Báo cáo khoa học: Efficient and targeted delivery of siRNA in vivo pdf

MINIREVIEW
Efficient and targeted delivery of siRNA in vivo
Min Suk Shim
1
and Young Jik Kwon
1,2,3
1 Department of Chemical Engineering and Materials Science, University of California, Irvine, CA, USA
2 Department of Pharmaceutical Sciences, University of California, Irvine, CA, USA
3 Department of Biomedical Engineering, University of California, Irvine, CA, USA
Introduction
RNA interference (RNAi) is a highly conserved biologi-
cal process among yeasts, worms, insects, plants and
humans [1]. A single strand of exogenously introduced
double-stranded small interfering RNA (siRNA; 20–30
nucleotides) guides an RNA-inducing silencing protein
complex to degrade the mRNA with the matching
sequence; thus, translation into the target proteins is
silenced [2–4]. RNAi has been of great interest not only
as a powerful research tool to suppress the expression of
a target gene, but also as an emerging therapeutic strat-
egy to silence disease genes [5]. Theoretically, siRNA
can interfere with the translation of almost any mRNA,
as long as the mRNA has a distinctive sequence,
whereas the targets of traditional drugs are limited by
types of cellular receptors and enzymes [6].
Cancer, viral infections, autoimmune diseases and
neurodegenerative diseases have been explored as
promising disease targets of RNAi [7,8]. Recent pro-
gress in clinical trials using siRNA to cure age-related
macular degeneration (bevasiranib; Opko Health, Inc.,
Miami, FL, USA; phase III) and respiratory syncytial

virus infection (ALN-RSV01; Alnylam, Cambridge,
MA, USA; phase II) have demonstrated the therapeu-
tic potential of RNAi [9]. Moreover, the first evidence
Keywords
administration routes; barriers in siRNA
delivery; chemically modified RNA; in vivo
disease models; nanoparticles; nonviral
carriers; nucleic acid therapeutics; RNA
interference; targeted delivery in vivo;
viral vectors
Correspondence
Y. J. Kwon, Department of Pharmaceutical
Sciences, 916 Engineering Tower,
University of California, Irvine, CA 92697,
USA
Fax: +1 949 824 2541
Tel: +1 949 824 8714
E-mail:
(Received 7 July 2010, accepted
26 August 2010)
doi:10.1111/j.1742-4658.2010.07904.x
RNA interference (RNAi) has been regarded as a revolutionary tool for
manipulating target biological processes as well as an emerging and prom-
ising therapeutic strategy. In contrast to the tangible and obvious effective-
ness of RNAi in vitro, silencing target gene expression in vivo using small
interfering RNA (siRNA) has been a very challenging task due to
multiscale barriers, including rapid excretion, low stability in blood serum,
nonspecific accumulation in tissues, poor cellular uptake and inefficient
intracellular release. This minireview introduces major challenges in achiev-
ing efficient siRNA delivery in vivo and discusses recent advances in over-

coming them using chemically modified siRNA, viral siRNA vectors and
nonviral siRNA carriers. Enhanced specificity and efficiency of RNAi
in vivo via selective accumulations in desired tissues, specific binding to
target cells and facilitated intracellular trafficking are also commonly
attempted utilizing targeting moieties, cell-penetrating peptides, fusogenic
peptides and stimuli-responsive polymers. Overall, the crucial roles of the
interdisciplinary approaches to optimizing RNAi in vivo, by efficiently and
specifically delivering siRNA to target tissues and cells, are highlighted.
Abbreviations
ApoB, apolipoprotein B; CPP, cell-penetrating peptide; FA, folic acid; GFP, green fluorescent protein; HER-2, human epidermal growth
factor 2; i.p., intraperitoneal; i.t., intratumoral; i.v., intravenous; 9R, nonamer arginine residues; RGD, Arg-Gly-Asp peptide; RNAi,
RNA interference; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.
4814 FEBS Journal 277 (2010) 4814–4827 ª 2010 The Authors Journal compilation ª 2010 FEBS
of targeted in vivo gene silencing for human cancer
therapy via systemic delivery of siRNA using transfer-
rin-tagged, cyclodextrin-based polymeric nanoparticles
(CALAA-01; Calando Pharmaceuticals, Pasadena, CA,
USA; phase I) has been recently announced [10].
Despite quite efficient and reliable gene silencing
in vitro, only limited RNAi has been achieved in vivo
because of rapid enzymatic degradation in combina-
tion with poor cellular uptake of siRNA [11]. There-
fore, novel delivery systems, which enable prolonged
circulation of siRNA with resistance against enzymatic
degradation, high accessibility to target cells via clini-
cally feasible administration routes and optimized
cytosolic release of siRNA after efficient cellular
uptake, are indispensably required [12]. In this mini-
review, major factors in determining overall RNAi
efficiency in vivo are introduced. Moreover, up-to-date

progress in achieving efficient and targeted siRNA
delivery in vivo, particularly by overcoming multiscale
hurdles using novel siRNA carriers, is discussed.
Challenges in RNAi in vivo
Design and in vivo delivery of siRNA
There are multiple key considerations in order to
achieve efficient RNAi in vivo by delivering exogenous
siRNA. siRNA has to be designed to target hybridiza-
tion-accessible regions within the target mRNA while
avoiding unintended (off-target) effects [13–15], which
is extensively reviewed in this series by Walton et al.
[16]. In addition, siRNA can also induce adverse
effects such as immune responses, as discussed by
Samuel-Abraham & Leonard [17]. siRNA may induce
interferon responses either through the double-
stranded RNA-activated protein kinase PKR [18] or
toll-like receptor 3 [19]. Therefore, a combination of
computer algorithms and experimental validation
should be employed to determine the optimized siRNA
sequences that are complementary to target mRNA
while inducing minimal immune responses [20].
Naked siRNA is relatively unstable in blood in its
native form and is rapidly cleared from the body (i.e.
short half-lives in vivo) via degradion by ribonucleases,
rapid renal excretion and nonspecific uptake by the
reticuloendothelial system [21]. The phosphorothioate
backbone, or various 2¢ positions in the sugar moiety
of siRNA, is conventionally modified to enhance its
stability and activity against nuclease degradation
[22,23], without affecting gene silencing activity [24].

siRNA is an anionic macromolecule and does not
readily enter cells by passive diffusion mechanisms. An
appropriate siRNA delivery system enhances cellular
uptake, protects its payload from enzymatic digestion
and immune recognition, and improves the pharmacoki-
netics by avoiding excretion via the reticuloendothelial
system and renal filtration (i.e. prolonged half-life
in vivo) [25–27]. In addition, targeted delivery systems
localize siRNA in the desired tissue, resulting in a
reduction in the amount of siRNA required for
efficient gene silencing in vivo , as well as minimized
side effects. Therefore, the development of effective
in vivo delivery systems is pivotal in overcoming the
challenges in achieving efficient and targeted siRNA
delivery in vivo. Major hurdles in siRNA delivery
in vivo and various approaches to overcoming them
are illustrated in Fig. 1.
Local versus systemic delivery
The types of target tissues and cells dictate the
optimum administration routes of local versus systemic
delivery. For example, siRNA can be directly applied to
the eye, skin or muscle via local delivery, whereas sys-
temic siRNA delivery is the only way to reach meta-
static and hematological cancer cells. Local delivery
offers several advantages over systemic delivery, such as
low effective doses, simple formulation (e.g. no
targeting moieties), low risk of inducing systemic
side effects and facilitated site-specific delivery [28].
Therefore, if applicable, local delivery is likely to be a
more cost-efficient strategy for siRNA delivery in vivo

than systemic administration. For example, initial clini-
cal trials for RNAi-based treatment of age-related mac-
ular degeneration have exclusively used local injections
of siRNA directly into the eye [10]. Other promising
local routes include intranasal siRNA administration
for pulmonary delivery [10,29–31] and direct injection
into the central nervous system [10,32,33].
Alternatively, systemic delivery via intravenous
(i.v.), intraperitoneal (i.p.) or oral administration is
widely applicable when the target sites are not locally
confined or not readily accessible. Metastatic tumors
are especially amenable for systemic delivery compared
with local administration. For example, human bcl-2
oncogene-targeting siRNA, which was complexed with
cationic liposomes and i.v. injected, effectively inhib-
ited tumor growth in a mouse liver metastasis model
[34]. Another study showed that siRNA encapsulated
in a lipid vesicle was able to impart efficient and per-
sistent antiviral activity after being injected into a
hepatitis B virus mouse model [35]. However, impor-
tantly, systemic siRNA delivery imposes several
additional barriers in comparison with local delivery.
siRNA should remain in its active form during circula-
tion and be able to reach target tissues after passing
M. S. Shim and Y. J. Kwon In vivo siRNA delivery
FEBS Journal 277 (2010) 4814–4827 ª 2010 The Authors Journal compilation ª 2010 FEBS 4815
through multiple barrier organs (e.g. liver, kidney and
lymphoid organs).
Extracellular and intracellular barriers in siRNA
delivery in vivo

Regardless of administration routes, the final desti-
nation of siRNA is the cytoplasm of the target cell,
where it incorporates into RNA-inducing silencing
protein complex and encounters target mRNAs. First,
siRNA that survives in the plasma and is transported
close to a target tissue must extravasate through the
tight vascular endothelial junctions. It has been
reported that microvascular transport of macromoel-
cules > 5 nm in diameter is significantly inhibited in
normal tissues [36]. However, transport of macromole-
cules across the tumor endothelium is more efficient
than that of normal endothelium because of its leaky
and discontinuous vascular structures with poor lym-
phatic drainage. Thus, tumor endothelium allows the
penetration of high molecular mass macromolecules
(> 40 kDa), which is also referred to as ‘enhanced
permeation and retention effect’ [37]. siRNA, in its
native form or formulated in a delivery carrier, must
then diffuse through the extracellular matrix, a dense
network of fibrous protein and carbohydrates
surrounding a cell [38], before accessing target cells.
siRNA or its complex adheres preferably to target cells
via receptor-mediated specific binding, followed by
cellular uptake. Even after it is internalized by a cell,
siRNA should be released from the endosome, while
avoiding entrapment and degradation [39,40]. Because
the condition in the endosome ⁄ lysosome is mildly
acidic, facilitated cytosolic release of siRNA using
acid-responsive delivery carriers has been a popular
strategy to overcome this intracellular hurdle [41,42].

Fusogenic peptides which undergo acid-triggered con-
formational changes have also been shown to acceler-
Fig. 1. Interdisciplinary approaches to achieving efficient and targeted RNAi in vivo by overcoming multiscale barriers in systemic siRNA
delivery. Detailed design parameters of an ideal siRNA carrier are depicted in Fig. 2.
In vivo siRNA delivery M. S. Shim and Y. J. Kwon
4816 FEBS Journal 277 (2010) 4814–4827 ª 2010 The Authors Journal compilation ª 2010 FEBS
ate endosomal escape of nucleic acids [43,44]. Finally,
siRNA delivered by a carrier should be decomplexed
in the cytoplasm [45]. A broad range of novel materials
that provide enhanced siRNA release have been devel-
oped (e.g. disulfide-based cationic polymers) [46].
Fig. 1 shows extracellular and intracellular barriers in
siRNA delivery with various approaches to overcom-
ing them.
Chemically modified siRNA for
enhanced RNAi in vivo
Various molecular positions in siRNA have been chem-
ically replaced or modified, mainly to resist enzymatic
hydrolysis. For example, phosphodiester (PO
4
) linkages
were replaced with phosphothioate (PS) at the 3¢-end,
and introducing O-methyl (2¢-O-Me), fluoro (2¢-F)
group or methoxyethyl (2¢-O-MOE) group greatly pro-
longed half-lives in plasma and enhanced RNAi effi-
ciency in cultured cells [47–51]. In addition, efficiency
enhancer molecules were conjugated to either the 5¢-or
3¢-end of the sense strand, without affecting the activity
of the antisense strand [52]. There are some potential
risks that chemically modifying siRNA may compro-

mise RNAi efficiency. For example, boranophospho-
nate modification at the central position of the
antisense strand of siRNA showed improved resistance
to nuclease degradation, but simultaneously reduced
RNAi activity [53]. In addition, non-natural molecules
produced upon the degradation of a chemically modi-
fied siRNA may generate metabolites that might be
unsafe or trigger unwanted effects. To date, cholesterol
and aptamers are the most promising siRNA conjugates
that have demonstrated efficient RNAi in vivo.
Cholesterol–siRNA conjugates
Improved pharmacokinetic and cellular uptake proper-
ties of cholesterol–siRNA conjugates silenced apolipo-
protein B (ApoB) in mice via i.v. administration [22].
By contrast, ApoB siRNA unconjugated with choles-
terol was unable to induce mRNA interference and
was rapidly cleared. The mechanisms of improved dis-
tribution and cellular uptake of siRNA through cho-
lesterol conjugation were demonstrated in a recent
study; cholesterol–siRNA conjugates seem to incorpo-
rate into circulating lipoprotein particles (i.e. improved
distribution in vivo) and are efficiently internalized by
hepatocytes via receptor-mediated processes (i.e. effi-
cient cellular uptake) [54]. Prebinding of cholesterol–
siRNA conjugates to lipoparticles dramatically
improved silencing efficacy in mice, and lipoparticle
types affected cholesterol–siRNA conjugate distribu-
tion in various tissues [54]. Using a transgenic mouse
model for Huntington’s disease, it was also demon-
strated that a single intrastriatal injection of choles-

terol–siRNA conjugates silenced a mutant Huntingtin
gene, attenuating neuronal pathology as well as
delaying the abnormal behavioral phenotype [55].
RNA aptamer–siRNA conjugates
RNA aptamers have been popularly used to selectively
deliver siRNA in vivo to target tissues and cells, such
as prostate cancer cells and tumor vascular endothe-
lium overexpressing prostate-specific membrane anti-
gen [56]. A key advantage of aptamer-mediated
targeted delivery systems is that RNA aptamers can be
facilely obtained by in vitro transcription reaction and,
therefore, avoid contamination by cell or bacterial
products. Promising in vitro and in vivo RNAi was
obtained using siRNA that was directly linked with
prostate-specific membrane antigen aptamers [57]. An
aptamer-based delivery system has also been used to
suppress HIV infection. Anti-gp120 RNA aptamers
were covalently conjugated with a strand of siRNA,
and the other siRNA strand was subsequently
annealed to the aptamer-conjugated strand. These
aptamer–siRNA conjugates were able to access HIV-
infected cells and silence viral replication in vitro [58].
Viral vectors: natural siRNA carriers
Various recombinant viral vectors have been shown to
be efficient in obtaining gene silencing for an extended
period in a wide range of mammalian cells [59]. For
example, an adenoviral vector encoding siRNA against
pituitary tumor transforming gene 1 significantly inhib-
ited the growth of the pituitary tumor transforming
gene 1-overexpressing hepatocellular carcinoma cells

in vitro and in vivo [60]. It was also demonstrated that
the herpes simplex virus type 1-based amplicon vectors
suppressed in vivo tumorigenicity of human polyomavi-
rus BK-transformed cells (pRPc cells) [61]. Recombi-
nant lentiviral vectors have also been frequently used to
achieve in vivo gene silencing. In particular, lentiviral
vectors containing the U6 promoter were found to be
efficient in green fluorescent protein (GFP) silencing
in vitro, resulting in  80% gene silencing at an average
of one integrated vector genome per target cell genome.
In addition, the U6 promoter was shown to be superior
to the H1 promoter in achieving in vivo gene silencing
and led to persistent GFP knockdown in the mouse
brain for at least 9 months [62]. This indicates that
lentivirus-mediated RNAi is a promising strategy for
long-term gene silencing in vitro and in vivo. Other viral
M. S. Shim and Y. J. Kwon In vivo siRNA delivery
FEBS Journal 277 (2010) 4814–4827 ª 2010 The Authors Journal compilation ª 2010 FEBS 4817
siRNA carriers such as retroviral vectors have not been
intensively explored for their use in vivo [63–65].
Although viral vectors provide excellent tissue-specific
tropism and high RNAi efficiency, safety concerns (e.g.
insertion mutagenesis and immunogenicity) and difficul-
ties with large-scale manufacture may limit the use of
viral vectors for siRNA delivery in clinical setting
[66,67]. Therefore, synthetic counterparts (nonviral
vectors) have been more and more intensively explored
as safe and effective alternatives that are easy to be
prepared and can deliver large payloads of siRNA.
Nonviral carriers: Trojan horses for

efficient, biocompatible and versatile
siRNA delivery in vivo
Delivery of siRNA in its unmodified form has several
advantages over using a chemically modified form.
Unmodified siRNA possesses untouched RNAi
capability (maximized RNAi per siRNA molecule) and
does not require potentially inefficient and time ⁄
labor-consuming modification processes (cost-effective
preparation). However, its highly anionic nature and
the macromolecular size of siRNA necessitates using
efficient carriers to overcome multiscale barriers.
Unlike viral vectors, which deliver siRNA in the form
of a viral genome, nonviral carriers deliver native
siRNA, generate low immunogenicity and offer high
structural and functional tunability. An ideally designed
nonviral siRNA carrier with its desirable structural
and functional multicomponents is depicted in Fig. 2.
Liposomes and lipoplexes
One of the most significant advances in RNAi in vivo
is successful knockdown of ApoB in nonhuman
primates by systemically delivered siRNA in stable
nucleic acid–lipid particles [68]. The siRNA–lipid
complexes showed significantly enhanced cellular inter-
nalization and endosomal escape of siRNA. ApoB
siRNA-carrying stable nucleic acid–lipid particles
greatly reduced ApoB expression and serum choles-
terol levels in monkeys when a clinically acceptable
single siRNA dose of 2.5 mgÆkg
)1
was injected i.v.

[68]. Importantly, expression of ApoB was silenced for
at least 11 days. With addressing the high toxicity
of the currently available liposomes for siRNA deliv-
ery in vitro and in vivo [69,70], cationic cardiolipin
analog-based liposomes carrying c-raf siRNA inhibited
the growth of breast tumor xenografts in mice [71].
Cationic liposomes formulated with anisamide-
conjugated poly(ethylene glycol) effectively penetrated
the lung metastasis of melanoma tumors in mice and
resulted in 70–80% gene silencing after a single i.v.
injection [72].
Further noticeable progress in siRNA delivery using
liposomes is the use of neutral lipids for systemic
siRNA delivery in order to address the toxicity of cat-
ionic lipids. For example, cyclin D1 (CyD1) siRNA
was efficiently encapsulated in neutral phospholipid-
based liposomes coated with hyaluronan [73]. The
resulting siRNA-carrying liposomes were stable during
circulation in vivo after i.v injection and suppressed
leukocyte proliferation and cytokine secretion by
type 1 T-helper cells. Another neutral dioleoyl phos-
phatidylcholine-based delivery system, which targets
EphA2 [74] and focal adhesion kinase [75], demon-
strated significantly inhibited tumor growth in an
orthotropic ovarian cancer model in mice. The same
type of liposome has also been reported to efficiently
silence neuropilin-2 expression and inhibit the growth
of colorectal cancer xenografts in the mouse liver [76].
Polymers and peptides
Nucleic acids such as siRNA are easily complexed

with synthetic cationic polymers e.g., polyethylenimine
Fig. 2. An ideally designed nonviral siRNA carrier for efficient and
targeted RNAi in vivo.
In vivo siRNA delivery M. S. Shim and Y. J. Kwon
4818 FEBS Journal 277 (2010) 4814–4827 ª 2010 The Authors Journal compilation ª 2010 FEBS
(PEI), biodegradable cationic polysaccharide (e.g.
chitosan) and cationic polypeptides [e.g. atelocollagen,
poly(l-lysine) and protamine], via attractive electro-
static interactions. For example, i.t. injection of siR-
NA– atelocollagen complexes silenced luciferase
expression in germ cell tumor xenografted in mice and
inhibited tumor growth [77]. In another study, vascular
endothelial growth factor (VEGF) siRNA–atelocolla-
gen complexes significantly suppressed tumor angio-
genesis and growth in a prostate tumor model in mice
[78]. Intravenous administration of chitosan–RhoA
siRNA complexes resulted in effective gene silencing in
subcutaneously implanted breast cancer cells in mice
[79]. In addition, intranasally administered chitosan–
siRNA complexes efficiently silenced GFP expression
in bronchiole epithelial cells in GFP-transgenic mice
[29]. Tumor necrosis factor expression in systemic mac-
rophages was silenced in mice after i.p. administration
of chitosan ⁄ siRNA complexes, thus downregulating
systemic and local inflammation [80].
Polyethylenimine is one of the most popularly inves-
tigated synthetic cationic polymers for nucleic acid
delivery in vitro and in vivo. Polyethylenimine is very
potent in transfection with its uniquely high buffering
capability at an endosomal pH (proton sponge effect)

which releases nucleic acid payloads into the cytoplasm
[39]. c-erbB2 ⁄ neu (HER-2) siRNA was delivered to
subcutaneous tumors via i.p. administration of siRNA ⁄
polyethylenimine complexes and resulted in a remark-
able reduction of tumor growth [81]. Pain receptors for
N-methyl-d-aspartate were effectively knocked-down by
intrathecal delivery of polyethylenimine-conjugated
siRNA in rats [82]. Inhibited viral propagation in the
lungs was also observed after deacetylated linear
polyethylenimine ⁄ siRNA complexes targeting influenza
nucleoprotein was retro-orbitally administered [83]. In
another study, polyethylenimine-conjugated siRNA
against secreted growth factor pleiotrophin reduced
tumor growth and cell proliferation with no toxicity or
abnormal animal behaviors after intracerebral adminis-
tration in an orthotopic glioblastoma mouse model [84].
Overall, polyethylenimine seems to be a promising
nonviral carrier for siRNA delivery in vivo, if its high
toxicity and limited biodegradability are appropriately
addressed.
Polypeptides, such as poly(l-lysine) and protamine,
have also commonly been used to deliver siRNA.
A sixth generation of dendritic poly(l-lysine) was
employed to systemically deliver siRNA to silence
ApoB expression without hepatotoxicity in hepatocytes
of apolipoprotein E-deficient mice [85]. Protamine, a
natural arginine-rich cationic polypeptide, condenses
negatively charged nucleic acids and has been used as
an efficient gene-delivery carrier [86]. An in vivo study
demonstrated that complexes of siRNA and low

molecular mass protamine, which possess membrane-
translocating potency, were accumulated in tumors via
i.p. administration and successfully inhibited the
expression of VEGF, thereby suppressing the growth
of hepatocarcinoma tumors in mice [87]. In addition,
no noticeable increase in inflammatory cytokines,
including interferon-a and interleukin-12, in serum was
observed when the low molecular mass protamine ⁄
siRNA complexes were administered, indicating
negligible immunostimulatory effects.
One of the fundamental concerns in using synthetic
polymers for siRNA delivery in vivo is dose-dependent
toxicity upon systemic administration. For example,
polyethylenimine and poly(l-lysine) have been shown
to trigger necrosis and apoptosis in a variety of cell lines
[88,89]. The toxicity can be ameliorated by conjugation
with biocompatible, hydrophilic polymers such as
poly(ethylene glycol) or by removing excess (i.e., uncom-
plexed) cationic polymers. In gneral, natural cationic
polymers (e.g. chitosan and protamine), which are bio-
compatible, biodegradable and nontoxic, are more desir-
able in siRNA delivery in vivo than synthetic polymers.
Targeted siRNA delivery in vivo
In order to achieve RNAi in vivo via systemic delivery,
it is crucial for siRNA to be efficiently located in
desired tissues ⁄ cells. This requires three important pro-
cesses: prolong circulation in the body, high accessibil-
ity to target tissues and specific binding to target cells.
Targeted siRNA delivery maximizes the local concen-
tration in the desired tissue (maximized and localized

silencing effects) and prevents nonspecific siRNA dis-
tribution (minimized unwanted effects in non-target
tissues). For example, recent studies have reported can-
cer-targeted siRNA delivery using nanoparticles that
specifically bind to cancer-specific or cancer-associated
antigens and receptors [90,91].
Folate-conjugated siRNA carriers
One of the most popular target molecules in cancer-
specific gene and drug delivery is the folate receptor
[92]. Folic acid (FA) is needed for rapid cell growth,
and many cancer cells overexpress folate receptors to
which FA and monoclonal antibodies specifically bind
[93]. FA can be easily conjugated onto the surface of
liposomal and polymeric siRNA carriers with or with-
out a poly(ethylene glycol) spacer [92]. For example,
FA-conjugated polyethylenimine showed enhanced
gene silencing via receptor-mediated endocytosis [94].
M. S. Shim and Y. J. Kwon In vivo siRNA delivery
FEBS Journal 277 (2010) 4814–4827 ª 2010 The Authors Journal compilation ª 2010 FEBS 4819
Chimeric survivin siRNA incorporated with bacterio-
phage phi29-encoded RNA and when further
conjugated with FA suppressed the growth of naso-
pharyngeal carcinoma in mice, whereas control
FA-free siRNA–phi29-encoded RNA hybrid did not
affect tumor development [95]. As described earlier,
RNA aptamer-mediated targeted siRNA delivery by
direct conjugation with siRNA or tethering onto
carriers has been a frequently adapted strategy.
Arg–Gly–Asp peptide-conjugated siRNA carriers
Arg–Gly–Asp (RGD) peptide targets tumor vascula-

ture expressing a
v
b
3
integrin. Poly(ethylene glycol)
ylated poly(ethylenimine) conjugated with RGD
peptides was developed to selectively deliver VEGF
siRNA to tumors [96]. In this study, i.v. injected poly-
ethylenimine-poly(ethylene glycol)-RGD ⁄ siRNA com-
plexes inhibited tumor angiogenesis and the growth of
integrin-expressing murine neuroblastoma tumors in
mice [96]. Systemic delivery (i.v. injection) and local
delivery of poly(ethylene glycol)-polyethylenimine-RGD
complexing VEGF siRNA also showed a significant
inhibitory effect on virus-induced angiogenesis as well
as the development of herpetic stromal keratitis lesions
[97].
Antibody-conjugated siRNA carriers
Many studies have suggested that antibodies are good
targeting modalities for targeted siRNA delivery
in vivo, when careful selection of target antigen is
made. Ideal antigens should be exclusively expressed
or substantially overexpressed on target cells. Exam-
ples of antigens that have been used for cancer-tar-
geted drug and gene delivery include HER-2 [98] and
epidermal growth factor receptor [99]. For example,
HER-2 siRNA-carrying liposomes decorated with
transferrin receptor-specific antibody fragments (i.e.
nanoimmunoliposome) silenced the HER-2 gene in
xenograft tumors in mice, significantly inhibiting

tumor growth [100]. An antibody fragment against an
HIV gp160 has also been used for targeted siRNA
delivery in vivo. siRNA linked to a protamine–anti-
body fusion protein, called F105-P, showed inhibited
HIV replication in infected primary T cells [101].
Moreover, i.t. or i.v. injection of F105-P ⁄ siRNA com-
plexes into mice successfully targeted gp160-expressing
B16 melanoma cells. A synthetic chimeric peptide,
which consists of nonamer arginine residues (9R)
added to the C-terminus of a rabies virus glycoprotein
peptide (29 amino acids) (RVG-9R), was able to spe-
cifically deliver siRNA to acetylcholine receptor-
expressing neuronal cells after i.v. administration [102].
In addition, treating mice with Japanese encephalitis
virus siRNA complexed with RVG-9R showed robust
protection of the animals from lethal infection.
Intracellular siRNA delivery
In many aspects, siRNA delivery is similar to that of
delivering other types of nucleic acids such as plasmid
DNA, because they share most extracellular and intra-
cellular barriers. However, several unique challenges in
siRNA delivery make achieving efficient RNAi difficult
compared with plasmid DNA delivery. First, the final
target destination of siRNA is the cytoplasm, whereas
plasmid DNA must be transported into the nucleus.
This implies that siRNA should be rapidly released
from its carrier upon endosomal escape. Second, overall
RNAi efficiency is proportional to the number of
siRNAs complexed with RNA-inducing silencing
protein complex, whereas a successfully delivered single

copy of plasmid DNA might be sufficient to express new
transgene proteins. In other words, the maximum possi-
ble number of siRNA needs to be delivered in the cyto-
plasm in order to achieve the desired biological effects.
Third, siRNA acts only once, whereas plasmid DNA
can be replicated or even can be incorporated into the
host chromosome [103] (short vs. permanent effects).
Cell-penetrating peptide-mediated siRNA delivery
Cell-penetrating peptides (CPPs), short cationic poly-
peptides with a maximum of 30 amino acids, have been
extensively used to obtain enhanced intracellular deliv-
ery of a wide range of macromolecules [104]. CPPs have
been shown to bind the anionic cell surface through elec-
trostatic interactions and rapidly induce cellular inter-
nalization through relatively unclear mechanisms,
although recent evidence shows that CPP-mediated
internalization might be an endocytosis-mediated pro-
cess [105,106]. Various CPPs, including TAT and MPG
proteins from HIV-1 [107–110], as well as penetratin and
polyarginine [111,112], have been employed for intracel-
lular delivery of various proteins and nucleic acids.
Oligoarginine (e.g. 9 arginine, 9R), the simplest
CPP, conjugated with cholesterol was shown to effi-
ciently deliver siRNA to a transplanted tumor in mice
[113]. It was also reported that HER-2 siRNA com-
plexed with short arginine peptide was localized in
perinuclear regions of the cytoplasm in vitro, further
significantly inhibiting tumor growth of ovarian cancer
xenografts [114]. Polyamidoamine dendrimer-TAT
conjugated with bacterial magnetic nanoparticles was

also used to deliver epidermal growth factor receptor
In vivo siRNA delivery M. S. Shim and Y. J. Kwon
4820 FEBS Journal 277 (2010) 4814–4827 ª 2010 The Authors Journal compilation ª 2010 FEBS
siRNA to human glioblastoma cells in vitro as well as
xenografts [115]. Another type of CPP, MPG-8, was
also used to complex cyclin B1 siRNA, and the result-
ing complexes were further decorated with cholesterol
for i.v. injection to the mice bearing human prostate
carcinoma and human lung cancer xenografts [116].
The results showed efficient siRNA delivery in vivo at
a low effective dose (0.5 mgÆkg
)1
), indicated by
inhibited tumor growth.
CPP-mediated cellular internalization via endocyto-
sis requires additional molecules for facilitated cyto-
solic release of siRNA. For example, it was found that
TAT–siRNA conjugates resulted in no gene silencing
because they were entrapped in the endosomes even
after efficiently entering cells [117]. Photostimulating
fluorescently labeled TAT efficiently released TAT–
siRNA conjugates from the endosome, resulting in
enhanced gene silencing efficiency. Chloroquine and
Table 1. siRNA delivery systems for RNAi in vivo. BCL-2, B-cell lymphoma 2; Cyb1, cyclin B1; CyD1, cyclin D1; DOPC, 1,2-dioleoyl-sn-glyce-
ro-3-phosphatidylcholine; DOPE, dioleoyl phosphatidylethanolamine; DOTAP, (N-[1-(2,3-dioleoyloxy)]-N-N-N-trimethyl ammonium propane);
DPPE, dipalmitoyl phosphatidylethanolamine; DSPE, distearoyl phosphatidylethanolamine; FAK, focal adhesion kinase; HST-1 ⁄ FGF-4,
fibroblast growth factor; i.c.v., intracerebroventricular; i.p., intraperitoneal; i.v., intravenous; MMP-2, matrix metalloproteinase-2; NMDA,
N-methyl-
D-aspartate; NR2B, NMDA-R2B receptor subunit protein receptors; PAMAM, polyamidoamine dendrimer; PLK-1, polo-like kinase 1;
PTTG1, pituitary tumor transforming gene 1; RVG, rabies virus glycoprotein; SNALP, stable nucleic acid-lipid particles; TNF-a, tumor necrosis

factor-a; VEGF, vascular endothelial growth factor.
Delivery system Target gene In vivo model
a
Delivery route Ref.
Cholesterol–siRNA ApoB ApoB transgenic mice i.v. 22
RNA aptamer–siRNA PLK-1, BCL-2 Prostate tumor xenograft i.t. 57
Adenoviral vector PTTG1 Hepatoma tumor xenograft i.t. 60
Lentiviral vector GFP GFP transgenic brain Stereotactic 62
Stable nucleic acid lipid particles (SNALP) ApoB Monkeys i.v. 68
Cardiolipin analog-based liposome c-Raf Breast tumor xenograft i.v. 71
DSPE–poly(ethylene glycol)
–DOTAP–cholesterol liposome
Luciferase B16F10 melanoma tumors i.v. 72
Hyaluronan–DPPE liposome CyD1 Gut inflammation i.v. 73
Neutral DOPC liposome EphA2 Ovarian cancer i.v. 74
Neutral DOPC liposome FAK Ovarian cancer i.p. 75
Neutral DOPC liposome Neuropilin-2 Colorectal tumor xenograft i.p. 76
Atelocollagen HST-1 ⁄ FGF-4 Luciferase Germ cell xenograft i.t. 77
Atelocollagen VEGF Prostate tumors xenograft i.t. 78
Chitosan EGFP Transgenic EGFP mice Intranasal 29
Chitosan RhoA Breast tumors xenograft i.v. 79
Chitosan TNF-a Mice i.p. 80
Polyethylenimine HER-2 Ovarian tumor xenograft i.p. 81
Polyethylenimine NR2B Nociception in rats Intrathecal 82
Polyethylenimine Influenza nucleoprotein Influenza virus infected-lung Retro-orbital 83
Polyethylenimine Pleiotrophin (PTN) Glioblastoma xenograft Intracerebral, i.p. 84
Poly(
L-lysine) ApoB Mice i.v. 85
Protamine VEGF Hepatocarcinoma xenograft i.p. 87
RGD–poly(ethylene glycol)–poly(ethylenimine) VEGF Neuroblastoma xenograft i.v. 96

RGD–poly(ethylene glycol)–poly(ethylenimine) VEGF Corneal neovascularization Subconjunctival, i.v. 97
HER-2-liposomes with histidine–lysine peptide HER-2 Pancreatic tumor xenograft i.v. 100
HIV antibody–protamine c-myc, MDM2, VEGF B16 melanoma cells expressing i.t. 101
HIV envelop i.v.
Arginine RVG Neuronal cells i.v. 102
Oligoarginine (9R) conjugated-water-soluble
lipopolymer (WSLP)
VEGF Colon adenocarcinoma xenograft i.t. 113
Oligoalginine (15R) HER-2 Ovarian tumor xenograft i.t. 114
TAT-PAMAM EGF receptor Glioblastoma xenograft i.t. 115
Cholesterol-MPG-8 CyB1 Prostate tumor xenograft i.t. 116
Lung tumor xenograft i.v.
DOPE-Cationic Lipid Luciferase Mouse brain i.c.v. 123
GALA peptide–poly(ethylene glycol)
–MMP-2 cleavable peptide-DOPE
Luciferase Fibrosarcoma xenograft i.t. 128
a
All the listed in vivo models involved a mouse model except Zimmermann et al. [68] and Tan et al. [82].
M. S. Shim and Y. J. Kwon In vivo siRNA delivery
FEBS Journal 277 (2010) 4814–4827 ª 2010 The Authors Journal compilation ª 2010 FEBS 4821
influenza virus-derived hemagglutinin peptide have also
been frequently used to destabilize the endosomal
membrane and enhance the cytosolic release of CPP-
conjugated macromolecules [118–120].
Fusogenic or pH-responsive intracellular delivery
of siRNA
Fusogenic peptides and lipids and pH-responsive lipo-
plexes and polyplexes have been used to ensure facili-
tated siRNA into the cytoplasm from the endosomes.
For example, the incorporation of polypeptides derived

from the endodomain of the HIV-1 envelope (HGP) or
influenza virus fusogenic peptide (diINF-7) signifi-
cantly promoted the liposomal fusion with the endoso-
mal membrane, enhancing siRNA escape into the
cytoplasm [40,121]. Similarly, equipping lipoplexes
with fusogenic lipids, such as dioleoyl phosphatidyl-
ethanolamine (DOPE), was shown to facilitate the
endosomal release of siRNA payload [122,123].
Stimuli-triggered macromolecule release from the
mildly acidic endosome (e.g. pH 5.0–6.0) has been
popularly investigated using a number of novel acid-
responsive polymers [124–126]. For example, poly(eth-
ylene glycol) shielding the surface of a highly fusogenic
phosphatidylethanolamine lipid vesicles was cleaved
upon acid hydrolysis of the vinyl ether bond, triggering
fusion with the endosomal membrane [127]. A matrix
metalloproteinase-cleavable and pH-sensitive GALA
peptide was also used to link poly(ethylene glycol) and
dioleoyl phosphatidylethanolamine (DOPE) lipid to
obtain enhanced siRNA delivery specifically into cancer
cells [128]. Highly efficient siRNA-mediated knockdown
of luciferase expression was achieved in human fibrosar-
coma cells in vitro and xenografted tumors using this
method. Acid-degradable ketalized linear polyethyl-
enimine significantly increased gene silencing efficiency
via efficient cytosolic release with high resistance to
serum and low cytotoxicity [129]. It was demonstrated
that ketalized linear polyethylenimine ⁄ siRNA poly-
plexes were efficiently released into the cytoplasm upon
acid-hydrolysis of ketal branches in the endosomes, fol-

lowed by enhanced siRNA disassembly from ketalized
linear polyethylenimine in the cytoplasm [129].
Conclusion
RNAi is an emerging therapeutic strategy and has
been widely investigated. Despite a few promising
clinical trials, effectively delivering siRNA in vivo
remains a pivotal challenge in translating RNAi in the
clinic as a conventional treatment option. A number of
delivery systems and strategies have been developed to
overcome multiscale extracellular and intracellular
barriers to siRNA delivery in vivo, as summarized in
Table 1. Chemically modified siRNA is stable against
enzymatic degradation but can be cleared easily, gener-
ating potentially hazardous metabolites. Viral siRNA
delivery raises several safety and preparation concerns
such as immune responses and limited large-scale pro-
duction. Nonviral siRNA carriers are efficient, safe
and versatile in tackling the barriers in siRNA circula-
tion, permeation into desired tissues, specific binding
to target cells and optimized intracellular trafficking.
Recent advances clearly indicate that interdisciplinary
approaches using biology, chemistry and engineering
play crucial roles in achieving efficient and targeted
siRNA delivery in vivo.
Acknowledgement
This work was supported by NSF CAREER Award
(DMR-0956091) and a Council on Research Comput-
ing and Libraries Research Grant (UC Irvine).
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