Lipid-Based Nanoparticles in The Systemic Delivery of siRNA

Abstract

RNAi therapeutics are believed to be the future of personalized medicine and have shown promise in early clinical trials. However, many physiological barriers exist in the systemic delivery of siRNAs to the cytoplasm of targeted cells to perform their function. To overcome these barriers, many siRNA delivery systems have been developed. Among these, lipid-based nanoparticles have great potential owing to their biocompatibility and low toxicity in comparison with inorganic nanoparticles and viral systems. This review discusses the hurdles of systemic siRNA delivery and highlights the recent progress made in lipid-based nanoparticles, which are categorized based on their key lipid components, including cationic lipid, lipoprotein, lipidoid, neutral lipid and anionic lipid-based nanoparticles. It is expected that these lipid nanoparticle-based siRNA delivery systems will have an enabling role for personalized cancer medicine, where siRNA delivery will join forces with genetic profiling of individual patients to achieve the best treatment outcome.

Full text

105
Review
IS SN 174 3- 58 8910.2217/NNM.13.192 © 2014 Future Med icin e Ltd Nanomedici ne (2 014) 9(1), 10 5 –120
Since the discovery of the RNAi in 1998 by Fire
et al. [1], the first validation of specific gene knock-
down in mammalian cells [2 ] and the first clinical
trial of siRNA for age-related macular degenera-
tion in 2004 [3], RNAi therapeutics gained the
world’s attention and became an attractive and
promising technique in personalized treatment
of a broad range of diseases, including cancer,
liver and immune-related diseases.
RNAi is found in the cytoplasm [4]. As illus-
trated in Figur e 1, siRNA, a double strand of RNA,
incorporates into the RNA-induced silencing
complex (RISC), causing unwinding of its dou-
ble strand. The sense strand of siRNA is then
removed from RISC, and the activated RISC
with the antisense strand serves as a template for
the binding of complementary mRNA, inducing
mRNA degradation. As siRNA only functions
when it reaches the cytoplasm of cells that pro-
duce the targeted gene, systemic siRNA delivery
encounters many barriers from its administration
all the way to reaching the target gene to be fully
functional.
Barriers in the systemic delivery
of siRNA
 Stability in the blood stream
After intravenous injection, the first aim is to
keep the siRNA stable in the bloodstream. Naked
siRNA is easily degraded by many endogenous
enzymes and aggregated by serum proteins in the
blood, which requires the siRNA delivery system
to be ‘cohesive’ enough to minimize degradation
or disintegration. The delivery system must also
be effective at minimizing nonspecific opsoniza-
tion, phagocytosis and immune activation, while
also presenting surface properties that promote
interaction with the desired cellular targets [5 ].
In addition, prolonging the blood circulation of
siRNA is also important for its effective delivery
as naked siRNA is eliminated from blood just
5 min after intravenous injection [4–8].
 Transport across the vascular
endothelium
The endothelium acts as a semiselective barrier
between the vessel lumen and surrounding tis-
sue, controlling the passage of materials into
tissues. As listed in Table 1, the normal capillary
endothelium can be divided into three types:
continuous endothelium, fenestrated capillaries
and discontinuous capillaries [9]. The size limita-
tion of normal endothelium structure suggests
that siRNA delivery systems should have small
sizes (≤150 nm) to readily cross the vascular
endothelial barrier. It is also known that the
regional structure of blood vessels changes in
the inflammation sites and solid tumors, where
the enhanced permeation and retention effect on
drug delivery has been widely observed. Small
nanoparticles (NPs) with diameter of less than
500 nm, usually less than 150 nm, showed signif-
icant enhanced permeation and retention effects
in ‘leaky’ tumor blood vessels [1 0] .
RNAi therapeutics are believed to be the future of personalized medicine and have shown promise in
early clinical trials. However, many physiological barriers exist in the systemic delivery of siRNAs to the
cytoplasm of targeted cells to perform their function. To overcome these barriers, many siRNA delivery
systems have been developed. Among these, lipid-based nanoparticles have great potential owing to
their biocompatibility and low toxicity in comparison with inorganic nanoparticles and viral systems.
This review discusses the hurdles of systemic siRNA delivery and highlights the recent progress made in
lipid-based nanoparticles, which are categorized based on their key lipid components, including cationic
lipid, lipoprotein, lipidoid, neutral lipid and anionic lipid-based nanoparticles. It is expected that these
lipid nanoparticle-based siRNA delivery systems will have an enabling role for personalized cancer
medicine, where siRNA delivery will join forces with genetic profiling of individual patients to achieve
the best treatment outcome.
KEYWORDS: lipid  lipoprotein  liposome  nanoparticle  RNAi  siRNA delivery
 systemic delivery barrier
Lipid-based nanoparticles in the systemic delivery
of siRNA
Qiaoya Lin1,2,3,
Juan Chen1,
Zhihong Zhang3
& Gang Zheng*1,2
1Ontario Can cer Instute & Techn a
Instute, Uni versity H ealth Netwo rk,
Toronto, ON, Cana da
2Department of Medic al Biophysi cs,
Universit y of Toronto, Toronto Medic al
Discover y Tower 5-363, 101 Col lege
Street, Toronto, ON, M 5G 1L7, Canada
3Brion Cha nce Center for B iomedical
Photonics, Wuhan Naona l Laborator y
for Optoelectronics, Huazhong
Universit y of Science & Technology,
Wuhan, China
*Author for correspondence:
gang.zheng@uhnres.utoronto.ca
part of
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Nanomedicine (20 14) 9(1)
106 future science group
Review Lin, Chen, Zhang & Zheng
 Diffusion through the extracellular
matrix
After crossing the vascular endothelium, the
siRNA delivery vehicle has to diffuse through
the extracellular matrix (ECM), which is com-
posed of gels of polysaccharides and fibrous
proteins, and acts as the structural support for
the animal cells. The tight structure of the ECM
hinders the diffusion of larger NPs, especially
in poorly permeable tumor tissue. For example,
Cabral et al. demonstrated that 30-nm micelles
could effectively penetrate deep tumor tissue of
Endosome
Nucleus
RNAi
Ta rget mRNA cleavage
RISC
siRNA
Cytoplasm
i
ii a
b
Nanomedicine © Future Medicine Ltd (2014)
Figure 1. In vivo siRNA systemic delivery barriers and the mechanism of RNAi. (A) Stability in
the blood stream; (B) transport across the vascular endothelial barrier; (C) diffusion through the
extracellular matrix; (D) delivery into the cy toplasm by (Di) endosomal escape and (Dii) direct
cytosolic delivery. (Dia) The siRNAs or siRNA nanoparticles were trapped in the endosome and
(Dib) the siRNAs were released from the endosome into the cytoplasm.

www.futuremedicine.com 107
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Lipid-based nanoparticles in the systemic delivery of siRNA Review
pancreatic adenocarcinoma while the 70-nm
micelles were retained around the vasculature,
indicating that only NPs smaller than 50 nm
can penetrate poorly permeable tumors [1 1].
In human glioblastoma (U87) and melanoma
(Mu89), it was demonstrated that an increase
of the particles’ molecular size decreased their
interstitial diffusion [1 2] . In addition, the trans-
mission electron microscopy results showed the
interfibrillar spacing between bundles of aligned
and compact fibrils in U87 mouse tumors was
20–42 nm [1 2] . Altogether, the small size of
nanocarriers is superior for effective delivery of
siRNA to poorly permeable tissues. In addition,
the charge of the NPs could also influence parti-
cle diffusion. Stylianopoulos et al. demonstrated
that neutral particles diffuse faster than charged
particles [13] .
 Delivery into the cytoplasm
After diffusion through the ECM, the siRNA
delivery system needs to transport through the
cell membrane, reach the cytoplasm and release
the siRNA payload. The naked siRNA with a
negative charge cannot readily cross the nega-
tively charged cell membrane. The siRNAs fer-
ried by nanocarriers usually enter into cells via
endocytosis pathways, such as macropinocytosis,
clathrin-mediated endocytosis (CME) and cave-
olae-/lipid raft-mediated endocytosis [14 , 15] . Mac-
ropinocytosis and CME usually drive the siRNA
carrier into the endosome (Fig ur e 1Dia), where
mature endosomes fuse easily with lysosomal
vesicles, resulting in enzymatic destruction of
siRNA. Therefore, escaping from the endosome
is further required for effective siRNA delivery
(Fig ur e 1 Dib ). The caveolae-/lipid raft-mediated
endocytosis is a lesser characterized pathway
compared with macropincytosis and CME [1 6 ,17].
However, it is has been demonstrated that some
lipid raft-related delivery could bypass the endo-
somal route to achieve direct cytosolic delivery
of drugs ( Figur e 1Dii) [18–20 ], opening a new avenue
for enhanced cytosolic siRNA delivery.
To overcome the barriers in the systemic
delivery of siRNA, variable nonviral siRNA
delivery vehicles have been developed, includ-
ing lipid-based NPs [21 –24 ], polymers [25] or lipid
polymer hybrid NPs [26 ], hydrogels [27], micro-
bubbles [2 8], inorganic NPs, such as silica [29],
gold [30], quantum dot [31], iron oxide [32 ,33]
and carbon nanotubes [3 4 ,35] , and other materi-
als, such as oligonucleotide NPs [3 6] and exo-
somes [3 7]. Among them, the lipid-based NPs
are in the most advanced stage of development
and have shown favorable biocompatible and
biodegradable properties in comparison with
inorganic carriers and viral vectors. This review
will focus on the development of lipid-based NPs
for the systemic delivery of siRNAs.
The lipid-based NPs reviewed here are divided
into the following types, based on their key
component lipids: cationic lipid-based NPs;
lipoprotein-related NPs; and other lipid NPs,
including lipid-like material-, neutral lipid- and
anionic lipid-based NPs.
Cationic lipid-based siRNA delivery
Cationic liposomes are the most common lipid-
based nanocarrier used for siRNA delivery.
siRNA loading onto the cationic liposome is
mainly attributed to the electrostatic interac-
tion between the anionic siRNA and cationic
lipids. The cationic lipids facilitate the par-
ticles’ intracellular uptake and further endo-
somal escape. Some cationic liposomes have
been widely used for siRNA delivery, such as
1,2-dioleoyl-3-trimethylammonium-propane
(DOTAP)–siR NA, N-(1-[2,3-dioleyloxy]-
propyl)-N,N,N-trimethylammonium chloride–
siRNA and Lipofectamine® 2000 (Life Tech-
nologies, MD, USA)–siRNA complexes [3 8 – 41] ,
which are simply formulated by cationic lipids
and siRNAs. However, these complexes gener-
ally have a high electric charge density on their
surface that readily induce nonspecific interac-
tions with serum proteins and immunogenic
response, resulting in their rapid removal from
the blood stream [4,42,43].
 Development of advanced cationic
lipid-based siRNA delivery system
Stable loading of siRNA
To stabilize the cationic lipid-based siRNA
delivery system, many neutral lipids, such as
cholesterol (Chol) or 1,2-dioleoyl-sn-glycerol-
3-phosphoethanolamine (DOPE) [4 4], have been
added to the complex formulation. These neutral
lipids not only stabilize the formation but also
enhance the cellular uptake of particles, which
will be discussed later (see the ‘Improving the
cellular uptake & enhancing siRNA release into
the cytoplasm’ section).
Table 1. Normal capillary endothelium.
Typ e Organs Size (nm)
Continuous Brain, skeletal, cardiac and smooth muscles, lung,
skin, subcutaneous and mucous membranes
≤1.8 –2.0
Fenestrated Kidney, small intestine and salivary glands ≤11
Discontinuous Liver, spleen and bone marrow Up to 150

Nanomedicine (20 14) 9(1)
108 future science group
Review Lin, Chen, Zhang & Zheng
In comparison with surface loading, siRNA
loading in the NP core should provide better
delivery stability. To improve the siRNA core
loading efficiency, many helper cationic poly-
mers, such as protamine, were introduced to pre-
condense siRNA into the core of a liposome. A
good example is the liposome–polycation–DNA
(LPD) NPs developed by Li et al. [45]. In this
NP, protamine interacts with the nucleic acid
to form a negatively charged compact core,
cationic liposomes composed of DOTAP/Chol
collapse onto the core via charge–charge inter-
action and 1,2-distearoyl-sn-glycero-3-phos-
phoethanolamine–PEG is then coated onto the
outer surface of the particles. Thus, the siRNA
delivery is supported and shielded by both the
cationic lipid bilayer and PEG coating [45].
To improve siRNA loading efficacy, many
efforts have been undertaken to optimize the
key component of liposome cationic lipids. San-
tel et al. synthesized a series of b-l-argi nyl-2,
3-l-diaminopropionic acid-N-palmityl-N-ole-
ylamide trihydrochloride (AtuFECT) cationic
lipids and validated that AtuFECT01 enables
enhancement of the siR NA-binding ability
when compared with commercial cationic lip-
ids, such as DOTAP or N-(1-[2,3-dioleyloxy]-
propyl)-N,N,N-trimethylammonium chloride
[7]. The ability of the AtuFECT-formulated
liposome to deliver siRNA (the resultant Atu-
FECT–liposome–siRNA complex is termed
as AtuPLEX) that inhibits CD31 and Tie2
in the vasculature of mice has also been dem-
onstrated [7] . Later, the same group extended
the application of the optimized AtuPLEX to
treat advanced solid tumor by targeting PKN3
(termed Atu027) and achieved significant
inhibition of tumor growth [46 ], lymph node
metastasis [46] and lung metastasis formation
[47]. Notably, a Phase I clinical trial of Atu027
for the treatment of advanced solid cancers has
been completed, and a further clinical test using
a combination of Atu027 and gemcitabine is
currently ongoing [201,202].
The stable loading of siRNA depends not
only on the formulation components but also on
siRNA-integrating methods. Buyens et al. pub-
lished a comprehensive review on this aspect,
which covers variable methods for siRNA load-
ing, such as simple mixing, direct hydration
of a lipid film by a siRNA-contained solution
and the ethanol dilution method [48]. Among
them, the simple mixing method usually gave
the poorest siRNA encapsulation efficacy [4 8].
Furthermore, the stable systemic deliv-
ery of siRNA can be improved by chemical
modifications of the siRNAs backbone, such
as 2´-O-methyl and 2´-fluoro RNA. Chol con-
jugation to the siRNA sequence also enhanced
siRNA loading efficacy on lipid-based NPs, and
exhibited higher biologic activity compared with
unmodified siRNA [49].
Improving siRNA delivery
pharmacokinetics
To improve the blood stability and the in vivo
pharmacokinetics properties, many other helper
lipids were introduced into the siRNA delivery
system, such as PEGylated lipid (PEG lipid) [48].
The PEGylation effect of prolonging the circula-
tion time of many carriers in the blood has been
reviewed by many groups [48,50,51], including,
but not limited to, the influence of acyl chain
length in PEG lipids and their molar percentage
in the liposome composition [52 ]. Sonoke et al.
found that the PEG lipids with long acyl chains
showed better siRNA delivery pharmacokinet-
ics and efficiency when compared with those
with the short or unsaturated chains [5 3]. The
percentage of PEG lipids in the siRNA delivery
system should consider both blood circulation
time and cell uptake efficiency. A higher percent-
age of PEG lipids usually gives a longer blood
circulation time but weakens the cellular uptake
and subsequent endosomal escape. Therefore, an
optimal density of PEG lipids is required [ 48 , 51] .
Yagi et al. developed a ‘wrapsome’ NP, com-
prised of a core of siRNA/cationic DOTAP that
was fully enveloped by a neutral lipid bilayer
containing egg phosphatidylcholine and PEG
lipid in a weight ratio of 24:14.8. They found
that the wrapsome could improve the stability
and systemic circulation of siRNA, thus result-
ing in enhanced specific gene knockdown and
significant anti-tumor activity in vivo [54 ].
To overcome the PEGylation-induced prob-
lem of cell uptake and endosomal escaping, Car-
mona et al. developed a pH-sensitive PEGylated
liposome by postcoupling a PEG-2000 dialde-
hyde on the surface of particles composed of
cationic cholesteryl polyamine–N1-cholester-
yloxycarbonyl-3, 7-diazanonane-1, 9-diamine,
neutral lipids (DOPE) and Chol–PEG350 ami-
noxy lipid via oxime linkage. The oxime link-
age of the NPs is stable at pH 7, but is prone to
decomposition in an acidic environment (pH 5.5
and below), which induces the release of PEG
coating from particles, thus granting acidic pH-
triggered cell uptake and endosome release of
nucleic acids. This formulation demonstrated
effective RNAi delivery for on the control of
hepatitis B virus (HBV) virus replication [55] .

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Lipid-based nanoparticles in the systemic delivery of siRNA Review
Hatakeyama et al. developed another PEG-
cleavable siRNA delivery system, termed a
‘multifunctional envelope-type nanodevice’
(MEND), composed of DOTAP, DOPE, Chol,
1,2-distearoyl-sn-glycero-3-phosphoethanol-
amine–PEG and a PEG–peptide–DOPE (PPD).
PPD has a peptide sequence-linked PEG and
DOPE lipid moiety together, so that the PEG is
removed when the peptide linker is cleaved by
the target molecule, such as MMP. It has been
demonstrated that PPD-formulated NPs acceler-
ated both cellular uptake and endosomal escape
in MMP-rich tumor environments, compared
with a conventional PEG fomulation, resulting
in potent silencing (~70%) activity in vivo with
no observed hepatotoxicity and innate immune
stimulation [56].
Notably, the PEGylation methods also influ-
ence the stability of siRNA delivery. For exam-
ple, simply mixing siRNAs with PEGylated lipo-
somes results in poor loading of siRNAs onto the
outer surface of particles, causing the premature
release of siRNA, whereas post-PEG-coating on
the siRNA liposome could enable more stable
siRNA delivery. This aspect has been discussed
and reviewed in detail by Buyens et al. [48].
Although the beneficial PEGylation effect on
siRNA delivery is well acknowledged, it has been
found that repeated injection of PEGylated NPs
could induce the loss of their long circulation
characteristics owing to the production of the
anti-PEG IgM [57]. The phenomenon of acceler-
ated blood clearance serves as a reminder of the
limitation of PEG-coated NPs.
In addition, other challenges in the context of
the pharmaceutical development of lipid-based
siRNA therapeutics have been reviewed exten-
sively by Gindy et al., including the development
of a robust manufacturing process, the setting of
appropriate product specifications and controls,
development of strategies to assess and ensure
product stability, and the evaluation of product
comparability throughout development [5 8].
Improving biocompatibility & reducing
immunotoxicity
To reduce the immunotoxicity of siRNA deliv-
ery, some biogenic materials were included in the
cationic lipid-based siRNA delivery system. For
example, hyaluronic acid, a biogenic component
distributed widely in the ECM, was introduced
into LPD-NPs by Chono et al. to develop a lipo-
some–protamine–hyaluronic acid (LPH)-NP
[59]. Hyaluronic acid provides LPH-NP multi-
valent charges to enhance the particle conden-
sation while containing no immunostimulatory
CpG motifs. This LPH-NP delivery of siRNA
induced a 80% silence of luciferase activity in
the metastatic B16F10 tumor in the lungs at a
low, single, intravenous injection dose of 0.15 mg
siRNA/kg [59 ], while not causing obvious
immunotoxicity at a dose range of 0.15–1.2 mg
siRNA /kg. Recently, they employed LPH-NP in
the delivery of siRNA for silencing of CD47, a
‘self-marker’ that is usually overexpressed on the
surface of cancer cells to enable them to escape
from immunosurveillance, and achieved effec-
tive inhibition of melanoma tumor growth and
lung metastasis [6 0].
To improve the biocompatibility of cationic
lipid delivery systems, Yang et al. developed a
lipid–polymer hybrid NP prepared by a single-
step nanoprecipitation of a formulation of cat-
ionic lipids, BHEM– Chol and amphiphilic
polymers. Such hybrid NPs exhibited excel-
lent stability in serum and showed significant
improvement on biocompatibility compared
with the pure BHEM–Chol particles [26 ]. A
similar toxicity reduction effect has also been
observed for other hybrid NPs with involvement
of polyglutamate [61– 63] .
Improving the cellular uptake
& enhancing siRNA release into the
cytoplasm
To enhance the cellular uptake and further
endosomal escaping of siRNA, some neutral
helper lipids, such as DOPE and 1,2-distear-
oyl-sn-glycero-3-phosphocholine were added
to the cationic lipid-based siRNA delivery sys-
tem. The role of such fusogenic lipids has been
reviewed by Wasungu et al. [4 4]. DOPE pro-
foundly affects the polymorphic features of the
liposome–siRNA complex (termed lipoplexes)
in that it promotes the transition from a lamel-
lar to a hexagonal phase, thus inducing fusion
and disruption of the membrane. The opti-
mized 3-b-(N-[N´,N´-dimethylaminoethane]
carbamoyl) Chol/DOPE-based lipoplexes have
been found to improve the transfection effi-
ciency of the lipoplex [64 ]. Khoury et al. pre-
pared a NP using the cationic lipid RPR209120
in combination with DOPE. This NP enables
effective systemic delivery of DNA, resulting
in successful silencing of TNF-a in collagen-
induced arthritis of mice, and complete cure of
collagen-induced arthritis via weekly intrave-
nous administration [65] . Since then, this for-
mulation for siRNA delivery to myeloid cells
has been successfully applied [66,67].
By replacing the protamine/DNA core of
the LPD NPs with a pH-sensitive calcium

Nanomedicine (20 14) 9(1)
110 future science group
Review Lin, Chen, Zhang & Zheng
phosphate (CaP) core, Yang et al. recently
developed a biodegradable lipid/calcium/phos-
phate NP (LCP-NP). CaP is biocompatible, bio-
degradable material and native to the body, as
it is the principle mineral component of teeth
and bones. It is hypothesized that the CaP core
could be rapidly dissolved at acidic endosome
pH, which will increase the osmotic pressure and
cause NP disassembly, endosome swelling and,
finally, siRNA release into the cytoplasm. The
study showed that the LCP-NP releases more
cargo to the cytoplasm compared with the LPD
formulation, leading to a significant (~40-fold
in vitro and ~fourfold in vivo) improvement in
siRNA delivery. More recently, they coformu-
lated three siRNAs against MDM2, c-myc and
VEGF in a LCP-NP. Such a formulation caused
simultaneous silencing of the three oncogenes in
metastatic nodules, and resulted in significant
inhibition of lung metastases (~70–80%) at a
relatively low dose (0.36 mg/kg) without any
observed toxicity [68].
Recently, to enhance endosomal escape,
Harashima et al. synthesized a pH-sensitive
cationic lipid, YSK05, which contains a tertiary
amine group for pH sensitivity. They incor-
porated this lipid into the above mentioned
MEND siRNA delivery system, and found
that the YSK05–MEND combination had a
better ability for endosomal escape than other
MEND-containing conventional cationic lip-
ids [69, 70]. Some other new cationic lipids have
also been reported for improving siRNA deliv-
ery, such as N´,N´-dioctadecyl-N-4, 8-diaza-
10-aminodecanoylglycine amide [7 1] a nd
1,2-dilinoleyloxy-3-dimethylaminopropane,
which will be the subject of later discussion [2 3].
Active targeting delivery
Incorporating an active target motif, such as
transferrin, RGD, EGF and various cell-penetra-
tion peptides, to the cationic lipid-based siRNA
delivery system could significantly improve the
intracellular uptake and endosomal release of
siRNA. Daka et al. [7 2] and Ogris et al. [73] have
reviewed this aspect extensively.
 Example of an advanced cationic
lipid-based siRNA delivery system:
stable nucleic acid lipid particles
& their application
Among the cationic lipid-based siRNA vehicles,
several advanced delivery systems have gained
worldwide attention owing to their high effi-
ciency in systemic siRNA delivery methods,
such as LPD, AtuPLEX, stable nucleic acid lipid
particle (SNALP) systems, LPD and AtuPLEX,
and have been well reviewed previously [74, 75].
Here, we take SNA LP as an example to highlight
its approach in overcoming barriers of siRNA
systemic delivery.
SNALP & its application
SNALP is constructed by three basic lipids:
an ionizable cationic lipid (1,2-dilinoleyloxy-
3-dimethylaminopropane), a neutral helper
lipid, including Chol and fusogenic lipids, and
a PEG lipid (Table 2). In SNALPs, the backbone-
modified siR NA is encapsulated within a closed
shell of a cationic-zwitterionic lipid bilayer, fur-
nished with an outer PEG shield (Figu re 2a). The
lipid bilayer contains a mixture of cationic and
fusogenic lipids to enable cellular uptake and
further endosomal escape owing to electrostatic
interactions between the negatively charge cell
Table 2. Stable nucleic acid lipid particle formulations.
Stable nucleic acid lipid particle formulations in systemic
delivery
Target siRNA and
modification (yes/no)
Animal model Ref.
DSPC:Chol:PEG–C–DMA:DLin– DMA (20:48:2:30) –siRNA Hepatitis B virus (yes) Mouse model of hepatitis B
virus replication
[23]
DSPC:Chol:PEG–C–DMA:DLin– DMA (10:48:2:40) –siRNA ApoB (yes) Cynomolgus monkeys [76]
DSPC:Chol:PEG–C–DMA:DLin– DMA (10:48:2:40) –siRNA Cell cycle proteins PLK1 (yes) Intrahepatic mouse tumor
models and s.c. tumor
models
[78–80]
DSPC:Chol:PEG–C–DMA:cationic lipid (10:40:10:40)–siRNA Factor VII (yes) C57BL / 6 mice [24]
DPPC:PEG–C–DMA:Chol:DLin–KC2–DMA (7.1:1.4:34.3:57.1)–siRNA ApoB, transthyretin (yes) Cynomolgus monkeys [24]
PEG–DMG:Chol:Octyl–CLinDMA (2:48:50) –siRNA Luciferase (yes) Rosa26-LSL-Luc mouse strain,
Adeno-liver-Luc mice
[77]
Chol: Cholesterol; DLin– DMA: 1,2-dilinoleyloxy-3-dimethylaminopropane; DLin–KC2–DMA: 2,2-dilinoleyl- 4-dimethylaminoethyl- (1,3)-
dioxolane; DMG: Dimyristoylglycerol; DPPC: Dipalmitoylphosphatidylcholine; DSPC: 1,2-distearoyl-sn- glycero-3-phosphocholine; Octyl–CLinDMA: 2-( [8- ([3b]-
cholest-5-en-3-yloxy)octyloxy) -N,N-dimethyl-3- ([9Z,12Z] -octadeca-9,12-dien-1-yloxy)propan-1-amine; PEG–C–DMA: 3-N- ([-methoxy PEG 2000] carbamoyl) -1,2-
dimyristyloxypropylamine; s.c.: Subcutaneous.

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Lipid-based nanoparticles in the systemic delivery of siRNA Review
membrane and cationic lipids. The coated PEG
provides a neutral, hydrophilic exterior to shield
the cationic bilayer, protect the nucleic acid core
against degradation by nucleases, sterically sta-
bilize the particles against disassembly in col-
lagen networks and prevent nonspecific bind-
ing to cells, thus making NPs ‘cohesive’ and
‘stealthy’ to prevent siRNA degradation and
rapid clearance in the bloodstream.
The SNALPs are approximately 70–150 nm
in diameter (Figure 2b), and their major accumula-
tion (±standard deviation) was found in mouse
liver (28 ± 1.7% of injected dose administered)
and spleen (8.2 ± 2.8% of injected dose admin-
istratered) [2 3]. Their biodistribution is related
to the property of NPs, such as size, lipid and
PEGylation features. In general, the PEGylated
NPs are readily navigated to the mononuclear
phagocyte systems, such as the liver and spleen.
The therapeutic efficacy of SNALP–siRNA
against HBV was validated in a HBV replication
mouse model by Morrissey et al. [23]. They found
that SNALP delivery prolonged the circulation
time of siRNA and achieved efficient inhibition
of serum HBV by daily intravenous injections
of 3 mg/kg/day of siRNA [23]. Zimmermann
et al. further demonstrated the efficient systemic
delivery of SNALP–siRNA in nonrodent spe-
cies. They administered intravenous injections of
SNALP–siRNA against the ApoB gene at single
doses of 1 or 2.5 mg/kg on cynomolgus monkeys
and achieved potent gene knockdown of ApoB
mRNA (>90%). Significant downregulation of
ApoB protein, serum Chol and low-density lipo-
protein (LDL) levels was also observed as early as
24 h after treatment and lasted for 11 days with
a 2.5-mg/kg siRNA dose [76].
More recently, Semple et al. found a high-
performance lipid, 2,2-dilinoleyl-4-dimethyl-
aminoethyl-(1,3)-dioxolane by screening vari-
ous ionizable cationic lipids, and demonstrated
the excellent activity of the 2,2-dilinoleyl-
4-dimethylaminoethyl-(1,3)-dioxolane-based
SNALP in effectively silencing the endogenous
Cytosolic siRNA–gold (%)
Hepatocytes HeLa
Time (h)
1. 51. 0
0.5
1. 5
1. 0
2.0 3.0 4.0 5.0 6.0
Neutral lipids Cationic lipids
siRNA PEG lipids
pH 5.5–6.5
Trapped in the
EE compartment
Transport through
the PM
Trapped in the
LE compartment
pH >5.5
Trapped in
the Lys
pH >4.5
Cytosolic release of siRNAs
M
LE
Golgi
500 nm 50 nm
In vitro HeLa cells
200 nm
200 nm
200 nm
200 nm
50 nm
50 nm
50 nm
50 nm
PM
PM
EE
EE
LE
LE
Lys
Golgi 0.5
50 nm
Figure 2. Stable nucleic acid lipid particle siRNA delivery system. (A) Stable nucleic acid lipid particle (SNALP) siRNA delivery
system. (B) Visualization of SNALP–siRNA –gold by electron microscopy. (C) Ultrastructural analysis of SNALP in vitro trafficking.
SNALP–siRNA–gold detected in HeLa cells in vitro, by electron microscopy. SNALP–siRNA–gold was found in the extracellular matrix
close to the PM and inside the EE compartment, LE compartment and Lys within cells. Magnified images (right panels) permit
appreciation of the subcellular localization of siRNA–gold. (D & E) Cytosolic release of siRNA. (D) siRNA–gold concentrates within the LE
compartment of HeLa cells in vitro and (E) the quantification of cytosolic siRNA– gold release kinetics in a liver section in vivo (solid line)
and in HeLa cells in vitro (dashed line). The error bars represent the standard error.
EE: Early endocytic; LE: Late endocytic; Lys: Lysosome; PM: Plasma membrane.
Adapted with permission from [82] .

Nanomedicine (20 14) 9(1)
112 future science group
Review Lin, Chen, Zhang & Zheng
hepatic gene at doses of siRNA as low as
0.01 mg/kg in rodents and 0.1 mg/kg in nonhu-
man primates in vivo [24] . Meanwhile, Tao et al.
developed another optimized SNALP system by
screening cationic lipids and adjusting PEG lipid
density, and achieved potent gene knockdown in
the liver (90%) of mice [77] .
Besides the application of SNALP in liver
diseases, Judge et al. demonstrated the success-
ful delivery of siRNA to solid tumors in mice
targeting the PLK1 gene, resulting in signifi-
cant inhibition of subcutaneous Hep3B tumor
growth [78].
Notably, the SNALP system encapsulated
with self-amplifying RNA has been reported as
a new vaccine platform by Geall et al., which
substantially increases immunogenicity by
eliciting broad, potent and protective immune
responses [79]. Altogether, it is not surprising that
the SNALP system has entered or completed
multiple clinical trials for systemic siRNA deliv-
ery, such as VEGF and KSP in liver cancer, ApoB
in liver disease, TTR in transthyretin amyloi-
dosis, PLK1 in cancer and Elola in Ebola virus
disease [75] .
The impact of physiological constrains, such
as tumor vasculature, on the efficiency of siRNA
delivery has also been investigated. Li et al. found
that SNALP predominantly delivered siRNA to
areas adjacent to functional tumor blood vessels
by analyzing the spatial distribution of local-
ized target knockdown within tumor sections
relative to tumor hypoxia [80] . This pheno-
menon suggests that it is probably not easy for
the SNALP–siRNA complex to cross the ECM
with a size of 70–150 nm; therefore, most of
them are trapped in the areas adjacent to blood
vessels [80]. To improve the transport of SNALP
across the ECM barrier, Rudorf et al. devel-
oped a small SNALP, called a ‘mono-NALPs’,
which was self-assembled by solvent exchange
from solution containing siRNA mixed with
the four lipid components DOTAP, DOPE,
1,2-dioleoyl-sn-glycero-3-phosphatidylcholine
and 1,2-d istearoyl-sn-glycero-3-phosphoetha-
nolamine-PEG-2000. The mono-NALP has a
similar core–shell structure as SNALP, but is
only 30 nm in diameter, which should facilitate
its transport in vivo across the ECM to achieve
deeper tissue penetration [8 1].
Recently, the mechanism of cellular uptake,
intracellular transport and the endosomal escape
of siRNA by SNALP delivery had been investi-
gated. It has been shown that SNALPs entered
cells by both constitutive and inducible pathways
in a cell type-specific manner using CME and
macropinocytosis (F igur e 2C) [82]. However, the
escape of siRNAs from the endosome into the
cytosol occurred at low efficiency (<5%), sug-
gesting further optimization of the SNALP for-
mulation is required to improve the endosomal
release of siRNA (Fi gure 2D ).
Lipoprotein-related siRNA delivery
Lipoproteins are naturally existing endogenous
serum proteins that play critical roles in lipid
transport in humans. Lipoproteins share a com-
mon structure consisting of an apolar core sur-
rounded by a shell composed of a phospholipid
monolayer containing unesterified Chol and
one or more apolipoproteins (Figu re 3a). Apoli-
poproteins function as structural components
of lipoprotein particles, cofactors for enzymes
and ligands for cell surface receptors. Accord-
ing to their size, density, lipid and apolipoprotein
components, lipoproteins are divided into high-
density lipoprotein (HDL; 8–12 nm), LDL
(18–25 nm), very LDL (30–80 nm) and chylomi-
cron (75–1200 nm). Being endogenous carriers,
lipoproteins escape recognition as foreign entities
by the human immune system and escape absorp-
tion by the reticuloendothelial system [83]. Their
favorable blood circulation characteristics pres-
ent them as viable alternate drug delivery systems
without the need for PEGylation. Thus, lipopro-
tein NPs may provide a solution to the biocom-
patibility problem associated with most synthetic
nanodevices. Among the lipoproteins, HDL and
LDL have been widely used for systemic delivery
of imaging agents and chemotherapeutics [8 4 ,85] .
With the controlled ultra-small size (<30 nm)
and suitable blood circulation (half-life in blood
is 13.5 and 15 h for native HDL and LDL, respec-
tively) they hold great potential for effective
siRNA systemic delivery [84].
 Natural lipoproteins for siRNA
delivery
In nature, both HDL and LDL play a vital role
in Chol transport. LDL functions as an extra-
cellular carrier for delivery of Chol to different
tissues via LDL receptor-mediated endocytosis,
while HDL is responsible for the reverse Chol
transport from peripheral tissues back to the
liver for catabolism [86]. Although the mecha-
nism of selective transport of lipids by HDL is
still unclear, it is generally accepted that HDLs
actively offload Chol esters and other lipids into
cells through SR-BI, via a nonendocytic pathway.
Therefore, LDL and HDL are attractive drug
delivery vehicles for potential cardio vascular
disease treatment. For example, Wolfrum et al.

www.futuremedicine.com 113
future science group
Lipid-based nanoparticles in the systemic delivery of siRNA Review
utilized HDL and LDL for siRNA delivery.
Unlike most of the cationic lipid-based NPs car-
rying siRNA by electrostatic interaction, HDL
or LDL stably load with siR NAs at a stoichio-
metry of 0.94 and 1.26 (molar proportion),
respectively, via interaction between lipoproteins
and lipophilic molecules on siRNA sequences,
such as Chol, long-chain fatty acids or bile acids
[22]. They found that LDL delivered most of the
Chol-labeled siRNA (Chol–siRNA) into the
liver, while HDL delivered Chol–siRNA into the
adrenal glands, ovaries, kidneys and liver. When
compared with naked Chol–siRNA–ApoB
(Chol–siRNA targeted to ApoB mRNA) treat-
ment, higher ApoB protein downregulation
(~8–15-fold more effective in the liver, gut
and blood) has been achieved by HDL deliv-
ery [2 2]. Interestingly, they also found that the
lipoprotein-associated Chol–siR NAs were taken
up by hepatocytes through a mechanism that
was dependent on lipoprotein-associated recep-
tors but independent of internalization of the
lipoprotein particles, suggesting the rapid uptake
of Chol–siRNAs compared with a much slower
cellular uptake of lipoprotein NPs.
Jin et al. has systematically studied the abil-
ity of LDL for Chol–siR NA delivery and dem-
onstrated that over 25 Chol–siRNAs could be
stably incorporated onto each LDL without
changing NP morphology [8 7]. The resulting
LDL–Chol–siRNA NPs were selectively taken
up by cells via LDL receptor-mediated endocy-
tosis, resulting in enhanced gene silencing com-
pared with naked Chol–siRNA (38 vs 0% gene
knockdown at 100 nM). However, most of the
Chol–siRNA was validated by being tracked in
Figure 3. Lipoprotein-based nanoparticle delivery system. (A) Lipoprotein or
lipoprotein-mimetic nanoparticle delivery system. (B) Transmission electron microscopy of
HPPS–siRNA nanoparticles. (C) Visualization of intracellular delivery of FITC-labeled siRNA in KB cells
(SR-BI receptor positive) by confocal imaging. (D) Quantitative measurement of the uptake of
FITC–Chol–siRNA –bcl-2 in different cell organelles via different delivery approaches (n = 3). The
results of (C & D) suggest the direct cytosolic delivery of siRNA by HPPS. Error bars represent the
standard deviation.
*p < 0.05; **p < 0.01.
Chol: Cholesterol; FITC: Fluorescein isothiocyanate; FITC–Chol–siRNA–bcl-2: FITC-labeled Chol-siRNA
targeted to bcl-2; HPPS: High-density lipoprotein-mimicking peptide–phospholipid scaffold.
Adapted with permission from [94 ].
FITC–Chol–
siRNA–bcl-2
Lipofectamine–
FITC–Chol–
siRNA–bcl-2
HPPS–FITC–
Chol–siRNA–
bcl-2
Intracellular uptake (%)
0
20
40
60
80
100
120
Cytosol
Nucleus and membrane
Other organelles
*
**
Phospholipid
siRNA
FITC–Chol–
siRNA–bcl-2
Alexa-Fluor®
transferrin Merged
FITC–Chol–
siRNA–bcl-2
Lipofectamine®–
FITC–Chol–
siRNA–bcl-2
HPPS–FITC–
Chol–siRNA–
bcl-2
20 µm 20 µm
20 µm20 µm
20 µm 20 µm
20 µm
20 µm
20 µm
100 nm
Hydrophobic cargo
Apolipoprotein/mimetic
peptide

Nanomedicine (20 14) 9(1)
114 future science group
Review Lin, Chen, Zhang & Zheng
the endolysosome. By using a photochemical
internalization strategy to trigger endosomal
escape of LDL–Chol–siRNA, further improved
gene knockdown (78%) was achieved.
Kuwahara et al. extended the utility of endog-
enous lipoprotein delivery of siRNA for neuro-
logical diseases treatment [88] . They found that
HDL successfully delivered the Chol–siRNA
targeting OAT3 mRNA (Chol–siOAT3) into
brain capillary endothelial cells and induced a
significant reduction of OAT3 mRNA expression,
whereas the naked Chol–siOAT3 showed poor
delivery efficacy to brain capillary endothelial
cells and had a negligible gene silencing effect.
They also validated that the efficient delivery of
Chol–siOAT3 by HDL was mainly mediated by
ApoE targeted to LDL receptor. In addition to
Chol modification, another lipophilic molecule,
vitamin E or a-tocopherol, has been used to mod-
ify siRNA for improved brain disease treatment.
The combination of vitamin E (a-tocopherol)-
conjugated siRNA with HDL caused dramatic
improvement of siRNA delivery to neurons,
resulting in specific knockdown of the targeted
BACE1 mRNA at both the mRNA and protein
level, especially in the parietal cortex [8 9].
 Lipoprotein-mimetic NPs
Beyond the naturally existing lipoproteins,
many reconstituted lipoproteins and lipoprotein-
mimetic NPs have been developed for siRNA
delivery, such as reconstituted HDL (rHDL) and
HDL-mimicking peptide–phospholipid scaf-
fold (HPPS) NPs. Their application has been
extended to RNAi cancer therapeutics, as many
cancer cells, particularly ovarian, colon, lung,
prostate and breast cancers, highly express the
SR-BI receptor [9 0 –92 ].
In 2009, Zhang et al. developed HPPS, com-
posed of cholesteryl oleate, phospholipid and an
18-amino acid ApoA-1-mimetic peptide (Figur e 3a)
[20], which closely mimics the structural and
functional properties of plasma-derived HDL.
The HPPS NP has a small particle size (<30 nm)
(Figu re 3b), long circulation half-life (15 h) and
excellent biocompatibility, with high tolerability
of systemic administration doses (>2000 mg/kg)
[93]. HPPS could stably carry a high payload of
Chol–siRNA (ten siRNAs per particle) and
deliver them directly into the cytosol of the tar-
get cells via the SR-BI receptor (Fig ure 3 C & D),
thereby bypassing the detrimental endosomal
trapping, resulting in enhanced gene silencing
efficacy [94] . Further studies demonstrated that
SR-BI-driven cytosolic delivery is predominately
mediated through a lipid raft/caveolae-like
pathway [95]. Furthermore, the successful sys-
temic delivery of bcl-2-targeting Chol–siRNA
by HPPS has been demonstrated in a xenograft
tumor mouse model, which resulted in bcl-2
mRNA knockdown, tumor cell apoptosis and
tumor growth inhibition, with no observable
adverse effects, thus demonstrating the ability
of HPPS as a safe and efficient nanocarrier for
RNAi therapeutics [90] .
McMahon et al. developed a rHDL by using a
gold NP (~5 nm) core as a template to assemble
a mixed phospholipid layer and adsorb ApoA-1,
forming a core–shell spherical structure [3 0].
These synthesized structures have the general
size (~16 nm) and surface composition of natural
HDL, and importantly, have shown high bind-
ing affinity to Chol. It has been further demon-
strated that the rHDL is capable of carrying a
high payload of Chol–DNA (over ten DNA pay-
load per particle) and mediated efficient cellular
nucleic acid delivery [3 0].
Recently, Ding et al. reported a rHDL-
based siRNA delivery system by integrat-
ing Chol–siRNA loading into rHDL particle
assembly. Briefly, a lipid film comprised of
soya bean phospholipids, Chol and choles-
teryl esters was prepared and hydrated with
an aqueous solution containing Chol–siRNA
to form lipoplexes/Chol–siRNA complexes,
then ApoA-1 protein was added to assemble
rHDL/Chol–siR NA NPs. Compared with
unmodified siRNA, Chol–siRNA showed sig-
nificantly higher loading efficiency in rHDL.
Although the size of rHDL/Chol–siRNA NPs
is approximately 90 nm, the cytosolic delivery of
Chol–siRNA was demonstrated using confocal
microscopy. In addition, enhanced tumor uptake
of rHDL/Chol–siRNA was observed in compari-
son with the lipoplexes/Chol–siRNA complex
(without ApoA-1 protein), resulting in improved
of anti-tumor efficacy in vivo [96] .
As well as using lipophilic modified siR NA
(e.g., with Chol) to help siRNA stably incorpo-
rate into lipoprotein NPs, other helper materials,
such as cationic polymer, were also introduced to
facilitate siRNA loading into the rHDL–siRNA
delivery system. Shahzad et al. developed an
rHDL–siRNA delivery system using unmodi-
fied siRNA/oligolysine to hydrate a lipid film
composed of Chol, cholesteryl oleate and egg
yolk phospholipids, followed by the ApoA-1 pro-
tein to assemble rHDL particles. The resulting
rHDL/siRNA NPs have a sma ll size of 12–18 nm,
which facilitates systemic delivery of siRNA
in vivo, evidenced by the therapeutic silencing
of two proteins that are key to cancer growth

www.futuremedicine.com 115
future science group
Lipid-based nanoparticles in the systemic delivery of siRNA Review
and metastasis (STAT3 and FAK) in orthotropic
mouse models of ovarian and colorectal cancer
[97]. Recently, Rui et al. prepared complexes of
siRNA with various cationic polymers including
protamine, polyethyleneimines or poly-l-lysine,
and then coated them with anionic lipid layers
containing ApoA-1 protein, lipid and sodium
cholate to form a rHDL–siRNA delivery sys-
tem. This formulation has shown minimum
cytotoxicity with improved gene silencing effi-
ciency in the hepatocellular carcinoma cell line
SMMC-7721 when compared with cationic poly-
mer delivery [9 8]. The confocal studies revealed
that intracellular uptake of the rHDL–iRNA
occurs via the HDL-receptor-mediated pathway.
Overall, HDL-based siRNA delivery possesses
many attractive features, including ultra-small
size, favorable surface properties (no PEGylation
is required for achieving suitable blood circula-
tion) and direct delivery of siRNA into the cyto-
plasm of cells via the SR-BI-mediated pathway,
thus offering a new avenue for effective siRNA
delivery.
 Apolipoprotein-incorporated
liposomes
In addition to lipoprotein-based nanoplat-
forms, many groups take the apolipoprotein
components of HDL or LDL, such as ApoA-1,
Apo-E and ApoB-100, as targeting motifs for
siRNA delivery. In 2007, Kim et al. incorpo-
rated ApoA-1 protein in cationic liposomes
formulation for siRNA delivery against HBV
and achieved a significant reduction of HBV
protein expression with high liver-targeting
specificity [99]. Nakayama et al. reported the use
of ApoA-1 or ApoE to enhance the targeting
efficacy of phosphatidylcholine-based liposomes
for Chol–siRNAs delivery [1 00] . The ApoE–lipo-
some also demonstrated higher efficiency in the
functional delivery of Chol–siRNA to mouse
liver than ApoA–liposomes. The delivery of
ApoE–liposomes was not significantly affected
by high endogenous levels of plasma LDL.
 Apolipoprotein-free lipoprotein
Recently, a cationic solid lipid NP derived from
apolipoprotein-free reconstituted LDLs was
reported for target-specific systemic treatment
of liver fibrosis [10 1].
Other siRNA delivery systems
 Lipid-like material lipidoid & its
application
As well as the use of cationic lipid-based NPs and
lipoprotein, another promising siR NA delivery
vehicle is lipid-like material (termed ‘lipidoids’)-
based NPs. Such NPs are structurally similar to
the SNALP, formulated with Chol and PEG
lipids, but with lipidoids instead of cationic lipids.
Akinc et al. synthesized a large library of over
1200 structurally diverse lipidoids. Among these,
the lipidoid 98N12-5 (five-tail) was identified as
performing optimally for in vivo siRNA deliv-
ery, as it could stably deliver siRNA to achieve
potent and specific gene therapeutic efficacy in
three different animal species: mice, rats and
nonhuman primates. Lipidoid 98N12-5–siRNA
delivery not only induced successful gene knock-
down of the Factor VII and ApoB gene in the
rodent liver, but also resulted in therapeutic effi-
cacy in lung tissue targeting respiratory syncytial
virus and in peritoneal macrophages against the
CD45 gene [21] . The same group further demon-
strated that in vivo biodistribution of the lipidoid
98N12-5 system was influenced by many factors,
including the formulation composition, nature
of PEG lipid, degree of drug loading and particle
size [10 2 ]. Akinc et al. finally developed a liver-
targeted lipidoid formulation with over 90% of
injected dose distribution in the liver [1 02].
In 2010, Love et al. developed a new generation
of lipidoid formulations using lipidoid C12-200,
1,2-distearoyl-sn-glycero-3-phosphocholine,
Chol and PEG lipid. Using this lipidoid for-
mulation, potent liver gene silencing has been
achieved in mice at a much lower injection dose
of siRNA (<0.01 mg/kg) compared with the
original lipidoid formulation (>1 mg/kg) [102 ,103].
The lipidoid formulation with a pool of five siR-
NAs also induced gene silencing of five specific
hepatic genes simultaneously via a single injec-
tion [10 3] . The formulation was further validated
in nonhuman primates, where significant knock-
down of the clinically relevant gene transthyretin
was observed at doses as low as 0.03 mg/kg, pro-
viding good potential for low-dose in vivo gene
silencing [10 3] .
The lipidoids system has also been applied in
ovarian tumor therapy by Huang et al. Follow-
ing intraperitoneal injection of lipidoid–siRNA
against the CLDN3 gene, a tight junction pro-
tein overexpressed in 90% of ovarian tumors,
obvious tumor growth inhibition and metastasis
suppression were observed in MISIIR/Tag trans-
genic mice and tumor-bearing mice derived from
mouse ovarian surface epithelial cells [1 0 4] .
Recently, the application of RNAi in the
immune system has been rapidly developed,
including using the lipidoids as a delivery sys-
tem [10 5] . The induction of innate immunity by
in vivo administration of siRNA was considered

Nanomedicine (20 14) 9(1)
116 future science group
Review Lin, Chen, Zhang & Zheng
as a major undesirable side effect of RNAi ther-
apy owing to the toxicities associated with exces-
sive cytokine release, such as TNF-a, IL-6 and
IFN release [42 ,106 –108]. Thus, it is essential to
control the undesirable immune activation dur-
ing application of the siR NA complex in treat-
ment of immune-related diseases. It has been
reported that certain chemical modifications to
the siRNA backbone, such as 2´-O-methyl- and
2´-fluoro-modified siRNA, could completely
abrogate its immunostimulatory activity [10 7] .
Nguyen et al. demonstrated that effective anti-
viral activity could be achieved without the
undesirable immune activation when lipidoid
98N12-5(1) was used to deliver 2´-O-methyl-
modified siRNA to the influenza ucleoprotein
gene [10 9] . After that, the same group optimized
the lipidoid system and demonstrated that the
lipidoid could be used as a vaccine adjuvant
to deliver immunostimulatory RNA to TLR-
expressing cells, inducing robust antiviral activ-
ity [105] . Recently, the lipidoid NPs were also
found to be able to deliver modified PD-L1
siRNA into Kupffer cells in vivo and enhance
natural killer and CD8+ T-cell-mediated hepatic
antiviral immunity [1 10] .
The intracellular uptake of siRNA by lip-
idoid-based NPs has been validated through
a macropinocytosis-mediated pathway [10 3] .
Recently, Sahay et al. further confirmed that
lipidoid C12-200 siRNA delivery is medi-
ated by Cdc42-dependent macropinocytosis,
and its internalization remains largely within
endosomes with little escape to the cytosol [111].
 Neutral lipid-based liposomes
Although cationic NPs offer many advantages
in systemic delivery of siRNAs, including
protecting siRNAs from enzymatic degrada-
tion and facilitating cellular uptake and effi-
cient endosomal escape, their positive charges
undergo nonspecific interactions with negatively
charged cellular components, often causing side
effects and toxicity in drug delivery. To over-
come this, Landen et al. developed a neutral
liposome composed of 1,2-dioleoyl-sn-glycero-
3-phosphatidylcholine for systemic delivery of
siRNA against the oncoprotein EphA2, and
demonstrated the successful knockdown of
EphA2 gene expression in vivo and significant
tumor growth inhibition in an orthotropic
mouse model of ovarian cancer [1 12 ]. This neutral
liposomal system was further applied for deliv-
ery of various siRNAs, such as FAK [11 3], TG2
[114 ], PAR-1 [115] , ATP7B [116] , PELP1 [117] and
survivin splice variant 2B [118], among others.
Among them, the system’s utility for EphA2
gene therapy is scheduled to be investigated in
a Phase I clinical trial according to the NIH
clinical trials database [20 3].
 Anionic lipid-based liposomes
Anionic liposomes offer another safer alternative
to cationic-based NPs. Koldehoff et al. utilized
an anionic liposome, composed of soya bean oil,
glycerol and phospholipids from egg, in delivery of
bcr–abl siRNA for treatment of chronic myeloid
leukemia and observed negligible side effects
[119]. However, anionic liposomes often have poor
siRNA encapsulation capability owing to the
electrostatic repulsion between negatively charged
liposomes and anionic siRNAs. To overcome this
hurdle a new formulation of siRNA–anionic lipo-
some has been developed based on physiologically
occurring anionic lipids, 1,2-dioleoyl-sn-glycero-
3-phospho-(1´-rac-glycerol), in which the anionic
liposomes integrate with siRNA through stable
calcium ion bridges. The optimal siRNA–anionic
liposomes showed good intracellular uptake,
efficient endosomal escape, and induced compa-
rable silencing efficiency with much lower toxic-
ity when compared with Lipofectamine® 2000
delivery [1 20] .
Conclusion
To overcome the obstacles in systemic siRNA
delivery, a number of NPs have been developed
with enhanced blood stability, decreased toxicity
and improved siRNA intracellular uptake to the
cytoplasm. This review covers recent progress
made in lipid-based NPs, which are categorized
according to their key lipid components, includ-
ing cationic lipid-, lipoprotein-, lipidoid-, neutral
lipid- and anionic lipid-based NPs. Compared
with inorganic and viral delivery systems, lipid
NPs in general have favorable biocompatible
and biodegradable properties. The advanced
cationic lipid NPs in combination with various
advantageous effects provided by different lipid
components, including the use of PEG lipids for
optimizing pharmacokinetics, cationic lipids
for improving cellular uptake and endosomal
escaping and neutral lipids for enhancing par-
ticle stability, enable achievement of significant
RNAi levels in the liver at a very low adminis-
tration doses (~0.01 mg/kg). The lipoprotein-
based NPs have shown superior advantages over
siRNA delivery owing to their excellent biocom-
patibility, favorable pharmacokinetics, nonim-
munogenicity, suitable small size and active tar-
geting features (built-in or redirected targeting)
[121,122]. Among them, the ultra-small HDL-like

www.futuremedicine.com 117
future science group
Lipid-based nanoparticles in the systemic delivery of siRNA Review
particles (<30 nm) with SR-BI-mediated direct
cytosolic delivery features hold great promise for
effective siRNA delivery to fulfill the vision of
personalized medicine.
Future perspective
Moving forward, we expect that lipid-based
NPs will have a key role in systemic applica-
tion of siRNA in the clinic. In particular, it will
have an enabling role for personalized cancer
medicine, where siRNA delivery will join forces
with genetic profiling of individual patients to
achieve the best treatment outcome. We expect
major advancements in efficient delivery of
siRNA into the tissue beyond the liver/spleen
by optimizing the particle size, charge and sur-
face properties. For poorly permeable tumors
(e.g., pancreatic cancer), decreasing particle
size (<30 nm) and including active targeting
will be beneficial. Future research is expected to
improve particle biocompatibility and address
the immuno stimulatory effects to improve
the therapeutic index. Finally, lipid NP-based
siRNA delivery systems have the potential to
achieve multi target RNAi or synergism with
other therapeutic modalities, leading to a more
effective treatment efficacy.
Financial & competing interests disclosure
The authors would like to thank the China– Canada Joint
Health Research Initiative (NSFC-30911120489, CIHR
CCI-102936), DLVR Therapeutics, Canadian Institutes
of Health Research, Ontario Institute for Cancer Research,
Natural Sciences and Engineering Research Council of
Canada, Canada Foundation for Innovation and the Joey
and Toby Tanenbaum/Brazilian Ball Chair in Prostate
Cancer Research for funding support. G Zheng is a scientific
cofounder of DLVR Therapeutics. The authors have no
other relevant affiliations or financial involvement with
any organization or entity with a financial interest in or
financial conflict with the subject matter or materials
discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of
this manuscript.
Executive summary
siRNA systemic delivery barriers
The ability to deliver siRNA systemically to its intended site of action is a challenge for the current field of RNAi therapy.
Lipid-based nanoparticles for siRNA delivery
Nanoplatforms have higher biocompatibility and lower toxicity in comparison with inorganic nanoparticles and viral vectors.
Function-engineerable nanoparticles in combination with the advantageous effects of various functional lipid components enable
achievement of enhanced blood stability, decreased toxicity and improved siRNA delivery to the cytoplasm.
Lipoprotein-based nanoparticles with excellent biocompatibility, favorable pharmacokinetics, nonimmunogenicity, suitably small size
(<30 nm) and active targeting features demonstrate great potential for siRNA delivery.
Lipid nanoparticle-based siRNA delivery enables us to fulfill the vision of personalized medicine.
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