with an increase in membrane per-
meability 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-nat-
ural molecules results into reduced RNAi activity and produc-
tion of toxic metabolites. As a result the delivery systems in
which unmodified siRNA will be loaded as a cargo for tar-
geted 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 interact-
ing 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 interactionsbetween 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-dioleoy-
loxy)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 for-
mulations like lipofectamine 2000 are used for in vitro trans-
fection [35]. Cationic liposomes have limited success in vivo,
they show dose-dependent toxicity and pulmonary inflamma-
tion can arise as a result of reactive oxygen intermediates
[36–38]. In a recent study, chemotherapeutics and MCL1-spe-
cific 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 diag-
nostic agent in cationic liposomes for theranostic purposes
[40].
Neutral nanoliposomes
The development of neutral 1,2-dioleoyl-sn-glycero-3-phos-
phatidylcholine (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], neu-
ropilin-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 thera-
peutically to mammals in vivo. In SNALPs, the siRNA is sur-
rounded by a lipid bilayer containing a mixture of cationic
and fusogenic lipids, coated with diffusible polyethylene gly-
col [46] (Figure 4(c)). The siRNA-lipid complexes showed con-
siderably enhanced cellular internalization and endosomal
escape of siRNA. Morrissey et al. suggested that HBV replica-
tion was inhibited through the delivery of a siRNA-SNALP
complex that targeted HBV RNA. Three daily intravenous
injections of 3mg/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 mon-
keys [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 pri-
mates and mice [51].
Polymer-based siRNA delivery system for cancer therapy
Cationic polymer-based delivery systems have been investi-
gated for the non-viral delivery of siRNAs. In polymer-based
delivery, the siRNA is condensed within different kinds of cat-
ionic polymers such as chitosan, cyclodextrin, PEI that form
nanoparticles, and the surface of the nanoparticles is deco-
rated 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.
Chitosan
Chitosan is one of the most widely investigated nonviral, nat-
urally derived polymeric gene delivery vectors. It is a cationic
polysaccharide composed of b-(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
278 A. SINGH ET AL.
and in vivo [59]. Chitosan-coated polyisohexylcyanoacrylate
nanoparticles have been studied for the delivery of RhoA-spe-
cific siRNA, efficiently inhibited the growth of aggressive xen-
ografted breast cancer in mice [52]. Self-assembling
a-tocopherol oligochitosan nanoparticles have been formu-
lated for the delivery of siRNA against MCL1 [53].
Cyclodextrins
Cyclodextrins are natural polymers, which can form water sol-
uble inclusion complexes with small and large molecules [60].
The cyclodextrin-containing polycation system for the tar-
geted delivery of siRNA was developed [61] . This system con-
sisted of a cyclodextrin – containing polymer, PEG for
stability, and human transferrin as the targeting ligand for
binding to transferrin receptors, which are often overex-
pressed 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 ribo-
nucleotide reductase (R2).
Polyethylenimine
Polyethylenimine (PEI), a commonly used synthetic cationic
polymer for the delivery of anticancer siRNAs [30]. PEI is avail-
able 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, access-
ible 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 deliv-
ery vectors. In this approach polycationic dendrimers, conju-
gated 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 den-
drimers 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. PPIden-
drimers 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 mole-
cules or PEG to improve stability, prolong circulation time,
and facilitate cellular uptake of siRNAs on in vivo administra-
tion 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 30-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
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 polyiso-
hexylcyanoacrylate
nanoparticles
RhoA – Human breast cancer MDA-MB-
231-xenograted mice
[52]
a-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]
ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY 279
efficacy by 8–15 fold in vivo [67]. For enhanced intracellular
delivery, siRNA may be conjugated with cell-penetrating pep-
tides or protein transduction domains [68]. Penetratin, trans-
portan 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, result-
ing 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 nanomedi-
cines 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 pur-
poses. 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 cap-
acity. 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 T
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280 A. SINGH ET AL.
(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 div-
ided into single-walled and multiwalled categories. Single
walled CNTs functionalised with –CONH–(CH2)6–NH3þCl� 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 posi-
tively 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 repre-
sents 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 inter-
disciplinary research is needed in this developing and promis-
ing 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.
Disclosure statement
The authors report no conflict of interest.
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ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY 283
	Advances in siRNA delivery in cancer therapy
	Introduction
	The history of RNAi
	Mechanism of RNA interference
	Barriers to siRNA delivery
	Potential systemic siRNA drug delivery system for cancer therapy
	Chemical modifications of siRNA for cancer therapy
	Lipid-based siRNA delivery system for cancer therapy
	Polymer-based siRNA delivery system for cancer therapy
	Conjugate siRNA delivery system for cancer therapy
	Co-delivery of siRNA and anticancer drugs
	Inorganic nanoparticles
	Conclusion and future perspectives
	Disclosure statement
	References