Galactose Derivative-Modified Nanoparticles for Efficient siRNA Delivery to Hepatocellular Carcinoma

Article abstract of DOI:10.1021/acs.biomac.8b00358

Successful siRNA therapy requires suitable delivery systems with targeting moieties such as small molecules, peptides, antibodies, or aptamers. Galactose (Gal) residues recognized by the asialoglycoprotein receptor (ASGPR) can serve as potent targeting moieties for hepatocellular carcinoma (HCC) cells. However, efficient targeting to HCC via galactose moieties rather than normal liver tissues in HCC patients remains a challenge. To achieve more efficient siRNA delivery in HCC, we synthesized various galactoside derivatives and investigated the siRNA delivery capability of nanoparticles modified with those galactoside derivatives. In this study, we assembled lipid/calcium/phosphate nanoparticles (LCP NPs) conjugated with eight types of galactoside derivatives and demonstrated that phenyl β-d-galactoside-decorated LCP NPs (L4-LCP NPs) exhibited a superior siRNA delivery into HCC cells compared to normal hepatocytes. VEGF siRNAs delivered by L4-LCP NPs downregulated VEGF expression in HCC in vitro and in vivo and led to a potent antiangiogenic effect in the tumor microenvironment of a murine orthotopic HCC model. The efficient delivery of VEGF siRNA by L4-LCP NPs that resulted in significant tumor regression indicates that phenyl galactoside could be a promising HCC-targeting ligand for therapeutic siRNA delivery to treat liver cancer.

Full text of DOI:10.1021/acs.biomac.8b00358

Article
pubs.acs.org/Biomac
Cite This: Biomacromolecules XXXX, XXX, XXX−XXX
Galactose Derivative-Modified Nanoparticles for Efficient siRNA
Delivery to Hepatocellular Carcinoma
Kuan-Wei Huang,†,‡ Yu-Tsung Lai,†,§ Guann-Jen Chern,‡ Shao-Feng Huang,§ Chia-Lung Tsai,§
Yun-Chieh Sung,‡,∥ Cheng-Chin Chiang,‡ Pi-Bei Hwang,‡ Ting-Lun Ho,‡ Rui-Lin Huang,‡
Ting-Yun Shiue,‡ Yunching Chen,*,‡,∥ and Sheng-Kai Wang*,§,∥
‡Institute of Biomedical Engineering, National Tsing Hua University, Hsinchu 30013, Taiwan
§Department of Chemistry, National Tsing Hua University, Hsinchu 30013, Taiwan
∥Frontier Research Center on Fundamental and Applied Sciences of Matters, National Tsing Hua University, Hsinchu 30013, Taiwan
S
* Supporting Information
ABSTRACT: Successful siRNA therapy requires suitable delivery systems with targeting moieties such as small molecules,
peptides, antibodies, or aptamers. Galactose (Gal) residues recognized by the asialoglycoprotein receptor (ASGPR) can serve as
potent targeting moieties for hepatocellular carcinoma (HCC) cells. However, efficient targeting to HCC via galactose moieties
rather than normal liver tissues in HCC patients remains a challenge. To achieve more efficient siRNA delivery in HCC, we
synthesized various galactoside derivatives and investigated the siRNA delivery capability of nanoparticles modified with those
galactoside derivatives. In this study, we assembled lipid/calcium/phosphate nanoparticles (LCP NPs) conjugated with eight
types of galactoside derivatives and demonstrated that phenyl β-D-galactoside-decorated LCP NPs (L4-LCP NPs) exhibited a
superior siRNA delivery into HCC cells compared to normal hepatocytes. VEGF siRNAs delivered by L4-LCP NPs
downregulated VEGF expression in HCC in vitro and in vivo and led to a potent antiangiogenic effect in the tumor
microenvironment of a murine orthotopic HCC model. The efficient delivery of VEGF siRNA by L4-LCP NPs that resulted in
significant tumor regression indicates that phenyl galactoside could be a promising HCC-targeting ligand for therapeutic siRNA
delivery to treat liver cancer.
growth factor (VEGF) from HCC cells in the tumor
microenvironment.9,10 Thus, silencing VEGF expression in
HCC cells by siRNA may serve as a potent antiangiogenic
approach as well as a promising anti-HCC therapy.
Despite being a promising therapeutic approach, efficient
delivery of siRNAs in vivo remains challenging due to the
following reasons: Naked siRNAs are rapidly degraded by
nucleases in the blood circulation and are eliminated by renal
filtration, and the anionic and hydrophilic properties of siRNAs
limit the efficacy of cellular uptake and endosome escape,
INTRODUCTION
■
Hepatocellular carcinoma (HCC) is a primary liver cancer with
high mortality and morbidity rates in East Asia.1,2 The current
treatment approaches for HCC, such as surgical resection,
chemoembolization, and tyrosine kinase inhibitor (TKI)
therapy, show poor prognosis or strong adverse effects due to
their complexities.3−5 Thus, developing an effective treatment
approach with reduced side effects is an urgent need. Small
interfering RNA (siRNA) therapy represents a potential
therapeutic strategy6,7 for mediating the downregulation of
pro-tumor molecules to suppress cancer progression and exerts
beneficial effects against various types of cancer.8 In HCC,
tumor progression and metastasis development significantly
rely on the formation of neovessels that can be promoted by
the secretion of angiogenic factors such as vascular endothelial
Special Issue: Biomacromolecules Asian Special Issue
Received: March 1, 2018
Revised: May 15, 2018
© XXXX American Chemical Society
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leading to poor bioavailability.11,12 Moreover, selective delivery
of siRNAs into cancer cells without inducing cytotoxicity in
normal cells is crucial to reducing the side effects.13 Thus,
successful siRNA therapy would require a suitable delivery
system with selectivity.11,14 In previous studies, we have
developed an LCP (lipid/calcium/phosphate) nanoparticle
(NP) as a multifunctional siRNA delivery system that
overcomes the above obstacles by encapsulating siRNA in a
pH stimuli-responsive calcium phosphate core enclosed in a
lipid bilayer shell.15 The LCP NPs protect siRNA from
degradation by nucleases and thus enhance its stability in the
blood circulation. Meanwhile, the CaP (calcium phosphate)
core of LCP NPs disassmbles at low pH in the endosome,
resulting in the release of nucleic acid cargo to the
cytoplasm.16,17 With these advantages, the remaining problem
toward an ideal delivery system is the selectivity for cancer cells.
More recently, specific delivery to HCC has been widely
explored using carbohydrate ligands. The advantages exploiting
carbohydrate−receptor interactions include their low toxicity,
high avidity, and expression.18,19 One major receptor
responsible for this specificity is the asialoglycoprotein receptor
(ASGPR), which is found predominately on hepatocytes and
HCC cells, but minimally on nonhepatic cells.19 Galactosylated
polymers or liposomes targeting ASGPR were developed to
serve as drug/gene carriers for the treatment of HCC.20,21
ASGPR is composed of two types of polypeptide subunits
(subunits 1 and 2), which form homo- and hetero-oligomers
that can coexist on the plasma membrane.22 Upon recognition
and binding of ligands, ASGPR is internalized and allows the
cargo to be contained in endosomes and then degraded in
lysosomes.23 ASGPR selectively binds to galactose (Gal) and
N-acetylgalactosamine (GalNAc) and weakly binds to
glucose.24,25 To improve the affinity of glycan ligands to
ASGPR, recent studies have introduced artificial moieties to
galactose. For example, the Ernst group has reduced the
anomeric hydroxyl group of GalNAc and extended aromatic
moieties on C-2 through a triazole ring.26 The Finn and
Mascitti groups have modified galactosides with broad types of
moieties on C-1, C-2, C-5, and C-6 to identify the
trifluoromethylacetamide derivative that significantly enhanced
ASGPR interaction.27 The same groups also developed bicyclic
bridged ketal as a GalNAc derivative to target ASGPR.28
In addition to modifying glycan ligands, another approach for
targeting ASGPR is through multivalent binding to this
oligomeric receptor on the plasma membrane. Despite the
unavailability of the ASGPR oligomer crystal structure, the
distances between carbohydrate binding sites have been
predicted to be 15−25 Å29,30 based on the binding response
of trimeric ligands on defined scaffolds.31 Immobilization of
ASGPR ligands on nanocarriers may provide multivalent
binding to the surface of HCC cells with elevated ASGPR
expression. In many cases, including nanoparticle32,33 and
micelle34 carriers, galactosides with a C-1 aromatic moiety are
employed as ASGPR ligands. To achieve more potent siRNA
delivery in HCC, we investigated the effect of the aromatic
moiety on ASGPR-based siRNA delivery in HCC. We
synthesized various galactoside derivatives and investigated
the siRNA delivery capability of LCP NPs modified with those
galactoside derivatives in HCC cells. Given the potent ASGPR-
targeting property of galactoside derivatives, we encapsulated
the therapeutic siRNA (VEGF siRNA) in the ASGPR-targeted
LCP NPs and investigated whether VEGF siRNA delivered by
ASGPR-targeted NPs suppressed HCC progression in murine
orthotopic HCC models.
EXPERIMENTAL SECTION
■
Materials. High-concentration and phenol red-free Matrigel and
phosphate-buffered saline (PBS) were purchased from Corning
(U.S.A.). Fetal bovine serum (FBS), trypsin, penicillin-streptomycin,
high-glucose Dulbecco’s modified Eagle’s medium (DMEM), MEM α
(minimum essential medium α), and F-12K medium (Kaighn’s
modification of Ham’s F-12 medium) were obtained from HyClone
(U.S.A.). Protein standard, polyacrylamide, and enhanced chemilumi-
nescence (ECL) were purchased from Bio-Rad (U.S.A.). Stellate cell
growth supplement was purchased from ScienCell Research
Laboratory (U.S.A.). Polyvinylidene fluoride membrane (PVDF) and
enhanced chemiluminescence (ECL) were acquired from GE
Healthcare Life Sciences (U.K.). HRP IgG conjugate was also
purchased from ImmunoReagents, Inc. (U.S.A.). Fluorescein amidite
(FAM) was conjugated to the 5′ end of the sense sequence. 5′-FAM-
labeled VEGF siRNA, sodium dodecyl sulfate (SDS), and bovine
serum albumin (BSA) were obtained from MDBio, Inc. (Taiwan).
Asialofeutin, coumarin 6, dimethyl sulfoxide (DMSO), β-actin
antibody, methylcellulose, paraformaldehyde, tocopheryl polyethylene
glycol succinate (TPGS), methanol, ethanol, acetone, and VEGF
siRNA with the sequence 5′-AUGUGAAUGCAGACCAAAGAA-3′
were purchased from Sigma-Aldrich (U.S.A.). RIPA buffer was
acquired from Bio Basic Canada, Inc. (Canada). LB broth was
purchased from FocusBio (Australia). LB Agar and tris(2-carbox-
yethyl) phosphine (TCEP) were obtained from ThermoFisher
Scientific (U.S.A.). Agarose and sodium chloride were purchased
from Avantor (U.S.A.). Optimal cutting temperature (OCT) was
purchased from Sakura Finetek (U.S.A.). Peptides were ordered from
Kelowna (Taiwan). Cholesterol, l,2-dioleoyl-sn-glycero-3-phosphate
(DOPA), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-
distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene
glycol)-2000] (DSPE-PEG), and 1,2-distearoyl-sn-glycero-3-phos-
phoethanolamine-N-[azido(polyethylene glycol)-2000] (Azido-DSPE-
PEG) were purchased from Avanti Polar Lipids (U.S.A.).
Cell Culture. Murine HCC cells (HCA-1), murine hepatocytes
(FL83B), and human HCC cells (Hep3B) were kindly provided by Dr.
Dan Duda (MGH, Boston). The HCA-1 and Hep3B cells were
cultured in Dulbecco’s modified Eagle’s medium (DMEM)/high
glucose and MEM α (minimum essential medium α), respectively.
The FL83B cells were cultured in F-12K medium (Kaighn’s
modification of Ham’s F-12 medium). All media contained 10% FBS
and 1% antibiotics. All cells are maintained at 37 °C in an incubator
supplied with 5% CO2.
Mice and Orthotopic Tumor Model Establishment. C3H/
HeNCrNarl mice were purchased from National Laboratory Animal
Center (Taipei, Taiwan). Mice aged 4−5 weeks were used for
pancreatic tumor establishment. HCA-1 cells (106), which were
resuspended with a mixture of medium-concentration, high-concen-
tration, and phenol red-free Matrigel, were orthotopically injected into
mice for the screening process. The remaining 105 of the tumor cells
were used otherwise. All animals received humane care, in compliance
with the “Guide for the Care and Use of Laboratory Animals”
published by the National Academy of Sciences.
Preparation of Nanoparticles. LCP NPs were prepared using a
modified protocol.16,35 Two separate microemulsions (3 mL each)
were prepared. To prepare the calcium-loaded microemulsion, siRNA
(15 μg) and 40 μL of 500 mM CaCl2 (pH 7) were added to the oil
phase of cyclohexane and Igepal-520 (7:3, v/v). To prepare the
phosphate buffer-loaded microemulsion, a Na2HPO4 solution (74 μL,
100 mM, pH 9) and 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA; 74
μL, 35 mM) were added to the oil phase of cyclohexane and Igepal-
520 (7:3, v/v). Two separate microemulsions were mixed using a
magnetic stir bar and stir plate at the speed of 700−1000 rpm for 20
min at room temperature. The emulsions were then mixed for 20 min
to form the condensed CaP cores of CaP/siRNA. Then, 6 mL of 100%
EtOH was added to disrupt the emulsion, and the mixture was
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Scheme 1. Synthesis Scheme of Alkynyl Galactosides Conjugating with Azido-PEG-DSPE for Assembly of NPs
centrifuged at 10000 × g for 20 min to remove free DOPA lipid. After
removing the supernatant solution, the precipitated CaP cores were
washed twice with 100% EtOH to remove the organic solvents with
emulsifying agents. The precipitate (LCP core) was then dried under
N2 and suspended in chloroform by sonication. A mixture of free lipids
(DOTAP/DSPE-PEG2000/cholesterol = 1:1:2 molar ratio) in
chloroform was added to the CaP cores (2.5:1 ratio of total free
lipids to DOPA) and then dried under N2. After evaporating the
chloroform, 160 μL of water was added to form LCP NPs with the
lipid-bilayer core structure followed by sonication. For galactoside-
decorated LCP NPs, the mixture of free lipids was changed to
(DOTAP/DSPE-PEG2000/cholesterol/glycolipid = 1:0.99:2:0.01
molar ratio).
Characterization of Nanoparticles. The NPs were formulated as
described above and were recovered in deionized water. The
morphology of the LCP NPs was characterized by transmission
electron microscopy (TEM; H-7500, Hitachi High-Tech, Tokyo,
Japan). The NPs were stained on dried Formvar-coated 100-mesh
copper grids at room temperature. All grids were further dried for 2
days before imaging. The particle size and surface charge were
examined using a Zetasizer system (Zetasizer nano zs, Malvern
Instruments Ltd., Worcestershire, U.K.) at room temperature.
Cellular Uptake. Cells and NPs were prepared as mentioned
above. The cells were incubated with the NPs for 4 h. The medium
was replaced with 4% paraformaldehyde for 10 min, followed by three
washes with PBS. The coverslips containing the cells were mounted
with 3 μL of DAPI, fixed on slides, and finally imaged using a confocal
microscopy (LSM 780, Zeiss, Germany). The images were analyzed
using Matlab.
of each of the isolated RNA samples was used to synthesize cDNA
using a High-Capacity cDNA Reverse Transcription kit (Applied
Biosystems, U.S.A.) in a Piko Thermal Cycler in a 24-well plate
(ThermoFisher Scientific) according to the manufacturer’s instruc-
tions. Quantitative PCR was carried out using SYBR Green PCR
Mastermix with gene-specific PCR primer pairs with a 7500 Real-Time
PCR System (Applied Biosystems, U.S.A.). The primer sequences
used were as follows: sense, 5′-CTGTGCAGGCTGCTGTAACG-3′
and antisense, 5′-GTTCCCGAAACCCTGAGGAG-3′ for VEGF-A;
sense 5′-TGAGAGGGAAATCGTGCGTG-3′ and antisense, 5′-
TTGCTGATCCACATCTGCTGG-3′ for β-actin; and sense, 5′-
CTGCCACCCAGAAGACTGTG-3′ and antisense, 5′-GGTCCTCA-
GTGTAGCCCAAG-3′ for GAPDH. Further validation was per-
formed using RT2 Profiler PCR Arrays Human Fibrosis (Qiagen).
cDNA samples along with SYBR Green PCR Mastermix were added
into wells preloaded with specific primers in the array. The PCR was
carried out according to the manufacturer’s instruction. The data were
subsequently analyzed and plotted using online software offered by the
manufacturer.
Tumor Growth Inhibition Study. Orthotopic HCA-1 tumor-
bearing mice were intravenously injected with different formulations
containing VEGF siRNA (0.7 mg/kg/dose, 3 doses per week). VEGF
siRNA in ASGPR-targeted LCP NPs was administered intravenously
to mice with orthotopic HCA-1 HCCs. Tumor size in the treated mice
was measured using clipper at 2 weeks after the first treatment and
estimated by using the following formula: tumor volume = length ×
width2/2.
Statistics. Data analyses were performed using Student’s t test and
were presented as mean
SEM. P-values less than 0.05 were
Competition Assay. Cells and NPs were prepared as mentioned
above. The free form of asialofetuin protein was added to medium 10
min prior to the treatment of the NPs. The cells were incubated with
the NPs for another 1 h. The medium was replaced with 4%
paraformaldehyde for 10 min, followed by three washes with PBS. The
coverslips containing the cells were mounted with 3 μL of DAPI, fixed
on slides, and finally imaged using a confocal microscopy (LSM 780,
Zeiss, Germany). The images were analyzed using Matlab.
qRT-PCR. To evaluate gene silencing by quantitative reverse
transcription polymerase chain reaction (qRT-PCR), the cells were
seeded in a 12-well plate, grown overnight with 24 or 48 h treatments
of various LCP formulations, and then washed with PBS. In total, 1 μg
considered statistically significant for all tests.
RESULTS AND DISCUSSION
■
Preparation of Galactoside-Decorated LCP Nano-
particles and Synthesis of Alkynyl Galactosides. To
optimize the affinity and specificity of the galactoside
derivatives to ASGPR on HCC, we first investigated the effect
of the aromatic moieties on the galactosides toward the uptake
of ASGPR-targeted NPs. We designed two phenyl and naphthyl
groups with different linkages and provided a galactoside ligand
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In addition, as GalNAc ligands generally initiate stronger
ASGPR interactions than their Gal analogs24,25,27 but take more
effort or resources to synthesize, we would also like to evaluate
the difference between these two families in our siRNA delivery
system. These galactosides with alkyne groups were conjugated
to commercial azido-PEG-DSPE through the robust Cu(I)-
catalyzed alkyne−azide cycloaddition (CuAAC) reaction. The
resulted glycolipids L1−L6 were employed to assemble the
LCP NPs for ASGPR-based delivery (Scheme 1 and Figures
S1−S33).
Scheme 2. Scheme of Alkynyl Galactoside Synthesis
The designed alkynyl galactosides can be prepared through
straightforward transformations from peracetylated galactose 7
or peracetylated GalNAc 8 (Scheme 2). Glycan 1a and 1b were
synthesized in a similar manner, which began from C-1
deacetylated intermediates of 7 and 8, respectively. Then, the
intermediates were transformed to trichloroacetimidate and
glycosylated with propargyl alcohol to give 1a and 1b after
deacetylation with sodium methoxide. Glycans 2a and 3 were
prepared with a similar approach by propargylamine coupling
and deacetylation to intermediates 9 and 10, which can be
carried out by following the reported methods.36 Glycan 2b was
obtained through glycosylation of the glycosyl bromide from 8
with 3-hydroxy-N-propargyl-benzamide and followed by
deacetylation. Galactosides 4−6 were prepared from the
coupling of 11−13 to pentynoic acid and then deacetylation.
The key reactant 11 was prepared by known methods,37 and 12
was prepared by phase transfer catalytic (PTC) glycosylation38
of galactosyl bromide of 7 with 5-amino-1-naphthol.39 Likewise,
galactoside 13 was prepared through the same PTC
glycosylation with 4-iodophenol, followed by reaction with 5-
nitroindole and by hydrogenation reduction of the nitro
group.39
In Vitro Cellular Uptake of ASGPR-Targeted NPs.
ASGPR-targeted NPs were prepared according to the methods
of previous studies.16,35 First, we utilized a reverse micro-
emulsion to encapsulate the nucleic acid (siRNA) in lipid-
coated CaP NPs. Next, the lipid-coated siRNA/CaP NPs were
allowed to form a self-assembled lipid-bilayer structure by
adding the outer leaflet lipids (DOTAP, cholesterol, DSPE-
PEG) to the siRNA/CaP cores. To achieve ASGPR targeting,
we conjugated galactoside derivatives into the outer leaflet
lipids (DSPE-PEG) and assembled the LCP NPs with
galactoside derivative-conjugated DSPE-PEG(L1-L6). We
further examined the physical properties of siRNA-loaded
ASGPR-targeted LCP NPs by transmission electron micros-
copy (TEM) and dynamic light scattering (DLS) analysis. The
TEM image showed that the ASGPR-targeted LCP NPs were
spherical (Figure 1A) with the average diameter of 126.3 0.8
nm and a zeta-potential of −2.72 mV measured by DLS
(Figures 1B and S34). The particle size of the ASGPR-targeted
LCP NP (L4-LCP) was similar to that of the nontargeted LCP
NP and Gal(L1a)-LCP NP. The stability of the LCP NPs was
also checked by measuring mean size and PDI after long
storage times (up to 14 days), at 4 °C in water. No significant
changes in the particle size and PDI were observed (Figure
S35). In addition, we also found the percentage of siRNA
encapsulation for the L4-LCP NP was 90%.
Figure 1. Morphology of L4-LCP NPs containing siRNA. (A)
Representative TEM images of L4-LCP NPs. (B) Sizes and zeta
potentials of nontargeted LCP, Gal-LCP, and L4-LCP NPs. The data
are the mean values the SD (n = 3). PDI, polydispersity index.
To investigate the cellular uptake of ASGPR-targeted LCP
NPs, FAM-siRNA was encapsulated in LCP NPs modified with
various galactose analogs, and the uptakes of FAM-siRNA
loaded NPs by HCC cells and normal hepatocytes were
examined 4 h after treatment (Figure 2). The results (Figure
2A,B) indicate that the simple galactose ligand (L1a) and the
without an aromatic group for comparison as well as extension
of an extra aromatic moiety from the phenyl group (Scheme 1).
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Figure 2. Cellular uptake of FAM-siRNA delivered by ASGPR-targeted LCP NPs. (A) Hep3B cells were treated with FAM-labeled siRNA (75 nM)
in different formulations for 4 h and observed with a Zeiss LSM 780 confocal microscope. FAM-siRNA was presented in green, and the nucleus was
presented in blue (DAPI). Scale bar = 25 μm. (B) The quantitative data of cellular uptake of FAM-siRNA delivered by ASGPR-targeted LCP NPs.
(C, D) Competitive inhibition was performed with excess free asialofetuin. The uptake of FAM-siRNA was imaged and quantified with a confocal
microscope. (E, F) L4-LCP NPs exhibited a superior siRNA delivery into murine HCC (HCA-1) cells compared to normal murine hepatocytes
(FL83B). Scale bar = 20 μm. The data are the mean values the SEM; *p < 0.05, **p < 0.01, ***p < 0.001. ns, not significant.
Figure 3. VEGF siRNA delivered by L4-LCP NPs downregulated VEGF expression in HCC cells in vitro. L4-LCP NPs containing VEGF siRNA (75
nM) significantly decreased VEGF expression in both human (Hep3B) and murine (HCA-1) HCC cells in a dose-dependent manner. VEGF
expression was detected by qRT-PCR (A, B) and Western blotting (C, D). The data are the mean values the SEM; *p < 0.05, ** p < 0.01.
GalNAc analog (L1b) on ASGPR-targeted LCP NPs only
slightly improved the cellular uptake of nanocarriers compared
with the nontargeted LCP NPs. However, the meta-benzamide
moiety introduced to the galactoside and GalNAc on L2a-LCP
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Figure 4. VEGF siRNA delivered by L4-LCP NPs suppressed angiogenesis in tumor microenvironment of murine orthotopic HCC models. (A)
Treatment schedule of VEGF siRNA (0.7 mg/kg) in different formulations in mice with orthotopic HCA-1 HCCs. (B−E) MVD was measured using
anti-CD31 antibodies. Representative immunofluorescence images of VEGF staining (B, C) and CD31 staining (D, E) in the tumor tissues of
orthotopic HCA-1 tumor models. L4-LCP NPs significantly decreased the expression of VEGF and inhibited the formation of vessels in HCC tumor
tissues. VEGF was presented in green, CD31 was presented in red, and the nucleus was presented in blue (DAPI). Scale bar = 50 μm. The data are
the mean value the SEM; *p < 0.05, **p < 0.01, ***p < 0.001.
and L2b-LCP NPs significantly enhanced the cellular uptake.
NPs modified with galactoside with a para-phenylamide moiety
(L4-LCP NPs) showed potent cellular uptake in HCC at 9-fold
higher than that of nontargeted LCP NPs in HCC cells.
Galactoside with naphthyl modification on L3-LCP and L5-
LCP NPs did not show improvement of HCC uptake when
compared with nontargeted LCP NPs. Notably, the indole
group extended from the para position of the phenyl moiety on
L6-LCP NPs also provided significant uptake improvement
over simple galactoside on Gal(L1a)-LCP NPs. Our data
indicated that the glycan ligand with phenyl group significantly
improved the uptake of siRNA-loaded NPs, and even larger
indole group at the para position can be tolerated. We also
showed that naphthyl moieties in two different linkages are
both not favored for NP delivery. It is also interesting that the
NPs modified with GalNAc ligand did not show improved
uptake in HCC compared to the NPs modified with their
galactose analogs. Among eight derivatives, we demonstrated
that the uptake of siRNA was greatest in HCC cells treated with
phenyl β-D-galactoside-decorated LCP NPs (L4-LCP NPs;
Figures 2 and S36). Furthermore, the uptake of L4-LCP NPs
was competitively inhibited by the addition of asialofetuin, a
specific ASGPR ligand (Figure 2C,D). These results indicated
that the cellular uptake of L4-LCP NPs was via ASGPR-
mediated endocytosis. However, from current understanding
on ASGPR, it is difficult to interpret the observed uptake
efficiency based on the receptor binding to different galactoside
structures. Recent structure−activity relationship investigation
on ASGPR H1 subunit by the Ernst, Finn, and Mascitti
groups26,27 have focused on galactosides with modifications at
2, 5, and 6 positions. Although an aromatic modification at the
anomeric position of GalNAc was also investigated, those were
C-glycosides and developed to increase the rigidity of the
molecule. With such purpose, a β-4-methoxyphenyl substituent
resulted in 5-fold loss of ASGPR H1 affinity compared to the β-
methyl substituent.27 As those C-glycoside ligands significantly
limit the motion of their anomeric substituent, they are very
different to our present set of galactoside ligands, so that the
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Figure 5. VEGF siRNA delivered by L4-LCP NPs suppressed HCC progression in murine orthotopic HCC models. (A) Treatment with VEGF
siRNA (0.7 mg/kg) delivered by L4-LCP NPs significantly reduced tumor sizes in the orthotopic tumor-bearing mice (n = 8). (B, C) Treatment
with VEGF siRNA delivered by L4-LCP NPs significantly reduced the number of lung metastatic nodules in the orthotopic tumor-bearing mice.
Scale bar = 200 μm. The data are the mean value the SEM; ***p < 0.001. ns, not significant.
effect of aromatic moiety cannot be directly compared.
Moreover, because the ASGPR is protein oligomer of H1 and
H2 subunits reported with varied stoichiometric ratios,40−42
and that recent studies indicated different ASGPR hetero-
oligomers forms affect the ligand specificity,16 the LCP
nanoparticle uptake activity arisen from glycan structural
modification is difficult to explain solely by the affinity to a
single subunit. Despite of the lack of a clear structure−activity
relationship explanation for the RNAi delivery efficiency, we
continued to investigate the capability of the most potent L4-
LCP NPs.
More interestingly, we demonstrated that L4-LCP NPs
exhibited a superior siRNA delivery into HCC cells compared
to normal hepatocytes (Figure 2E,F). The enhanced uptake of
ASGPR-targeted NPs in HCC cells may be due to the
upregulation of ASGPR expression in HCC cells compared
with normal hepatocytes (Figure S37). With these observations,
L4 was chosen as a targeting moiety for HCC in the following
studies.
VEGF siRNA Loaded in ASGPR-Targeted NPs Silences
VEGF Expression in HCC In Vitro and In Vivo. In an
attempt to block angiogenesis in HCC effectively and
specifically, we encapsulated VEGF siRNA in different
formulations and examined the gene silencing effect and
capability to suppress angiogenesis in HCC. We first performed
qRT-PCR to validate VEGF mRNA expression in HCC cells
after treatment of VEGF siRNA loaded in different
formulations. As shown in Figure 3A,B, VEGF siRNAs
delivered by L4-LCP NPs showed significant inhibition of
VEGF mRNA expression. As expected, VEGF expression
remained unchanged or slightly changed when cells were
treated with VEGF siRNA in the nontargeted LCP NPs or Gal-
LCP NPs, respectively. Furthermore, we also observed a
significant decrease in VEGF protein expression in the HCC
cells treated with VEGF siRNA-loaded L4-LCP NPs, as
detected by using a Western blot (Figure 3C,D).
For the in vivo evaluation, we next established a syngeneic
orthotopic murine HCC model with HCA-1 cells intrahepati-
cally implanted into C3H mice. After HCC tumors were
established, VEGF siRNAs in different formulations were given
at a dose of 0.7 mg/kg, 3 doses per week for 2 weeks of
treatment (Figure 4A). The systemic injections of VEGF
siRNA in L4-LCP NPs significantly suppressed VEGF
expression in HCA-1 tumors, whereas VEGF siRNA delivered
by nontargeted LCP only moderately decreased VEGF
expression in HCA-1 tumors (Figure 4B,C). Our results
indicated that L4-LCP NPs efficiently delivered VEGF siRNAs
into HCC and achieved a significant gene silencing effect in
vitro and in vivo. The delivery efficacy and the silencing activity
of siRNAs loaded in NPs were ligand (L4) dependent.
VEGF siRNA Loaded in ASGPR-Targeted NPs Demon-
strated Potent Antiangiogenic Activity in HCA-1 Tumors
and Suppressed Primary HCC Growth and Distal
Metastasis. We next evaluated the antiangiogenic effects of
VEGF siRNAs in L4-LCP NPs in orthotopic HCC models.
After the HCA-1 tumor was established, VEGF siRNAs loaded
in L4-LCP NPs were I.V. (intravenous) administered into
HCA-1 tumor-bearing mice. VEGF siRNAs loaded in L4-LCP
NPs significantly decreased the mean vessel density (MVD)
compared to VEGF siRNA delivered by nontargeted LCP NPs
(Figure 4D,E). Given that VEGF siRNAs delivered by ASGPR-
targeted LCP NPs demonstrated potent antiangiogenic effects,
we further examined whether the treatment could lead to tumor
growth inhibition. Indeed, tumor growth inhibition was
observed after treatment with VEGF siRNA-loaded L4-LCP
NPs in the HCA-1 orthotopic model (Figure 5A), while mice
receiving VEGF-siRNAs from Gal-LCP NPs or nontargeted
LCP NPs showed moderate or nonsignificant HCC growth
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Figure 6. Schematic illustration of the anti-HCC mechanisms of VEGF siRNA-loaded ASGPR-targeted LCP NPs, which deliver VEGF siRNA into
HCCs cells and suppress angiogenesis in HCC. Therefore, systemic treatment with VEGF siRNA delivered by ASGPR-targeted LCP NPs
significantly inhibits both primary and distal HCC progression and serves as a potent therapeutic strategy to treat HCC.
inhibition, respectively (Figure 5). Furthermore, VEGF siRNAs
delivered by L4-LCP NPs significantly inhibited distal lung
metastasis in the orthotopic HCA-1 model (Figure 5B,C). Mice
receiving VEGF siRNAs in nontargeted LCP NPs did not show
a reduction in metastasis formation compared to the control
group. Our results demonstrated that L4-LCP NPs delivering
therapeutic VEGF siRNAs significantly suppressed primary
tumor progression and metastasis formation in the orthotopic
HCC models.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.bio-
mac.8b00358.
MALDI-TOF mass spectra of commercial N3-PEG-
DSPE, L1a, L2a, L3, L4, L5, L6, L1b, and L2b; The
intensity size distributions from LCP NP, Gal-LCP NP,
and L4-LCP NP; The stability of the LCP NPs; Cellular
uptake of FAM-siRNA delivered by ASGPR-targeting
LCP NPs; The upregulation of ASGPR expression in
HCC cells (PDF).
CONCLUSIONS
■
In summary, we developed a highly efficient siRNA delivery
carrier targeting HCC. β-phenyl galactoside modified PEG-
DSPE (L4) was incorporated into the gene carrier LCP NP to
facilitate the delivery of siRNA into HCCs and to achieve an
enhanced gene inhibition effect in HCC in vitro and in vivo.
Significant suppression of VEGF expression led to MVD
reduction in the tumor microenvironment. Finally, systemic
treatment with VEGF siRNA delivered by LCP NPs modified
with β-phenyl galactoside could serve as a potent anticancer
treatment to suppress HCC progression (Figure 6). The
ASGPR-targeted NPs may serve as a platform to encapsulate
various RNA therapeutic agents for the treatment of HCC or
other liver diseases in the future.
AUTHOR INFORMATION
■
Corresponding Authors
*Phone: +886-3-571-5131, ext: 35503. E-mail: yunching@mx.
nthu.edu.tw.
*Phone: +886-3-571-5131, ext: 33360. E-mail: skwang@mx.
nthu.edu.tw.
ORCID
Yunching Chen: 0000-0001-6228-5169
Sheng-Kai Wang: 0000-0002-3827-7983
Author Contributions
†These authors contributed equally to this work.
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Notes
The authors declare no competing financial interest.
(9) Hanahan, D.; Folkman, J. Patterns and emerging mechanisms of
the angiogenic switch during tumorigenesis. Cell 1996, 86 (3), 353−
64.
ACKNOWLEDGMENTS
■
This study was supported by the Ministry of Science and
Technology (MOST 104-2628-B-007-001-MY3, 105-2628-E-
007-007-MY3 for Y.C.; MOST 105-2633-M-007-002, 106-
2633-M-007-004 for S.K.W.) and by the National Institute for
Health Research (NHRI-EX107-10609BC). This work was
also supported by the Frontier Research Center on
Fundamental and Applied Sceinces of Matters from The
Featured Areas Research Center Program within the framework
of the Higher Education Sprout Project by Ministry of
Education (MOE) in Taiwan.
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ABBREVIATIONS
■
ASGPR: asialoglycoprotein receptor; BSA: bovine serum
albumin; CaP: calcium phosphate; CuAAC: Cu(I)-catalyzed
alkyne−azide cycloaddition; DAPI: 4′,6-diamidino-2-phenyl-
indole; DMEM: Dulbecco’s modified Eagle’s medium; DMSO:
dimethyl sulfoxide; DLS: dynamic light scattering; DOPA: 1,2-
dioleoyl-sn-glycero-3-phosphate; DOTAP: 1,2-dioleoyl-3-trime-
thylammonium-propane; DSPE-PEG: 1,2-distearoyl-sn-glycero-
3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000];
azido-DSPE-PEG: 1,2-distearoyl-sn-glycero-3-phosphoethanol-
amine-N-[azido(polyethylene glycol)-2000]; ECL: enhanced
chemiluminescence; EtOH: ethanol; FAM: fluorescein amidite;
FBS: fetal bovine serum; GAPDH: glyceraldehyde 3-phosphate
dehydrogenase; Gal: galactose; GL: glycolipids; HCC:
hepatocellular carcinoma; HRP: horseradish peroxidase; I.V.:
intravenous; LCP: lipid/calcium/phosphate; MVD: mean
vessel density; NP: nanoparticle; GalNAc: N-acetylgalactos-
amine; PBS: phosphate-buffered saline; PBST: phosphate-
buffered saline with Tween 20; PDI: polydispersity index; PEG:
polyethylene glycol; PTC: phase transfer catalytic; PVDF:
polyvinylidene fluoride; RT-qPCR: reverse transcription
quantitative polymerase chain reaction; SDS: sodium dodecyl
sulfate; SEM: standard error of the mean; siRNA: small
interfering ribonucleic acid; TKI: tyrosine kinase inhibitor;
TEM: transmission electron microscopy; VEGF: vascular
endothelial growth factor
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