Cyclam-Modified PEI for Combined VEGF siRNA Silencing and CXCR4 Inhibition to Treat Metastatic Breast Cancer

Article abstract of DOI:10.1021/acs.biomac.7b01487

Chemokine receptor CXCR4 plays an important role in cancer cell invasion and metastasis. Recent findings suggest that anti-VEGF therapies upregulate CXCR4 expression, which contributes to resistance to antiangiogenic therapies. Here, we report the develop

Full text of DOI:10.1021/acs.biomac.7b01487

Article
pubs.acs.org/Biomac
Cite This: Biomacromolecules XXXX, XXX, XXX−XXX
Cyclam-Modified PEI for Combined VEGF siRNA Silencing and CXCR4
Inhibition To Treat Metastatic Breast Cancer
Yiwen Zhou,†,§ Fei Yu,‡,§ Feiran Zhang,† Gang Chen,† Kaikai Wang,† Minjie Sun,† Jing Li,‡
and David Oupicky*,†,‡
́
†State Key Laboratory of Natural Medicines, Department of Pharmaceutics, China Pharmaceutical University, Nanjing, 210009, China
‡Center for Drug Delivery and Nanomedicine, Department of Pharmaceutical Sciences, University of Nebraska Medical Center,
Omaha Nebraska 68198, United States
ABSTRACT: Chemokine receptor CXCR4 plays an important role in
cancer cell invasion and metastasis. Recent findings suggest that anti-
VEGF therapies upregulate CXCR4 expression, which contributes to
resistance to antiangiogenic therapies. Here, we report the develop-
ment of novel derivatives of polyethylenimine (PEI) that effectively
inhibit CXCR4 while delivering anti-VEGF siRNA. PEI was alkylated
with different amounts of a CXCR4-binding cyclam derivative to
prepare PEI-C. Modification with the cyclam derivatives resulted in a
considerable decrease in cytotoxicity when compared with unmodified
PEI. All the PEI-C showed significant CXCR4 antagonism and the
ability to inhibit cancer cell invasion. Polyplexes of PEI-C prepared
with siVEGF showed effective silencing of the VEGF expression in
vitro. In vivo testing in a syngeneic breast cancer model showed
promising antitumor and antimetastatic activity of the PEI-C/siVEGF
polyplexes. Our data demonstrate the feasibility of using PEI-C as a carrier for simultaneous VEGF silencing and CXCR4
inhibition for enhanced antiangiogenic cancer therapies.
breast cancer has a poor prognosis as currently there is no
effective treatment for patients diagnosed with this disease.13
Recent studies have suggested that breast cancers over-
expressing CXCR4 have a tendency to metastasize to distant
sites where SDF-1 levels are high, including lung, liver, lymph
nodes, and bones.14 CXCR4 regulates the chemotactic function
via activation of several intracellular signaling transduction
pathways, including PI3K, MAPK, and Erk1/2 upon binding
and interaction of the receptor and the ligand.15 Blockade of
such interactions using CXCR4 antagonists like AMD3100
(Plerixafor) have shown significant reduction of cancer
metastasis.16,17
RNA interference (RNAi) has emerged as a potential tool for
treating various genetic and acquired diseases such as
cancer.18−21 Targeted delivery of siRNA to cancer cells is a
complicated and challenging process and the lack of efficient
and safe delivery platforms remains a major hurdle for
successful clinical application of RNAi.22 Cationic polymers
have been extensively explored for delivering siRNA because
they are able to efficiently complex siRNA and protect it from
degradation and facilitate siRNA transport across cellular
membranes.23,24 Polyethylenimine (PEI) is one of the most
studied nonviral nucleic acid carriers due to its high transfection
1. INTRODUCTION
Vascular endothelial growth factor (VEGF) is one of the most
critical regulators of tumor-induced angiogenesis, which
facilitates tumor growth and survival. In breast cancer, VEGF
overexpression markedly increases intratumoral lymphangio-
genesis, resulting in significantly enhanced metastasis to
regional lymph nodes and to the lung.1 Antiangiogenic therapy
with monoclonal antibodies has been successfully used clinically
in the treatment of various cancers. Gene silencing with siRNA
has been used in preclinical studies as an alternative approach
to VEGF inhibition by antibodies and has been reported to
control tumor growth, inhibit metastasis, and prolong
survival.2,3 Despite the clear benefits of VEGF inhibition, the
improvement of survival has often been modest. Recent reports
suggested that many anti-VEGF therapies upregulate expression
of the CXCR4 chemokine receptor as well as the expression of
its ligand stromal derived factor-1 (SDF-1).4,5 Available
evidence shows that VEGF and CXCR4 expression are highly
correlated and might synergistically promote lymphatic meta-
stasis in various cancers.6−9
Chemokines and chemokine receptors form a complex
network involved in a variety of homeostatic and pathological
activities. Among the family of over 40 chemokines and their
receptors, SDF-1 (also known as CXCL12) along with its
receptor CXCR4 are highlighted in regulating tumor metastasis,
proliferation, survival, and angiogenesis in various types of
metastatic cancers including breast cancer.10−12 Metastatic
Received: October 16, 2017
Revised: January 3, 2018
© XXXX American Chemical Society
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efficacy both in vitro and in vivo.25,26 PEI is known to efficiently
facilitate endosomal escape of the polyplexes, in part due to its
high charge density which contributes to its membrane-active
properties. However, such high charge density also leads to
high cytotoxicity, which has been a major concern for PEI that
limits its development. Multiple approaches have been
advanced to overcome the toxicity issue, including changing
molecular weight and architecture of the polymer,27,28
modification of amines with hydrophobic moieties,29 introduc-
tion of biodegradable bonds to the PEI backbone,30−32 and
combining PEI with lipid components to form hybrid
polycation liposomes.33,34 We have previously developed a
series of polycationic CXCR4 antagonists that can not only
limit cancer metastasis but also deliver therapeutic nucleic acids
like siRNA.35−38 In this study, we present a novel strategy to
modify PEI with a cyclam-based CXCR4 antagonist. Toxicity,
CXCR4 antagonism, and siVEGF delivery in vitro have been
evaluated. The potential of PEI-C/siVEGF polyplexes as
antitumor and antimetastatic treatment strategy that benefi-
cially combines VEGF silencing with concordant CXCR4
inhibition was tested in a syngeneic mouse model of breast
cancer.
Hydrodynamic diameter and zeta potential of the polyplexes were
determined by Zeta Plus (Brookhaven Instruments Corp, Holtsville,
NY).
2.4. Cell Culture. 4T1 cells (ATCC, Manassas, VA) were cultured
in RPMI-1640 supplemented with 10% FBS and 1% Pen-Strep.
Human epithelial osteosarcoma U2OS cells stably expressing func-
tional EGFP-CXCR4 fusion protein (Fisher Scientific) were cultured
in DMEM supplemented with 2 mM L-Glutamine, 10% FBS, 1% Pen-
Strep, and 0.5 mg/mL G418. Mouse lung fibroblasts L929 cells were
cultured in DMEM, supplemented with 10% fetal bovine serum and
1% Pen-Strep. All the cells were cultured at 37 °C in 5% CO2
atmosphere.
2.5. Cytotoxicity. Cytotoxicity of the polymers was measured by
MTT assay in 4T1, U2OS, and L929 cells. The cells were seeded in
96-well plates at a density of 8000 cells per well for 24 h and then
incubated with serial dilutions of PEI and PEI-C for 24 h. Twenty
microliters of MTT reagent was added and the cells were further
incubated for 4 h. The medium was removed and 200 μL of DMSO
was then added to each well. The absorbance [A] at 490 nm was
measured using a microplate reader. The cell viability (%) was
calculated as ([A]sample − [A]blank)/([A]untreated − [A]blank) × 100%.
2.6. Cellular Uptake and Intracellular Trafficking. Confocal
microscopy was used to observe cellular uptake and intracellular
trafficking of the polyplexes using FAM labeled siRNA. 4T1 cells were
seeded into glass-bottom dishes at a density of 50,000 cells per dish.
The cells were incubated for 18 h prior to use. The culture medium
was then discarded and replaced by polyplexes in 1 mL of serum-free
medium. The final concentration of siRNA was 100 nM. After 2 h
incubation, the cells were washed three times with PBS, fixed with 4%
paraformaldehyde for 20 min at room temperature, and the nuclei
were stained by DAPI for 10 min. Cells were then visualized under a
LSM700 confocal fluorescence microscope.
For determination of endosomal escape, cells were washed 3 times
with PBS and stained with LysoTracker Red for 40 min after 2 or 6 h
incubation with PEI-C/FAM-siRNA polyplexes. The cells were then
washed 3 times with PBS and imaged by confocal microscopy.
2.7. CXCR4 Antagonism. U2OS cells stably expressing EGFP-
CXCR4 fusion protein were seeded in 96-well black plates with optical
bottom. Cells were washed twice with 100 μL of assay buffer (DMEM
supplemented with 2 mM L-glutamine, 1% FBS, 1% Pen-Strep, and 10
mM HEPES) and treated with polymers (2 μg/mL) or AMD3100
(300 nM) for 30 min. Ten nanomolar SDF-1 was then added to each
well and incubated for an additional 1 h. Cells were fixed with 4%
paraformaldehyde for 20 min, washed with PBS for 4 times, and the
nuclei were stained with 1 μM Hoechst 33258 for 30 min. Cells were
imaged using EVOS fl microscope.
2. MATERIALS AND METHODS
2.1. Materials. Cyclam (1,4,8,11-tetraazacyclotetradecane) was
purchased from Vesina Industrial (Tianjin, China). Branched
polyethylenimine (PEI, Mw 10 kDa) was obtained from Polysciences
(Warrington, PA). Solvents (certified ACS) were purchased from
Fisher (Fair Lawn, NJ) without further purification. AMD3100 (base
form) was from Biochempartner (Shanghai, China). Fluorescently
labeled FAM-siRNA (5′-UUCUCCGAACGUGUCACG UTT-3′),
scrambled siRNA (siScr) and VEGF siRNA (5′-AUGUGAAUGC-
AGACCAAAGAA-3′) were purchased from GenePharma (Shanghai,
China). Dulbecco’s phosphate buffered saline (PBS), trypsin,
penicillin/streptomycin (Pen-Strep), RPMI-1640, Dulbecco’s modified
Eagle medium (DMEM), and fetal bovine serum (FBS) were from
Hyclone (Waltham, MA). LysoTracker Red was from the Beyotime
Institute of Biotechnology. Human SDF-1α was from Shenandoah
Biotechnology, Inc. (Warwick, PA). VEGF mouse antibody, β-tubulin
mouse antibody, and goat antimouse lgG-HRP were purchased from
Santa Cruz Biotechnologies (Dallas, TX). All other reagents were from
Nanjing Wanqing Chemical Glassware Instrument unless otherwise
stated.
2.8. Cell Invasion. The transwell inserts in 24-well plates were
coated with 40 μL of diluted Matrigel (1:3 v/v with serum-free
medium) and then placed in 37 °C incubator for 2 h. Thirty thousand
4T1 cells were trypsinized, washed, and resuspended in serum-free
media containing PEI-C (2 μg/mL), PEI-C/siRNA polyplex (w/w 3)
or AMD3100 (300 nM) for 30 min before adding to the insets. SDF-1
(20 nM) was applied as the chemoattractant and added to the
companion 24-well plates. After 24 h of incubation, the noninvaded
cells on the top side of the insets were removed using a cotton swab,
and the migrated cells at the bottom side were fixed with 100%
methanol and stained with 0.2% crystal violet for 10 min. Cells were
imaged and counted using EVOS xl microscope. Results were
expressed as percent of invaded cells SD (n = 3) using untreated
cells as a control.
2.9. VEGF Silencing by Western Blot. 4T1 cells were seeded
into 12-well plates (1 × 105 cells per well) 1 day prior to the
transfection. The cells were incubated with PEI-C37/siVEGF or PEI-
C37/siScr (final siRNA concentration 100 nM) in serum-free medium
for 4 h. The polyplexes were then removed and the cells were further
cultured in fresh culture medium for 48 h. To measure VEGF protein
level after siRNA transfection, cells were first washed twice with cold
PBS, and then lysed with RIPA lysis buffer containing protease and
phosphatase inhibitors. After centrifugation at 12 000× g for 15 min,
the protein concentration was quantified by BCA assay kit (Beyotime,
2.2. Synthesis and Characterization of PEI-C. The synthesis of
tri-tert-butyl-11-(4-(chloromethyl)benzyl)-1,4,8,11-tetraazacyclotetra-
decane-1,4,8-tricarboxylate (cyclam derivative) was previously re-
ported.36 The conjugation of the cyclam derivative to PEI was
achieved by nucleophilic substitution. The typical conjugation
procedure is described as follows. Branched PEI (Mw 10 kDa, 258.6
mg), tri-tert-butyl-11-(4-(chloromethyl)benzyl)-1,4,8,11-tetraazacyclo-
tetradecane-1,4,8-tricarboxylate (3.088 g, 4.8 mmol) and potassium
carbonate (1.66 g, 12 mmol) were suspended in acetonitrile (20 mL)
and refluxed for 16 h. The resulting product was filtered and
evaporated. Trifluoroacetic acid (20 mL) was added and stirred
overnight to perform deprotection. The final product was dialyzed
against HCl (pH 3) for 2 days and water for an additional day before
lyophilization. The typical yield was ∼75%. The composition of the
polymer was characterized by 1H NMR (Bruker 500 MHz) and
analyzed by TopSpin 3.5pl6 software.
2.3. Preparation and Characterization of Polyplexes.
Polyplexes were prepared by mixing equal volume of siRNA solution
(20 μg/mL in 10 mM HEPES, pH 7.4) with polymers to achieve the
desired w/w ratios. The mixture was vigorously vortexed for 30 s and
incubated at room temperature for 30 min. To evaluate the ability of
PEI-C to condense siRNA, polyplexes prepared at different PEI-C-to-
siRNA weight ratios were run on 2% agarose gel containing JelRed at
100 V for 30 min and then visualized under UV illumination.
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China). Collected protein in the cell lysate was then separated by 10%
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) at 120 V for 2 h,
and transferred to a nitrocellulose membrane. After 1 h of blocking
procedure in 5% nonfat milk at room temperature, the membrane was
probed with mouse primary anti-VEGF antibody overnight at 4 °C.
Following incubation with the secondary antimouse IgG-HRP
antibody for 2 h, the membrane was washed and visualized after
incubating with Pierce ECL Western Blotting Substrate (Thermo
Scientific, U.S.A.).
Scheme 1. Mechanism of Action of PEI-C/siVEGF
Polyplexes
2.10. Anticancer Activity in Vivo. Female BALB/c mice (7
weeks old, 20−25 g) were supplied by the Experimental Animal
Centre of Yangzhou University (Yangzhou, China). All animals were
treated following protocols approved by the ethical committee of
China Pharmaceutical University. 4T1 cells (105) suspended in 100 μL
of PBS were injected into the mammary fat pad of the mice and
allowed to grow for 10 days. The mice were then randomized into
three experimental groups (n = 5): saline, PEI-C/siScr, and PEI-C/
siVEGF polyplexes. The polyplexes were prepared at w/w 6 and
administered by direct intratumoral injection (20 μg siRNA/mouse).
The day of the first administration was designated as day 0. Polyplexes
were given for a total 4 doses at a 3-day interval (Day 0, 3, 6, and 9).
Body weight and tumor size of mice were monitored and recorded
every other day. All the animals were sacrificed at Day 16, and tumors
and lungs were collected and processed for H&E staining and
immunohistochemistry (IHC) analysis.
3. RESULTS AND DISCUSSION
PEI-C polymers with increasing cyclam content were
synthesized and named based on the conjugation ratio. The
conjugation ratio and cyclam content (mol %) in feed and in
the product of all the PEI-C were summarized in Table 1. In
general, the cyclam content in the polymer increased with
increasing feed content of (1). However, when the feed ratio
doubled in the case of PEI-C37 when comparing with PEI-C33
(80 vs 40 mol %), the cyclam content in the synthesized
polymer only increased slightly (25 vs 27 mol %). This is most
likely due to the hyperbranched architecture of the PEI and the
related steric hindrance, which reduced accessibility of the
amines for alkylation with (1).
3.2. Cytotoxicity. Despite its superior gene delivery
efficiency, PEI is also notorious for its cytotoxicity and
hemolytic activity.40−42,44 Before we proceeded to evaluate
the biological activity of PEI-C, MTT assay was used to
examine the cytotoxicity of PEI-C in mouse breast cancer 4T1
cells, EGFP-CXCR4 + U2OS (model cell line to study CXCR4
antagonism) and normal mouse lung fibroblasts L929 cells. PEI
was included as a control. The cell viability curves and
calculated IC50 values of all the polymers are summarized in
Figure 2. All the PEI-C polymers showed reduced cytotoxicity
when compared with PEI in all cell lines as indicated by the
increased IC50 values. In addition, the PEI-C cytotoxicity
decreased with increasing cyclam content in the polymer. PEI-
C37, which contains the highest cyclam content, showed the
highest IC50 values among all the tested polymers. The
observed IC50 of PEI-C37 was ∼23-fold higher than PEI in
4T1 cells and ∼10-fold higher than PEI in U2OS cells. In L929
cells, used as an example of noncancerous normal cells, the cell
viability remained above 80% even after treatment with 100 μg/
mL PEI-C. The IC50 of PEI-C was above 600 μg/mL. This
result suggested that PEI-C polymers may show selective
cytotoxicity in tumor cells due to their CXCR4-mediated
effects.
We have previously reported the development of poly(amido
amine) polycations for simultaneous delivery of nucleic acids
and CXCR4 inhibition to treat metastatic cancer. We have
shown that using small-molecule cyclam CXCR4 antagonists as
the polymer building blocks equips the polymers with strong
antimetastatic activity, while preserving the ability to form
polyplexes with nucleic acids. We have reported synthesis of
both degradable and nondegradable poly(amido amine)s.35,36,39
In the original design, the cationic cyclam moieties provided
both the CXCR4 inhibition and nucleic acid binding, thus
shouldering the burden of providing both biological functions
and potentially compromising the overall activity. In this study,
we designed and developed a new class of CXCR4-inhibiting
polycations by conjugating our recently developed cyclam
CXCR4 antagonist to PEI, a well-studied polycation for
delivering therapeutic nucleic acids. We hypothesized that the
proposed polymer design will allow us to better combine
CXCR4 antagonism and siRNA delivery in a single molecule,
thus representing a simple way to introduce an additional
antimetastatic function to existing carrier (Scheme 1).
3.1. Synthesis of PEI-C. A series of cyclam modified PEI
(PEI-C) were synthesized by nucleophilic substitution of PEI
with the developed cyclam derivative (1) (Scheme 2). To
synthesize the Boc-protected cyclam derivative (tri-tert-butyl-
11-(4-(chloromethyl)benzyl)-1,4,8,11-tetraazacyclotetradecane-
1,4,8-tricarboxylate) we have followed our previously published
procedure.36 To prepare PEI-C, we selected branched PEI with
molecular weight 10 kDa due to its favorable safety profile.40−43
In a typical conjugation procedure, PEI was refluxed with (1) in
acetonitrile in the presence of K2CO3. The resulting product
was collected by filtration and the Boc protecting groups from
cyclam were removed by trifluoroacetic acid. The final PEI-C
product was then obtained as a hydrochloride salt by
lyophilization after dialyzing against HCl-acidified water. We
obtained typical yield around 75%. The content of cyclam in
Every third atom in the PEI chemical structure is a
protonizable amine, giving the polymer high charge density
and outstanding buffering capacity over a wide pH range. Such
high cationic charge density, however, becomes a double-edged
1
PEI-C was calculated using H NMR, by comparing the signal
of the protons on the phenylene ring (δ 6.7−7.6) in (1) to the
signal of the PEI ethylene groups (δ 2.1−2.9) (Figure 1). Three
C
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a
Scheme 2. Synthesis of PEI-C
a
Could be any primary, secondary, or tertiary amine.
Figure 1. Typical 1H NMR spectrum of PEI-C (PEI-C37 in D2O) used in the determination of the cyclam content. Integration of the proton on the
aromatic ring (a and a′) and on the unconjugated ethylenimine (b,c) were used for the calculations of the conjugation ratio.
sword. The high protonation degree of PEI at physiological pH
provides high binding and complexation with negatively
charged nucleic acids due to electrostatic interactions but also
leads to nonspecific interactions and perturbations of lipid
bilayers in cell membranes, thus causing toxicity.45−47 Cyclam,
on the other hand, has a complex and unusual protonation
behavior as suggested by the pKa of the four amines: 11.29,
10.19, 1.61 and 1.91.48 Only two out of the four amines can be
protonated at physiological pH. After cyclam modification, the
fraction of protonated amines in PEI-C thus decreases
significantly, resulting in decreased overall cationic charge
density of the polymer. Such reduction in charge density also
becomes more pronounced when the cyclam content is
Table 1. Characterization Summary of PEI-C
cyclam content
(mol %)
a
polymer conjugation ratio (mol %) in feed in polymer Mw (kDa)
PEI
0
22
33
37
0
30
40
80
0
18
25
27
10.0
20.8
24.1
25.0
PEI-C22
PEI-C33
PEI-C37
a
Conjugation ratio (mol %) is defined as the ratio of conjugated
ethylenimines to unconjugated ethylenimines based on 1H-NMR.
D
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Figure 2. Cytotoxicity of PEI-C in U2OS, 4T1, and L929 cells. Cells were incubated with increasing concentrations of polymers for 24 h before
measuring cell viability by MTT assay. IC50 values were calculated.
Figure 3. CXCR4 antagonism of PEI-C. U2OS overexpressing EGFP-CXCR4 cells were treated with PEI-C for 30 min before incubation with 10
nM SDF-1. AMD3100 (300 nM) was used as a positive control.
increased. The specific binding of cyclam moiety and cell
surface CXCR4 receptors might also alter the cellular uptake
pathways and contribute to the reduced toxicity.
CXCR4/SDF-1 axis using CXCR4 antagonists has been
reported to significantly abrogate cell migration and reduce
tumor metastasis in breast cancer.16,49,51 After we confirmed
the CXCR4 antagonistic activity of PEI-C, the efficiency to
inhibit CXCR4/SDF-1 axis modulated cancer cell migration
was further evaluated using 4T1 mouse breast cancer cells. A
Boyden chamber cell migration setting was applied and SDF-1
(20 nM) was used as the chemoattractant. As shown in Figure
4, AMD3100 exhibited marked inhibition of cell migration
(75%). All the PEI-C (2 μg/mL) achieved enhanced inhibitory
activity (88−92% cell migration inhibition) when compared
with AMD3100. PEI-C polyplexes (w/w 3) showed slightly
improved cell migration inhibition (91−93%) compared with
PEI-C polymer due to significant amount of free polycation.
PEI (2 μg/mL) showed minimal inhibition of cell migration
(∼25%), which might be related to nonspecific interactions
between PEI and cell membrane receptors involved in cell
migration.
3.5. Preparation and Characterization of the Poly-
plexes. The ability of PEI-C to complex siRNA was
determined by agarose gel electrophoresis. The polyplexes
were prepared at increasing PEI-C to siRNA w/w ratios. As
shown in Figure 5A, unmodified PEI could fully complex the
siRNA at w/w ratios as low as 1. All the PEI-C polymers
exhibited complete siRNA complexation at w/w 1.5, while
leaving traces of free siRNA at w/w 1. This again could be
3.3. CXCR4 Antagonism. After the safe dosing range of
PEI-C was determined, we investigated the CXCR4 antago-
nistic ability of the polymers using a phenotypic CXCR4
receptor redistribution assay.35 This assay can be applied to
track and visualize the translocation of EGFP-tagged CXCR4
receptors on the cell membrane to endosomes upon ligand
(SDF-1) stimulation, which is a typical behavior for G-protein
coupled receptors. As shown in Figure 3, untreated cells
exhibited CXCR4 translocation as indicated by the enhanced
green fluorescent signals inside the cells with minimal visible
signal on the plasma membrane. Cells that were treated with
CXCR4 antagonist AMD3100 exhibited a diffused pattern of
green fluorescence, indicating the inhibition of CXCR4
translocation after SDF-1 stimulation. All the PEI-C (2 μg/
mL) demonstrated strong CXCR4 inhibition when compared
with unmodified PEI. PEI-C37 with the highest cyclam content
appears to show the best CXCR4 inhibition. Our results
confirmed that PEI-C function as CXCR4 antagonists after
nondegradable conjugation of the cyclam moiety onto the PEI
polymer.36,39
3.4. Cell Invasion. Binding of SDF-1 and CXCR4 initiates
downstream signaling that leads to intracellular calcium flux,
chemotaxis, cell survival, and proliferation.49−51 Blocking the
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polyplexes were evaluated by dynamic light scattering at w/w
3, 6, and 9 (Figure 5B,C). The sizes of all the PEI-C/siRNA
polyplexes were around 100 nm in diameter (ranging from 62
to 114 nm) with positive surface charge (ranging from 25 to 53
mV). No substantial differences in either size or surface charge
were observed between PEI-C and PEI.
3.6. Cellular Uptake. Small molecule CXCR4 antagonists
like AMD3100 exert their biological activity by competitively
binding with the CXCR4 at the plasma membrane to prevent
them from internalization and triggering intracellular signaling
cascade. However, for polymeric CXCR4 antagonists to achieve
dual functions as both CXCR4 inhibitors and efficient delivery
vectors of nucleic acids, the polyplexes are required to be
internalized inside the cells. Here we used fluorescently labeled
siRNA (FAM-siRNA, green) to monitor the cell uptake of the
PEI-C/siRNA polyplexes. The 4T1 cells were treated with PEI-
C/FAM-siRNA prepared at w/w 3 for 2 h before visualization
by confocal microscopy. Cell nuclei were stained with DAPI
(blue). As shown in Figure 6A, a significant amount of green
fluorescent signal was observed in the cytoplasm for all the
polyplexes compared with naked FAM-siRNA. No notable
differences were observed between the PEI-C and PEI
polyplexes, which confirmed the delivery function of PEI was
preserved after cyclam modification with no compromise in the
cellular uptake of siRNA.
We then determined the endosomal escape of PEI-C/FAM-
siRNA using confocal microscopy. The 4T1 cells were
incubated with the polyplexes for 2 and 6 h and lysosomes
were stained with LysoTrackerRed. After incubation for 2 h, the
green fluorescence of FAM-siRNA overlaid with the red
LysoTracker signal, suggesting that most of the endocytosed
FAM-siRNA was localized in the lysosomes. After 6 h, notable
separation between green fluorescence of FAM-siRNA and the
red signal of LysoTracker Red was clearly noted, indicating that
FAM-siRNA had successfully escaped from the endosomes.
3.7. Transfection and VEGF Silencing in Vitro. Cyclam
modification of PEI has shown no negative effect on cellular
uptake of siRNA. Only marginal differences were observed in
Figure 4. Inhibition of cancer cell invasion. (A) 4T1 cells were treated
with PEI-C (200 nM), PEI-C polyplexes (w/w 3), and allowed to
invade through Matrigel for 24 h. AMD3100 (300 nM) was used as a
positive control and PEI was used as a negative control. (B) The
percent of invaded 4T1 cells. Results shown as mean percent of
invaded cells using untreated cells as 100% SD (n = 3). (**p < 0.01,
***p < 0.001 vs AMD3100).
attributed to the unique protonation constants of the cyclams.
Hydrodynamic sizes and zeta potential of PEI-C/siRNA
Figure 5. Physicochemical characterization of PEI-C/siRNA polyplexes. (A) siRNA complexation by agarose gel electrophoresis. (B) Hydrodynamic
size and (C) zeta potential of polyplexes at various w/w ratios. Data shown as mean SD (n = 3).
F
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demonstrated robust VEGF silencing in 4T1 cells, giving 75%
knockdown of VEGF protein expression when compared with
negative control PEI-C37/siScr. These results confirmed
efficient siVEGF delivery in vitro and provided support for
testing in vivo anticancer activity.
3.8. Anticancer Activity in Vivo. The anticancer and
antimetastatic activity of PEI-C37/siVEGF polyplexes was
evaluated using orthotopic metastatic breast cancer mouse
model. A total of 105 4T1 cells were injected subcutaneously
into the mammary fat pad of female Balb/C mice and allowed
to form palpable tumors. Tumor size was monitored closely
until reaching 100 mm3. All the tumor-bearing mice were then
randomized into three different groups (n = 5) and
intratumorally administered with (i) saline, (ii) PEI-C37/
siScr, and (iii) PEI-C37/siVEGF. As shown in Figure 8A, no
significant body weight loss was observed throughout the
treatment, indicating minimal toxicity of the polyplexes. PEI-
C37/siVEGF exhibited marked decrease in tumor growth
compared with the saline group (Figure 8B). Treatment with
PEI-C37/siScr also slowed down the primary tumor growth,
which could be attributed to the CXCR4 antagonism of PEI-
C37 as inhibition of CXCR4 regulates cell growth and
proliferation. At the concluding day of the experiment, primary
tumors (Figure 8C) and lungs (Figure 8D) were harvested. As
shown in Figure 8D, compared with the saline-treated group,
which showed extensive metastasis in the lung, no significant
metastasis was observed in the animals treated with either the
PEI-C37/siScr or PEI-C37/siVEGF. H&E staining of the lung
sections was further performed (Figure 8F) and the number of
lung metastatic lesions was counted (Figure 8E) to validate the
antimetastatic activity of PEI-C37. To investigate the in vivo
siRNA delivery efficiency by PEI-C37, tumor tissue slides were
stained with CD31, an endothelial cell-specific surface marker
to detect disorganized vascular endothelium and angiogenesis
in the tumor. Tumors treated with the PEI-C37/siVEGF
polyplexes showed marked reduction in tumor vasculature
compared with saline and PEI-C37/siScr groups, indicating
significant inhibition of tumor angiogenesis. These results
provide important in vivo evidence that PEI modified with
cyclam-based CXCR4 antagonists is capable of simultaneously
delivering therapeutic siVEGF and inhibiting CXCR4-regulated
tumor growth and metastasis, thus achieving beneficial
combinational anticancer activity.
Figure 6. (A) Cellular uptake of polyplexes in 4T1 cells by confocal
microscopy after 2 h of incubation using FAM-siRNA (green). The
nuclei are stained with DAPI (blue). (B) Endosomal escape of PEI-C
polyplexes (w/w 3) in 4T1 cells for 2 and 6 h incubation. Lysosomes
were stained with Lysotracker Red.
the CXCR4 antagonism and cell migration inhibition among all
the PEI-C polymers. Therefore, we selected PEI-C37, which
exhibited the lowest cytotoxicity to further investigate siRNA
transfection and VEGF silencing effect in vitro. The 4T1 cells
were transfected with PEI-C37/siVEGF and PEI-C37/siScr
polyplexes at w/w 3 (final siRNA concentration 100 nM), and
VEGF expression was analyzed by Western blot 48 h post-
transfection. As shown in Figure 7, PEI-C37/siVEGF
CONCLUSIONS
■
In this study, we developed a new polycationic CXCR4
antagonists based on PEI (PEI-C) that can simultaneously
deliver VEGF siRNA and inhibit CXCR4/SDF-1 axis to achieve
combination anticancer therapy in metastatic breast cancer.
Our results confirmed that conjugation of cyclam-based
CXCR4 antagonists to PEI is able to (i) retain the siRNA
delivery efficiency of PEI, (ii) decrease the cytotoxicity of PEI,
and (iii) exert CXCR4 antagonism and inhibition of CXCR4-
mediated cancer cell invasion. The PEI-C/siVEGF polyplexes
demonstrated combination antimetastatic and anticancer
activity due to the simultaneous CXCR4 inhibition and
VEGF silencing. The reported PEI modification represents a
simple approach to introducing a new pharmacologic function
to existing siRNA delivery vectors in order to achieve enhanced
delivery and therapeutic efficiency for potential antitumor and
antimetastatic applications.
Figure 7. VEGF protein silencing by PEI-C37/siVEGF polyplexes in
4T1 cells by Western blot. Quantification of the bands was performed
using ImageJ software.
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Figure 8. Anticancer activity of PEI-C37/siRNA polyplexes in 4T1 metastatic breast cancer mouse model. (A) Body weight change of the mice after
different treatments. (B) Tumor growth curves of different treatment groups. Results are shown as average of tumor volume SD (n = 5, ***p <
0.001). (C) Photograph of primary tumors harvested after sacrificing the animals at the end of the experiment. (D) Photograph of representative
lungs from each treatment group. Visible surface metastases were indicated by black arrows. (E) CD31 immunohistochemistry staining of primary
tumors and H&E staining of lungs. (F) Average number of metastases on lung tissue sections. Results are shown as average number of lung
metastases SD (n = 5, ***p < 0.001).
(3) Kim, S. H.; Jeong, J. H.; Lee, S. H.; Kim, S. W.; Park, T. G. Local
and systemic delivery of VEGF siRNA using polyelectrolyte complex
micelles for effective treatment of cancer. J. Controlled Release 2008,
129 (2), 107−116.
(4) Duda, D. G.; Kozin, S. V.; Kirkpatrick, N. D.; Xu, L.; Fukumura,
D.; Jain, R. K. CXCL12 (SDF1α)-CXCR4/CXCR7 Pathway
Inhibition: An Emerging Sensitizer for Anticancer Therapies? Clin.
Cancer Res. 2011, 17 (8), 2074−2080.
AUTHOR INFORMATION
Corresponding Author
*E-mail: david.oupicky@unmc.edu.
ORCID
■
Minjie Sun: 0000-0003-0582-6189
David Oupicky: 0000-0003-4710-861X
́
Author Contributions
§Y.Z. and F.Y. contributed equally to the manuscript.
(5) Jung, K.; Heishi, T.; Incio, J.; Huang, Y.; Beech, E. Y.; Pinter, M.;
Ho, W. W.; Kawaguchi, K.; Rahbari, N. N.; Chung, E.; Kim, J. K.;
Clark, J. W.; Willett, C. G.; Yun, S. H.; Luster, A. D.; Padera, T. P.;
Jain, R. K.; Fukumura, D. Targeting CXCR4-dependent immunosup-
pressive Ly6Clow monocytes improves antiangiogenic therapy in
colorectal cancer. Proc. Natl. Acad. Sci. U. S. A. 2017, 114 (39), 10455−
10460.
(6) Barone, A.; Sengupta, R.; Warrington, N. M.; Smith, E.; Wen, P.
Y.; Brekken, R. A.; Romagnoli, B.; Douglas, G.; Chevalier, E.; Bauer,
M. P.; et al. Combined VEGF and CXCR4 antagonism targets the
GBM stem cell population and synergistically improves survival in an
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9811−9822.
(7) Liu, C.-H.; Chan, K.-M.; Chiang, T.; Liu, J.-Y.; Chern, G.-G.;
Hsu, F.-F.; Wu, Y.-H.; Liu, Y.-C.; Chen, Y. Dual-Functional
Nanoparticles Targeting CXCR4 and Delivering Antiangiogenic
siRNA Ameliorate Liver Fibrosis. Mol. Pharmaceutics 2016, 13 (7),
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(8) Liu, J.-Y.; Chiang, T.; Liu, C.-H.; Chern, G.-G.; Lin, T.-T.; Gao,
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was financially supported by the National Science
and Technology Major Project (2017YFA0205400), Chang-
jiang Scholar program from the Chinese Ministry of Education,
University of Nebraska Medical Center, China National Science
Foundation (81373983, 81573377), China Postdoctoral
Science Foundation (No. 2016M601923), and the National
Institutes of Health (EB020308).
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