Dual-Function Polymeric HPMA Prodrugs for the Delivery of miRNA

Article abstract of DOI:10.1021/acs.molpharmaceut.6b00999

An HPMA-based polymeric prodrug of a CXCR4 antagonist, AMD3465 (P-SS-AMD), was developed as a dual-function carrier of therapeutic miRNA. P-SS-AMD was synthesized by a copolymerization of HPMA with a methacrylamide monomer in which the AMD3465 was attached via a self-immolative disulfide linker. P-SS-AMD showed effective release of the parent AMD3465 drug following treatment with intracellular levels of glutathione (GSH). The AMD3465 was released in the cells and exhibited functional CXCR4 antagonism, demonstrated by inhibition of the CXCR4-mediated cancer cell invasion. Due to its cationic character, P-SS-AMD could form polyplexes with miRNA and mediate efficient transfection of miR-200c mimics to downregulate expression of a downstream target ZEB-1 in cancer cells. The combined P-SS-AMD/miR-200c polyplexes showed improved ability to inhibit cancer cell migration when compared with individual treatments. The reported findings validate P-SS-AMD as a dual-function delivery vector that can simultaneously deliver a therapeutic miRNA and function as a polymeric prodrug of CXCR4 antagonist.

Full text of DOI:10.1021/acs.molpharmaceut.6b00999

Article
pubs.acs.org/molecularpharmaceutics
Dual-Function Polymeric HPMA Prodrugs for the Delivery of miRNA
Zheng-Hong Peng,† Ying Xie,† Yan Wang, Jing Li, and David Oupicky*
́
Center for Drug Delivery and Nanomedicine, Department of Pharmaceutical Sciences, University of Nebraska Medical Center,
Omaha, Nebraska 68198, United States
S
* Supporting Information
ABSTRACT: An HPMA-based polymeric prodrug of a CXCR4 antagonist, AMD3465 (P-SS-AMD), was developed as a dual-
function carrier of therapeutic miRNA. P-SS-AMD was synthesized by a copolymerization of HPMA with a methacrylamide
monomer in which the AMD3465 was attached via a self-immolative disulfide linker. P-SS-AMD showed effective release of the
parent AMD3465 drug following treatment with intracellular levels of glutathione (GSH). The AMD3465 was released in the
cells and exhibited functional CXCR4 antagonism, demonstrated by inhibition of the CXCR4-mediated cancer cell invasion. Due
to its cationic character, P-SS-AMD could form polyplexes with miRNA and mediate efficient transfection of miR-200c mimics to
downregulate expression of a downstream target ZEB-1 in cancer cells. The combined P-SS-AMD/miR-200c polyplexes showed
improved ability to inhibit cancer cell migration when compared with individual treatments. The reported findings validate P-SS-
AMD as a dual-function delivery vector that can simultaneously deliver a therapeutic miRNA and function as a polymeric
prodrug of CXCR4 antagonist.
KEYWORDS: polyplexes, miRNA delivery, polymeric prodrug, CXCR4 antagonist, self-immolative linker
them, including liposomes, PEI, PDMAEMA, polylysine, and
polyarginine, have also been used for miRNA delivery.13−19
The combination of therapeutic miRNAs and small molecule
drugs has emerged as a promising strategy in cancer treatment
to overcome adaptive multidrug resistance and target multiple
disease pathways.20−23 However, to achieve successful
codelivery of both therapeutics requires development of an
appropriate delivery platform. Not only must the vector be safe
and efficient, it also needs to be versatile to accommodate the
two therapeutics with distinct physicochemical characteristics.
Most of the miRNA and small molecule codelivery systems
simply encapsulate the drug via hydrophobic interactions in a
hydrophobic part of the carrier and then incorporate the
miRNA by electrostatic interactions.14,24−28 One major
disadvantage of this combination is that it is difficult to achieve
a synergistic release profile of the drug and miRNA. Another
INTRODUCTION
■
miRNAs are a large group of small noncoding RNAs of 20−24
nucleotides that regulate gene expression by suppressing
mRNA translation and reducing mRNA stability.1 An increasing
number of miRNAs have been identified as direct regulators of
cancer progression and metastasis and proposed as potential
therapeutics.2,3 There are two major approaches to utilizing
miRNA as therapeutics. First, miRNA mimics, which provide
the same sequences as the mature endogenous target miRNA,
can be delivered into cancer cells to increase the expression of
tumor-suppressive miRNAs. The second approach is to use
miRNA antagonists, which contain sequences complementary
to the target miRNA mature strand to inhibit the activity of
oncogenic miRNAs.4,5 However, miRNA faces the same
delivery challenges as other nucleic acids, which are unable to
easily penetrate cell membranes due to their polar character and
negative charge. Synthetic carriers such as cationic liposomes
and polycations, including poly(ethylenimine) (PEI), poly(2-
dimethylaminoethyl methacrylate) (PDMAEMA), guanidi-
nium-rich polymers, and cyclodextrin-based polycations, have
been developed for siRNA and DNA delivery.6−12 Some of
Special Issue: Bioconjugate Therapeutics
Received: November 3, 2016
Revised: December 30, 2016
Accepted: January 14, 2017
© XXXX American Chemical Society
A
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disadvantage is that most of the polycations are nondegradable
and produce unwanted systemic toxicities.29
Scheme 1. Proposed Mechanism of Action of P-SS-AMD/
miR-200c Polyplexes
Self-immolative spacers represent a class of linkers that can
undergo spontaneous and irreversible intramolecular reaction
upon stimulation.30 They have attracted interest in the
development of small-molecule and polymeric prodrugs
especially in situations where the linker is sterically inaccessible
or the rates of the linker cleavage and drug release are too
slow.31,32 Disulfide-based self-immolative linkers are among the
most attractive linkers for developing polymers suitable for
delivering drugs and imaging agents because they can be
cleaved by the thiol−disulfide reaction in the intracellular
reducing environment.33−35 So far, there are no reports on the
use of self-immolative disulfide spacers in miRNA delivery.
Existing evidence highlights the critical role of chemokine
receptor CXCR4 and its ligand CXCL12 in cancer metastasis.36
CXCR4 is associated with more than 23 types of human
cancer.37 CXCR4 inhibition by small molecule antagonists
based on cyclam, such as plerixafor (AMD3100), can
successfully block the intracellular signaling cascades that lead
to cancer cell migration and invasion.38 Monocyclam
AMD3465 is another promising CXCR4 antagonist in clinical
trials, with a 22-fold higher potency than AMD3100.39,40 The
protonated multiple amines in AMD3465 at physiological pH
are also capable of interacting with nucleic acids, which makes it
a suitable building block to design a polycationic prodrug to
deliver drug and nucleic acid combinations to treat metastatic
cancers.
anhydrous Na2SO4. The solvent was then removed under
reduced pressure, and the residue was purified by flash
chromatography on silica gel to obtain pure product 2 (4.46
g, 71.4%). H NMR (500 MHz, CDCl3, δ): 1.45 (s, 27H),
1.64−1.78 (m, 2H), 1.82−1.98 (m, 2H), 2.60−2.64 (m, 2H),
2.78−2.84 (m, 2H), 3.22−3.50 (m, 12H).
Here we report a novel polymeric AMD3465 prodrug named
P-SS-AMD that not only delivers therapeutic nucleic acids, but
also inhibits the CXCR4/CXCL12 chemokine axis. CXCR4
antagonist AMD3465 was attached to a hydrophilic and
nontoxic N-(2-hydroxypropyl) methacrylamide (HPMA)41
copolymer via a self-immolative disulfide linker. P-SS-AMD
was expected to function dually to deliver therapeutic miR-200c
mimic and to release the parent drug AMD3465 following
internalization into cancer cells to achieve combination cancer
therapy (Scheme 1).
1
Synthesis of Tri-tert-butyl 11-(4-(Chloromethyl)benzyl)-
1,4,8,11-tetraazacyclotetradecane-1,4,8-tricarboxylate (3).
To a solution of 2,2′-dichloro-p-xylene (875.3 mg, 5 mmol)
in acetonitrile (12.5 mL) was added anhydrous K2CO3 (172.8
mg, 1.25 mmol). A solution of compound 2 (500.7 mg, 1
mmol) in acetonitrile (10 mL) was added slowly into the above
mixture. After stirring at reflux temperature for 6 h, the solution
was filtered and concentrated. The residue was dissolved in
DCM and purified by flash chromatography to obtain product
MATERIALS AND METHODS
■
Materials. Azobis(isobutyronitrile) (AIBN), L-glutathione
(GSH), 2,2′-dichloro-p-xylene, 2-picolylamine, 2,2′-dithiodie-
thanol, methacryloyl chloride, triphosgene, and ethylenedi-
amine tetraacetic acid (EDTA) were purchased from Sigma-
Aldrich. HPMA was purchased from Amadis Chemical
(Hangzhou, China). Human CXCL12 was from Shenandoah
Biotechnology, Inc. (Warwick, PA). miR-200c mimic (mature
miRNA sequence: 5′-UAAUACUGCCGGGUAAUGAUGGA-
3′) and negative control miR-NC (mature miRNA sequence:
5′-UCACAACCUCCUAGAAAGAGUAGA-3′) were pur-
chased from GE Dharmacon (Lafayette, CO). Oligofectamine
(OligoFT) was from ThermoFisher Scientific. All the other
regents were purchased from Fisher Scientific unless specifically
noted.
1
3 (530.1 mg, 83%). H NMR (500 MHz, CD2Cl2, δ): 1.41 (s,
18H), 1.45 (s, 9H), 1.67 (m, 2H), 1.87 (m, 2H), 2.38 (m, 2H),
2.59 (m, 2H), 3.20−3.38 (m, 12H), 3.53 (s, 2H), 4.59 (s, 2H),
7.28−7.29 (d, J = 5 Hz, 2H), 7.32−7.33 (d, J = 5 Hz, 2H).
Synthesis of Tri-tert-butyl 11-(4-(((Pyridin-2-ylmethyl)-
amino)methyl)benzyl)-1,4,8,11-tetraazacyclotetradecane-
1,4,8-tricarboxylate (4). Anhydrous K2CO3 (207.3 mg, 1.5
mmol), acetonitrile (5 mL), compound 3 (639.3 mg, 1 mmol),
and 2-picolylamine (1.08 g, 10 mmol) were added successively
into a round-bottom flask and stirred at room temperature
overnight. Then the solid was filtered and the filtrate was
concentrated. The remaining residue was dissolved in DCM,
and the solution was washed with water and brine and dried
with anhydrous Na2SO4. After concentration, the crude product
was purified by flash chromatography and pure product 4
Synthesis of MA-SS-AMD Monomer. Synthesis of Tri-
tert-butyl-1,4,8,11-tetraazacyclotetradecane-1,4,8-tricarbox-
ylate (2). Cyclam (1) (2.5 g, 12.5 mmol) and dichloromethane
(DCM) (500 mL) were added to a round-bottom flask and
cooled to 0 °C. A solution of Boc2O (8.18 g, 37.5 mmol) in
DCM (162.5 mL) was added slowly into the above solution,
and the reaction mixture was allowed to warm to room
temperature and stirred overnight. The mixture was then
concentrated, washed with water and brine, and dried with
1
(579.4 mg, 81.5%) was obtained. H NMR (500 MHz, d6-
DMSO, δ): 1.30 (s, 18H), 1.40 (s, 9H), 1.60 (m, 2H), 1.81 (m,
2H), 2.30 (m, 2H), 2.52 (m, 2H), 3.16−3.25 (m, 12H), 3.48 (s,
2H), 3.71 (s, 2H), 3.78 (s, 2H) 7.20−7.22 (d, J = 10 Hz, 2H),
7.24 (d, 1H), 7.26−7.28 (d, J = 10 Hz, 2H), 7.44 (d, 1H),
7.73−7.76 (d, 1H), 8.5 (d, 1H).
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Synthesis of 2-((2-Hydroxyethyl)disulfanyl)ethyl Methacry-
late (5). 2,2′-dithiodiethanol (6.16 g, 40 mmol), anhydrous
tetrahydrofuran (THF) (200 mL), and triethylamine (TEA)
(6.07 g, 60 mmol) were added to a 1 L round-bottom flask and
cooled to 0 °C. Then methacryloyl chloride (4.18 g, 40 mmol)
in 100 mL of anhydrous THF was added dropwise into the
above solution. The reaction mixture was warmed to room
temperature and stirred overnight. The precipitation was
filtered, and the collected solution was concentrated. The
residue was dissolved in ethyl acetate, washed with water and
brine, and dried over anhydrous Na2SO4. The crude product
was purified by silica gel column chromatography using ethyl
acetate/hexane as the eluent. Pure product 6 (5.62 g) was
was increased from 5% buffer B to 95% buffer B (buffer A, 0.1%
TFA in water; buffer B, 0.1% TFA in acetonitrile) in 20 min,
and the flow rate was 1 mL/min. The experiment was repeated
three times. AMD3465 was used as the reference.
Gel Retardation Assay. Polyplexes were prepared by
adding a predetermined volume of P-SS-AMD or AMD3465 to
a miRNA solution (20 μM in 10 mM HEPES pH 7.4) to
achieve the desired w/w ratio and vigorously vortexed for 10 s.
Polyplexes were then incubated at room temperature for 30
min before further use. Some polyplexes were also incubated
with 10 mM GSH for 24 h. Each sample (7.5 μL) was then
mixed with 1.5 μL of a loading buffer (Ambion, USA) before
loading to 2% agarose gel. The gel was developed at 120 V for
30 min in a running buffer (40 mM Tris-HCl, 1 v/v% acetic
acid, 1 mM EDTA) containing 0.5 μg/mL ethidium bromide.
The miR-200c bands were detected using an EL LOGIC100
imaging system (Kodak, USA).
1
obtained with a yield of 63.2%. H NMR (500 MHz, CDCl3,
δ): 1.95 (s, 3H), 2.88−2.90 (t, J = 5 Hz, 2H), 2.97−2.99 (t, J =
5 Hz, 2H), 3.89−3.92 (t, J = 5 Hz, 2H), 4.41−4.43 (t, J = 5 Hz,
2H), 5.60 (s, 1H), 6.14 (s, 1H).
Synthesis of Tri-tert-butyl 11-(4-(13-Methyl-3,12-dioxo-2-
(pyridin-2-ylmethyl)-4,11-dioxa-7,8-dithia-2-azatetradec-13-
en-1-yl)benzyl)-1,4,8,11-tetraazacyclotetradecane-1,4,8-tri-
carboxylate (MA-SS-AMD, 6). Compound 5 (855.2 mg, 3.85
mmol), triphosgene (380.5 mg, 1.28 mmol), and TEA (537
μL) in DCM (10 mL) were stirred at −65 °C for 2 h. Then, a
solution of compound 4 (2.735 g, 3.85 mmol) and TEA (537
μL) in DCM (10 mL) was added and stirred overnight. The
mixture was washed with water and brine and dried with
anhydrous Na2SO4. After concentration under reduced
pressure, the crude product was purified by flash chromatog-
raphy (DCM/MeOH) to give the pure product 7 (2.36 g,
64%). The calculated mass of [M + H]+ was 959.5, and the
Polyplex Size and Surface Charge. The average
hydrodynamic diameter and surface charge of polyplexes were
determined by dynamic light scattering (Zetasizer Nano Series,
Malvern). miR-200c mimic (4 μL, 100 μM) was mixed with P-
SS-AMD at different amine-to-phosphate (N/P) ratios in
HEPES buffer (10 mM, pH 7.4) for 30 min and then diluted to
1 mL with the same HEPES buffer. Some polyplexes were
incubated with 10 mM GSH for 24 h. All measurements were
conducted in automatic mode at room temperature at 173°
scattering angle.
Cell Culture. Human osteosarcoma cells (U2OS) stably
expressing human CXCR4 receptor fused to the N-terminus of
enhanced green fluorescent protein (EGFP) were purchased
from Fisher Scientific and cultured in Dulbecco’s modified
Eagle’s medium high glucose medium supplemented with 10%
fetal bovine serum (FBS), 2 mM L-glutamine, 1% Pen-Strep,
and 500 μg/mL G418 in an incubator at 37 °C and 5% CO2.
Cytotoxicity. In vitro cytotoxicity of P-SS-AMD in U2OS
cells was examined using the CellTiter-Blue cell viability assay
(Promega). The cells were plated in 96-well microplates at a
density of 5000 cells/well and treated with P-SS-AMD diluted
in culturing medium for 24 h. The medium was then removed
and replaced with a mixture of 100 μL of serum-free medium
and 20 μL of CellTiter-Blue reagent. After 2 h of incubation,
the fluorescence intensity [I] was measured using Spectra-
MaxM5e Multi-Mode microplate reader (Molecular Devices,
CA) at 560Ex/590Em. Relative cell viability (%) was calculated as
[I]treated/[I]untreated × 100%. To confirm that the polyplex
formulation used in transfection and cell migration is within the
safe dosing range, cells were plated as described before and
treated with PBS, OligoFT/miR-NC, OligoFT/miR-200c, P-
SS-AMD/miR-200c, or P-SS-AMD/miR-200c. After 48 h of
incubation, the medium was removed and cell viability was
measured as described before.
1
found mass was 959.2. H NMR (500 MHz, CD2Cl2, δ): 1.41
(s, 18H), 1.44 (s, 9H), 1.62−1.68 (m, 2H), 1.82−1.99 (m, 2H),
1.91 (s, 3H), 2.3−2.4 (m, 2H), 2.52−2.62 (m, 2H), 2.85−3.02
(m, 4H), 3.18−3.38 (m, 12H), 3.52 (s, 2H), 4.30−4.44 (m,
4H), 4.46−4.54 (m, 4H), 5.56 (s, 1H), 6.08 (s, 1H), 7.15−7.25
(m, 6H), 7.62−7.66 (dd, 1H), 8.50−8.51 (d, 1H).
Synthesis and Characterization of P-SS-AMD. HPMA
(103.6 mg, 0.72 mmol), MA-SS-AMD (6) (347 mg, 0.36
mmol), AIBN (11.3 mg), and DMF (2 mL) were added into a
5 mL ampule. After bubbling with nitrogen for 30 min, the
ampule was sealed and the mixture was heated at 50 °C for 24
h. The polymer was precipitated into cold diethyl ether,
centrifuged, and dried in air for 4 h. The dried polymers were
mixed with trifluoroacetic acid (10 mL) and stirred at room
temperature for 3 h. After trifluoroacetic acid was removed
under reduced pressure, the remaining polymer solution was
precipitated by dropwise addition into cold diethyl ether. The
polymer was isolated by filtration, dried in vacuum, and then
dissolved in water and dialyzed against 1 M HCl followed by
final lyophilization. The molecular weights and molecular
weight distribution of the polymers were determined by size-
exclusion chromatography (TSK-GEL PWXL column, Toshoh
Bioscience) equipped with a miniDAWN TREOS multi-angle
static light scattering detector (Wyatt) and Optilab T-rEX
differential refractive index detector (Wyatt) using 0.5 M
sodium acetate buffer (pH 5) as the mobile phase.
CXCR4 Antagonism. The U2OS cells were seeded at a
density of 8,000 cells per well 24 h prior to treatment. Cells
were washed twice with PBS and incubated in HEPES-buffered
DMEM medium containing 1% FBS with AMD3465 and P-SS-
AMD (with or without 24 h of GSH pretreatment) for 30 min
before exposure to CXCL12 (10 nM) for another 1 h. The cells
were then fixed with 4% paraformaldehyde and washed four
times with PBS, and nuclei were stained with 1 μM Hoechst
33258 solution for 30 min before imaging. Images were taken
by EVOS FL microscope at 20× magnification. High-content
analysis was applied to quantify the CXCR4 antagonistic
activity based on the internalization of the EGFP-CXCR4
Drug Release Kinetics. P-SS-AMD (2.5 mg) was dissolved
in 1.5 mL of phosphate buffer (100 mM potassium phosphate,
1 mM EDTA, pH 7.4) and incubated with or without 10 mM
GSH at 37 °C. The released AMD3465 in the solution was
measured at 254 nm on an analytical HPLC column (Eclipse
plus C18, 5 μm, 4.6 × 150 mm) every 30 min during the first
24 h and every 12 h in the following 36 h. The elute gradient
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Scheme 2. Synthesis of P-SS-AMD Polymeric Prodrugs
receptors from plasma membrane into the cells. Cellomics
Arrayscan VTI fluorescent microscope imager (Thermo Fisher)
was used for the imaging and analysis.42 Untreated cells
stimulated with CXCL12 were used as negative control (0%
CXCR4 antagonism), and cells treated with 300 nM AMD3100
were used as positive control (100% CXCR4 antagonism).
Cellular Uptake. U2OS cells were seeded in 6-well plates at
a density of 500,000 cells per well and cultured for 24 h prior to
treatment. The cells were incubated with polyplexes prepared at
different N/P ratios using P-SS-AMD and AlexaFluor555
labeled double-stranded oligonucleotides Alexa Red-Oligo
(BLOCK-iT Alexa Fluor Red Fluorescent Control, catalog
number 14750100, ThermoFisher Scientific) in serum-free
medium for 4 h. The cells were then detached from the plate
after being incubated with TrypLE (ThermoFisher Scientific)
for 3 min and collected for flow cytometry analysis using BD
FACSCalibur. Data were processed and analyzed using FlowJo
software V7.6.1.
miRNA Transfection. U2OS cells were seeded 24 h prior
to transfection to reach logarithmic growth phase. On the day
of transfection, cells were incubated with polyplexes prepared
with either miR-200c or miR-NC at various N/P ratios in
serum-free medium. After 4 h incubation, polyplexes were
completely removed and replaced with culture medium with
10% FBS for 48 h prior to RT-PCR analysis. Total RNA was
extracted from the transfected U2OS cells using mirVana
miRNA isolation kit (Ambion, USA) according to the
manufacturer’s protocol. The quantification of mature miR-
200c using TaqManMicroRNA assay included a two-step RT-
PCR. First, 10 ng of RNA from each sample was converted to
cDNA using TaqMan microRNA reverse transcription cDNA
synthesis kit (Applied Biosystems, California) with specific
primers for miR-200c or internal reference Z30 (Applied
Biosystems, California) according to the manufacturer’s
protocol. Second, the PCR amplification of target cDNA was
performed on Rotor-Gene Q qPCR instrument (QIAGEN)
with specific primers for miR-200c or Z30 using AmpliTaq
Gold enzymes. The expression of miR-200c level was
normalized to internal miRNA reference Z30 using comparative
CT method.
Western Blot. The ZEB1 levels in U2OS cells were
quantified with Western blot. After transfection with the miR-
200c or miR-NC polyplexes for 48 h, the cells were washed
twice with PBS and lysed in radioimmunoprecipitation assay
buffer (RIPA) buffer containing 1× protease inhibitor cocktail
(Thermo Scientific) for 10 min on ice. The mixture was
centrifuged, and the supernatant was collected. After measuring
the protein concentration with bicinchoninic acid protein assay
(Pierce), the total protein samples (20 μg) were separated on a
10% SDS−PAGE gel run at 120 V for 2 h. Then the proteins
were transferred to the nitrocellulose membrane at a constant
current of 300 mA for 2 h. After confirming the transfer
efficiency and blocking the membranes with 5% nonfat milk,
the membrane was probed by human ZEB1 mAb (Cell
Signaling Technology, USA) followed by incubation with
secondary rabbit IgG HRP-linked antibody (Cell Signaling
Technology, USA). After the membrane was incubated with
Pierce ECL Western blot detection reagent (Thermo Scientific,
USA) for 1 min, the film was developed on the medical film
processor (Konica Minolta Medical & Graphic Inc.). Finally,
the film was scanned and the intensity of the protein bands was
quantified by ImageJ software (NIH).
Cell Migration Assay. U20S cells (2 × 105) were seeded
into 6-well plates and cultured in complete DMEM for 24 h
before transfection. The cells were subsequently treated with
PBS, OligoFT/miR-NC, Oligofectamine/miR-200c, P-SS-
AMD/miR-NC, and P-SS-AMD/miR-200c. After 48 h
incubation, the cells were trypsinized and suspended in medium
without serum. Subsequently, 3 × 104 cells were seeded in the
top chambers in 300 μL of serum-free medium and 500 μL of
complete medium containing 10% FBS was added to the lower
Transwell chambers. After 12 h, the nonmigrated cells in the
top chamber were removed with a cotton swab. The migrated
cells were fixed in 100% methanol and stained with 0.2%
Crystal Violet solution for 10 min at room temperature. The
images were taken by EVOS XL microscope. Three 20× visual
fields were randomly selected for each insert, and each group
was conducted in triplicate.
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RESULTS AND DISCUSSION
■
Synthesis of Polymeric Prodrug Based on AMD3465
(P-SS-AMD). The key procedure to synthesize the polymeric
prodrug P-SS-AMD with the self-immolative linker was to
develop the reactive monomer MA-SS-AMD (Scheme 2). We
designed a concise synthetic scheme that protects the amines
on the cyclam ring because of their critical function in binding
with the CXCR4 receptor.39 Three of the four cyclam (1)
amines were protected with di-tert-butyl dicarbonate to give 2
with 71% yield.43 The protected cyclam (2) was reacted with
α,α′-dichloro-p-xylene to afford the benzyl chloride (3) at 83%
yield. The benzyl chloride in 3 was subsequently substituted
with 2-picolylamine to prepare Boc-protected AMD3465 (4) in
82% yield. Methacryloylation of 2-hydroxyethyl disulfide
afforded the corresponding alcohol (5),34 which was then
reacted with triphosgene and 4 to give the desired monomer
1
MA-SS-AMD (6). The monomer was analyzed by H NMR
(Figure S1) and mass spectroscopy. The calculated mass of the
MA-SS-AMD monomer (6) ([M + H]+) was 959.5, and the
found mass was 959.2.
Figure 1. (A) Proposed release mechanism and (B) cumulative release
The presence of the disulfide bond in the monomer MA-SS-
AMD has the potential to interfere with reversible addition−
fragmentation transfer polymerization due to the thiol−
disulfide exchange.44 Thus, we have used conventional free
radical polymerization to prepare the polymer prodrug P-SS-
AMD. As shown in Scheme 2, P-SS-AMD was synthesized by a
two-step reaction. First, MA-SS-AMD was copolymerized with
HPMA in DMF at 50 °C for 24 h. Following isolation of the
polymer by precipitation in diethyl ether, the Boc protecting
groups were removed from the cyclams by trifluoroacetic acid.
The final polymer was dialyzed against water acidified with 1 M
HCl to remove residual monomers and other low molecular
weight impurities. Acidified water was used to enhance
solubility of the monomers during dialysis and to convert the
final polymer into hydrochloride salt. The molecular weight and
molecular weight distribution of the synthesized copolymer
were determined by size exclusion chromatography. The
number-average molecular weight (Mn) was 27.8 kDa, and
the polydispersity index (PDI, Mw/Mn) was 2.2. The calculated
mol % of the MA-SS-AMD comonomer in the polymer was
31%, which was close to the 33% in the feed. The weight
content of AMD3465 in the P-SS-AMD was 22.1%, which was
of AMD3465 from P-SS-AMD with 10 mM GSH.
1B) shows that over 90% of AMD3465 was released from the
polymer upon incubation with GSH within 24 h. In contrast, in
the absence of GSH there was less than 1% of the AMD3465
released even after 60 h.
Cytotoxicity. Before evaluating miRNA delivery and
biological activity of P-SS-AMD, we tested the cytotoxicity to
determine the safe concentration range for subsequent studies
in U2OS cells (Figure S5). The cells were treated with
increasing concentrations of P-SS-AMD in culture medium
before measuring cell viability, and the estimated IC50 for P-
SS-AMD was 82 μg/mL. Safe concentrations of P-SS-AMD
were selected and applied in all the subsequent cell-based
experiments.
CXCR4 Antagonism. We have shown in Figure 1 that the
parent drug AMD3465 can be successfully released from P-SS-
AMD. It was then important to test if the intracellularly
released drug retains its CXCR4 antagonism. We examined the
CXCR4 antagonism using U2OS cells that are stably expressing
CXCR4 receptor tagged with EGFP. High-content analysis was
applied to quantify the antagonism activity and calculate the
EC50 as described in our previous publications.42 Control small
molecule CXCR4 antagonist AMD3100 (300 nM) was used as
a positive control and set as 100%. To comprehensively assess
the CXCR4 activity of the polymeric prodrug, we constructed
dose−response curves and determined EC50 values for
AMD3465 and P-SS-AMD (with and without GSH) (Figure
2A). AMD3465 exhibited EC50 of 0.3 nM, indicating high
potency in CXCR4 inhibition. As expected, P-SS-AMD
demonstrated lower activity in CXCR4 inhibition with an
EC50 of 54.9 nM (expressed as molar concentration of
AMD3465 units in the polymer). When P-SS-AMD was
pretreated with GSH before incubation with the cells, however,
the EC50 was decreased to 5 nM, confirming enhanced potency
for CXCR4 antagonism when exposing the receptor directly
from the extracellular space. We then assessed CXCR4
inhibition at a concentration expected to be used in the
subsequent miRNA delivery experiments. As shown in Figure
2B, AMD3465 exhibited complete CXCR4 inhibition at 300
nM concentration. Even though P-SS-AMD showed a some-
what lower inhibitory activity when tested at the equivalent
1
calculated based on the H NMR data (Figure S2).
AMD3465 Release from P-SS-AMD. We have selected the
disulfide-based self-immolative linker because of the specific
cellular location of its activation. Disulfide bonds are
preferentially cleaved by GSH in the reducing intracellular
environment typically found in the cytoplasm (Scheme 1). The
cytoplasmic location of the linker cleavage is ideally suited for
delivery and release of therapeutic miRNA because cytoplasm is
the site of miRNA activity. The mechanism of GSH-triggered
drug release is shown in Figure 1A. Reaction with GSH thiolate
initiates a release cascade of the parent drug AMD3465 from P-
SS-AMD via thiolactone formation and subsequent amide bond
cleavage. The kinetics of the linker cleavage was measured by
incubating the polymer with 10 mM GSH at 37 °C to mimic
the elevated intracellular GSH levels in cancer cells. The drug
release was monitored by an analytical HPLC system. The
HPLC profile of polymer P-SS-AMD is shown in Figure S3A. A
new peak appeared at 5.36 min in Figure S3B, which was
confirmed by mass spectrometry to be the released parent
product AMD3465 (Figure S4). The release kinetics (Figure
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Figure 3. Physicochemical properties of P-SS-AMD/miR-200c
polyplexes. (A) Gel retardation assay of polyplexes prepared at
different N/P ratios of miR-200c mimic and P-SS-AMD or free
AMD3465 without and with 10 mM GSH. (B) Heparin- and GSH-
induced miRNA release from P-SS-AMD/miRNA (N/P 10).
Polyplexes were incubated with increasing concentrations of heparin
either with or without 20 mM GSH for 30 min. (C) Hydrodynamic
diameters and PDI of P-SS-AMD/miR-200c polyplexes (N/P 10). (D)
Zeta potential of P-SS-AMD/miR-200c polyplexes (N/P 10).
Figure 2. CXCR4 antagonism of P-SS-AMD by CXCR4 redistribution
assay. (A) Dose−response curve of P-SS-AMD3465 ( GSH) and
AMD3465. (B) % CXCR4 antagonism at 300 nM AMD3465 equiv.
AMD3465 concentration, it still achieved nearly complete
CXCR4 inhibition (85%). When P-SS-AMD was pretreated
with GSH before incubation, the antagonistic activity further
increased to ∼95%. It is worth noting that despite the decrease
of activity in the polymer, P-SS-AMD still exhibited CXCR4
inhibition within nM range, which falls well below the doses to
be used in the miRNA delivery. These findings suggest that
AMD3465 released from P-SS-AMD in the intracellular space is
likely released from the cells and binds cell surface CXCR4 or
binds the CXCR4 receptor as it recycles through the cell. When
combined with our previously published findings, the results
with GSH pretreatment also point to the possibility that P-SS-
AMD exhibits CXCR4 inhibition both as AMD3465 prodrug
and as a polymeric drug (i.e., P-SS-AMD itself binds and
inhibits CXCR4).42,45
Reversible Complexation of miRNA with P-SS-AMD.
After confirming that P-SS-AMD retained the CXCR4 activity
of AMD3465, we set to evaluate the ability of the polymer to
condense miRNA. Condensation of nucleic acids by poly-
cations is the first requirement for their successful use as
delivery vectors. The ability of P-SS-AMD to complex miR-
200c into polyplexes was first evaluated by agarose gel
retardation assay. Polyplexes were prepared by adding P-SS-
AMD to miRNA solution at increasing N/P ratios as shown in
Figure 3A. The migration of miR-200c in the gel was
completely retarded at N/P ratios greater than 5. Control
small molecule AMD3465 failed to complex the miRNA even
at N/P 10. The reversibility of the complexation was assessed
by the addition of 10 mM GSH to the polyplexes (Figure 3A).
After 24 h incubation in the reducing GSH conditions, the
polyplexes prepared at low N/P ratios (N/P ≤ 3) disassembled
because of the cleavage of the self-immolative linker and free
miRNA was released completely. Polyplexes prepared at higher
N/P ratios displayed only partial miRNA release, indicating
incomplete disulfide cleavage in P-SS-AMD/miRNA poly-
plexes. Because of the partial release, we performed the release
study also upon exposure to higher concentration GSH (20
mM) and heparin to assist in fully disassembling the polyplexes.
As shown in Figure 3B, in the absence of GSH, the polyplexes
were stable and released no miRNA until the heparin
concentration exceeded 80 μg/mL. However, when incubated
with 20 mM GSH, rapid disulfide reduction destabilized the
polyplexes, which resulted in the partial release of miRNA even
without the addition of heparin and complete release with the
addition of heparin as low as 40 μg/mL. These results
confirmed that P-SS-AMD can form polyplexes with miRNA
and that the miRNA can be released from the polyplexes after
the disulfide bonds between AMD3465 and the HPMA
backbone were cleaved by GSH.
The hydrodynamic diameters of P-SS-AMD/miR-200c
polyplexes prepared at N/P = 1, 5, and 10 were 116.6 2.4,
139.4
19.9, and 218.2
2.7 nm, respectively, with
polydispersity indices of 0.203, 0.220, and 0.179, respectively
(Figure 3C). Particle size is a key parameter that determines the
in vivo fate of polyplexes. We know from previous studies that
nanoparticles with sizes less than 200 nm are regarded as
suitable for tumor delivery. Thus, the size of the P-SS-AMD
polyplexes is expected to be suitable for miRNA delivery in
vivo, although other parameters, like zeta potential, will also
influence the efficacy of the miRNA delivery. The zeta potential
of polyplexes increased from −11.1 1.6 to 2.4 0.3 and to
15.8 0.6 (mV) when the N/P ratio of P-SS-AMD to miR-
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200c increased from 1 to 10 (Figure 3D). These results further
confirmed that the P-SS-AMD can form nanosized polyplexes
with miRNA.
Cellular Uptake of miRNA Polyplexes. Polyplexes with
positive surface charge can facilitate cellular uptake of a wide
range of nucleic acids. The ability of P-SS-AMD polyplexes to
deliver nucleic acids in U2OS cells was evaluated by flow
cytometry and confocal microscopy using double-stranded
RNA oligomer Alexa Red-Oligo labeled with AlexaFluor555.
We have used the double-stranded RNA oligomer as a model of
miRNA because of its commercial availability and similar
structure and molecular weight with the miR-200c mimic.
Polyplexes were prepared at different N/P ratios up to 10,
established as safe in the cytotoxicity studies. As shown in
Figure 4, cells treated with P-SS-AMD polyplexes exhibited
Figure 5. Transfection efficiency of P-SS-AMD/miR-200c polyplexes
in U2OS cells. (A) Cells were transfected with miR-200c mimics using
free AMD3465 and polymer P-SS-AMD at N/P 10. (B) Cells were
transfected with different N/P ratios of P-SS-AMD polyplexes with
miR-200c or miR-NC. Data are shown as mean SD (n = 3).
prepared at N/P 10, P-SS-AMD exhibited a more than 21,000-
fold increase in the miR-200c levels over background cellular
expression, while the AMD3465/miR-200c treatment had no
significant effect. In addition, as shown in Figure 5B, increasing
the N/P ratios of P-SS-AMD/miR-200c polyplexes resulted in
significant enhancement in miR-200c levels, which was
consistent with the cell uptake results above. Treatment with
P-SS-AMD/miR-NC polyplexes had no effect on miR-200c
expression.
After confirming that P-SS-AMD can effectively deliver
miRNA, we further evaluated the effect of the delivered miR-
200c on one of its important downstream targets, ZEB1.
Western blot was used to analyze the cellular levels of ZEB1
after treatment with P-SS-AMD/miR-200c polyplexes. As
shown in Figure 6, delivery of miR-200c with P-SS-AMD
polyplexes at N/P 10 resulted in a substantial downregulation
(53%) of ZEB1 in the U2OS osteosarcoma cells. In contrast,
control miR-NC polyplexes showed negligible effect on ZEB1
protein expression. At lower N/P ratio (N/P 5), the extent of
ZEB1 downregulation by P-SS-AMD/miR-200c polyplexes was
less pronounced as only 28% downregulation was observed.
Figure 4. Cellular uptake of polyplexes. (A) Fraction of cells that
internalized the polyplexes and (B) mean fluorescent intensity (MFI)
per cell. U2OS cells were treated with polyplexes prepared with
different N/P ratios of P-SS-AMD and Alexa Red-Oligo. Data are
presented as mean SD (n = 3).
significant cell uptake, indicated by both enhanced fraction of
fluorescent cells (Figure 4A) and mean fluorescent intensity
(MFI) per cell (Figure 4B). The use of polyplexes prepared at
higher N/P ratios resulted in higher cell uptake. At the highest
N/P tested, nearly all cells (>97.5%) showed internalization of
the polyplexes. In contrast, small molecule AMD3465 failed to
facilitate any appreciable cell uptake of the fluorescently labeled
oligonucleotide.
miRNA Transfection. Transfection efficiency of P-SS-
AMD/miR-200c polyplexes was evaluated by measuring the
levels of miR-200c in U2OS cells using RT-PCR (Figure 5).
Control polyplexes prepared with negative control miRNA
(miR-NC) were also included to exclude any nonspecific effects
of miRNA and the polymer on miR-200c expression. miR-200c
mimics were selected as the therapeutic miRNA to test delivery
efficiency of P-SS-AMD as miR-200c was found to be
significantly downregulated in metastatic osteosarcoma tissue
specimens when compared to normal bone samples.46 The
downregulation of miR-200c resulted in the high expression of
Zinc-finger E-box-binding 1 (ZEB1), which is a transcription
repressor factor that has been identified as one of the powerful
inducers of epithelial−mesenchymal transition and cancer
metastasis.47,48 Studies have demonstrated that successful
delivery of miR-200c mimics can lead to downregulation of
ZEB1 followed by inhibition of cancer migration and
invasion.45,49,50 First, our results showed that cells transfected
with P-SS-AMD/miR-200c polyplexes exhibited highly elevated
miR-200c levels when compared with cells treated with a
mixture of AMD3465 and miR-200c (Figure 5A). When
Figure 6. Effect of P-SS-AMD/miR-200c polyplexes on ZEB1 protein
expression. The cells were transfected with polyplexes prepared at N/P
5 and 10 and ZEB1 levels quantified by Western blot (one-way
ANOVA, *p < 0.05; **p < 0.01).
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This result further confirmed that miR-200c could be efficiently
delivered by the P-SS-AMD polyplexes into the U2OS cells and
released successfully into the cytoplasm to regulate the miRNA
downstream signaling pathways.
prepared with excess polycation to fully condense the nucleic
acids. As a result, polyplex formulations are a mixture of the
actual polyplexes and excess free polymer. Based on our
previous studies and findings in Figure 2, we propose that both
the polyplex and free polymer P-SS-AMD antagonize the
CXCR4 receptor. There is also the possibility that a small
amount of the polymer is dissociated from the polyplexes upon
binding to the cell surface and that partial GSH-mediated
reduction occurs in the extracelullar space as well. Further, to
rule out the effect of cytotoxicity on the observed cell migration
data, we examined the cytotoxicity of all the formulations
tested, and no cytotoxicity was observed 48 h post-transfection
(Figure S6).
Effect of Combined CXCR4 Inhibition and miR-200c
Delivery on Cancer Cell Migration. Both CXCR4 and
downstream miR200c targets are involved in cancer metastasis.
Thus, we conducted Transwell cell migration assay to examine
whether the successful delivery of miR-200c using the dual-
functional P-SS-AMD and its CXCR4 antagonistic activity can
inhibit the migration of U2OS cells. Cells were treated with
either P-SS-AMD/miR-200c polyplexes or commercial trans-
fection agent Oligofectamine (OligoFT) for 48 h before
conducting Transwell cell migration assay. As shown in Figure
7, FBS induced a significant amount of cell migration.
CONCLUSION
■
We have developed a novel dual-function polymeric prodrug
using self-immolative linker chemistry and CXCR4 antagonist
AMD3465. The dual-function polymers could effectively inhibit
the CXCR4 chemokine receptor and degrade into safe HPMA
copolymers. Due to their polycationic character, the polymeric
prodrug could form nanosized polyplexes with miRNA and
effectively deliver the nucleic acid to cancer cells. The
developed delivery methodology may provide a new direction
for developing approaches for drug−nucleic acid combination
therapies.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.molpharma-
ceut.6b00999.
1H NMR, HPLC, MS, and cytotoxicity results (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail: david.oupicky@unmc.edu.
■
ORCID
Zheng-Hong Peng: 0000-0001-9783-5108
David Oupicky: 0000-0003-4710-861X
́
Author Contributions
†Z.-H.P. and Y.X. contributed equally.
Figure 7. Inhibition of cancer cell migration by P-SS-AMD/miR-200c
polyplexes. U2OS cells were transfected with either miR-200c or miR-
NC polyplexes and allowed to migrate through porous membrane for
12 h toward 10% FBS. (A) Representative images of invaded cells at
20× magnification. (B) Average number of invaded cells per 20×
imaging view (n = 3, ANOVA, **p < 0.01, ***p < 0.001, ****p <
0.0001).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported in part by the University of Nebraska
Medical Center and in part by grants from the National
Institutes of Health (EB015216, EB020308, EB019175).
Support from the China Scholarship Council for Ying Xie is
gratefully acknowledged.
OligoFT/miR-200c showed about 42% inhibition of migration,
while OligoFT/mi-NC showed no significant effect, confirming
the antimetastatic potential of miR-200c. In contrast, P-SS-
AMD/miR-NC polyplexes exhibited 56% inhibition of cell
migration due to the CXCR4 antagonistic activity of the
polymer prodrug. The combined P-SS-AMD/miR-200c poly-
plexes further enhanced the migration inhibition to 77%.
The observed inhibitory effect could be attributed to the
combined activity of the CXCR4 antagonism of P-SS-AMD and
the effect of miR-200c on ZEB1, which plays a critical role in
inhibiting cancer cell migration. For the mechanism of action of
the polyplex formulations in this study, we propose that the
polyplex and free polymer P-SS-AMD both contribute to
CXCR4 antagonism (Scheme 1). Typical polyplexes are
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DOI: 10.1021/acs.molpharmaceut.6b00999
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