Block ionomer complexes consisting of siRNA and aRAFT-synthesized hydrophilic-block-cationic copolymers: The influence of cationic block length on gene suppression

Abstract

Block ionomer complex (BIC) dissociation and the subsequent effects on gene “knockdown” are reported. Aqueous reversible addition-fragmentation chain transfer (aRAFT) polymerization was utilized to prepare a statistical macro chain transfer agent (macroCTA) consisting of N-(2-hydroxypropyl)methacrylamide (HPMA) and N-(3-aminopropyl)methacrylamide (APMA); HPMA confers water stability, and APMA provides a facile pathway for the conjugation of folic acid, a cellular targeting moiety. This macroCTA was chain extended with N,N-(3-dimethlyaminopropyl)methacrylamide (DMAPMA), thus preparing hydrophilic-block¬-cationic copolymers with varying repeat units of DMAPMA. After end-group removal and folic acid conjugation, these hydrophilic-block-cationic copolymers were complexed with small interfering RNA (siRNA) and GLuc DNA, a double stranded DNA (dsDNA) analogue of siRNA. These complexes were prepared at a nitrogen-to-phosphate ratio (N:P) = 1, and complexes prepared with siRNA and GLuc DNA were demonstrated to be comparable; the hydrodynamic radii (Rh) and changes to the secondary structure were identical, while ζ-potential and gel electrophoresis confirmed complex neutrality. Analytical ultracentrifugation (AUC) was utilized to ascertain binding constants and stoichiometry. Increased DMAPMA block length (cationic length) caused an increase in the binding constant, but the stoichiometry remained constant at 1:1. Solution differential scanning calorimetry (DSC) was conducted to investigate the BIC stability. Similar to AUC, the melting temperature (Tm) increased with increasing cationic block length, and overall, a shift in Tm of ~ 40 °C was observed, indicating that increasing the DMAPMA length confers greater BIC stability. Furthermore, complex dissociation was not observed. Gene down-regulation was monitored in KB cells expressing Gaussia Luciferase, and the time for maximum gene knockdown to occur increased with increasing DMAPMA block length. Given the large binding constants and increased stability with increased BICs, it can be concluded that in vitro complex dissociation occurs via an ion exchange mechanism.

Full text

Polymer Chemistry c4py00940a
1
Block ionomer complexes consisting of siRNA and
aRAFT-synthesized hydrophilic-block-cationic
copolymers: the influence of cationic block length
on gene suppression
Andrew C. Holley, Keith H. Parsons, Wenming Wan,
Daniel F. Lyons, G. Reid Bishop, John J. Correia,
Faqing Huang and Charles L. McCormick*
Mechanisms of BIC dissociation are presented for neutral
BICs comprised of oligonucleotides and well-defined
hydrophilic-block-cationic copolymers.
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PAPER
Cite this: DOI: 10.1039/c4py00940a
Received 8th July 2014,
Accepted 28th August 2014
DOI: 10.1039/c4py00940a
www.rsc.org/polymers
Block ionomer complexes consisting of siRNA and
aRAFT-synthesized hydrophilic-block-cationic
copolymers: the influence of cationic block length
on gene suppression
Q1 †
Andrew C. Holley,
a
Keith H. Parsons,
a
Wenming Wan,
a
Daniel F. Lyons,
b
G. Reid Bishop,
c
John J. Correia,
b
Faqing Huang
d
and Charles L. McCormickQ2 *
a,d
Block ionomer complex (BIC) dissociation and the subsequent effects on gene “knockdown”are reported.
Aqueous reversible addition–fragmentation chain transfer (aRAFT) polymerization was utilized to prepare
a statistical macro chain transfer agent (macroCTA) consisting of N-(2-hydroxypropyl)methacrylamide
(HPMA) and N-(3-aminopropyl)methacrylamide (APMA); HPMA confers water stability, and APMA provides
a facile pathway for the conjugation of folic acid, a cellular targeting moiety. This macroCTA was chain
extended with N,N-(3-dimethylaminopropyl)methacrylamide (DMAPMA), thus preparing hydrophilic-
block-cationic copolymers with varying repeat units of DMAPMA. After end-group removal and folic acid
conjugation, these hydrophilic-block-cationic copolymers were complexed with small interfering RNA
(siRNA) and GLuc DNA, a double stranded DNA (dsDNA) analogue of siRNA. These complexes were pre-
pared at a nitrogen-to-phosphate ratio (N : P) = 1, and complexes prepared with siRNA and GLuc DNA
were demonstrated to be comparable; the hydrodynamic radii (R
h
) and changes to the secondary struc-
ture were identical, while ζ-potential and gel electrophoresis confirmed complex neutrality. Analytical
ultracentrifugation (AUC) was utilized to ascertain binding constants and stoichiometry. Increased
DMAPMA block length (cationic length) caused an increase in the binding constant, but the stoichiometry
remained constant at 1 : 1. Solution differential scanning calorimetry (DSC) was conducted to investigate
the BIC stability. Similar to AUC, the melting temperature (T
m
) increased with increasing cationic block
length, and overall, a shift in T
m
of ∼40 °C was observed, indicating that increasing the DMAPMA length
confers greater BIC stability. Furthermore, complex dissociation was not observed. Gene down-regulation
was monitored in KB cells expressing Gaussia Luciferase, and the time for maximum gene knockdown to
occur increased with increasing DMAPMA block length. Given the large binding constants and increased
stability with increased BICs, it can be concluded that in vitro complex dissociation occurs via an ion
exchange mechanism.
Introduction
The discovery of siRNA,
1
the effector molecule in the RNA
interference (RNAi) pathway in 1998, has resulted in extensive
interventive therapy research giving the potential to regulate
nearly any gene of interest.
2–4
However, in vitro delivery of
siRNA alone is limited because of enzymatic degradation and
lack of cell specificity. As a result, numerous approaches to
more efficient delivery have been investigated.
5–7
One promis-
ing approach capitalizes on the formation of interpolyelectro-
lyte complexes (IPECs) between the anionic groups along the
phosphodiester backbone and a hydrophilic cationic
polymer.
8,9
These IPECs stabilize the RNA from nucleases;
10
however, the molar ratio of cationic to phosphodiester repeat-
ing units should be ≈1. Nitrogen-to-phosphate (N : P) values
†Electronic supplementary information (ESI) available: Determination of folic
acid content by
1
H NMR and UV/Vis, determination of D
m
and the autocorrela-
tion functions, polymer and oligonucleotide/complex scan rate dependence,
heating and cooling DSC scans of polymers and polymer/oligonucleotide com-
plexes and plots of polymer cytotoxicity. See DOI: 10.1039/c4py00940a
a
Department of Polymer Science and Engineering, The University of Southern
Mississippi, Hattiesburg, MS 39406, USA
b
Department of Biochemistry, The University of Mississippi Medical Center, Jackson,
MS 39211, USA
c
Department of Chemistry and Biochemistry, Belhaven University, Jackson,
MS 39202, USA
d
Department of Chemistry and Biochemistry, The University of Southern Mississippi,
Hattiesburg, MS 39406, USA. E-mail: charles.mccormick@usm.edu
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greater than one lead to non-specific transfection and those
less than one to reduced cellular uptake. Recent advances in
controlled polymerization techniques including RAFT,
11–13
aRAFT,
14,15
ATRP,
16
and ROMP
17
now allow the formation of
well-defined cationic block copolymers in which block length
and stoichiometry along the IPEC can be “tuned”. These com-
plexes are often referred to as block ionomer complexes (BICs)
and possess a balance of non-complexing hydrophilic segment
(s) and binding segments. A potential advantage of BICs is the
reduction of off-target effects and toxicity.
18
The formation of IPECs has been extensively studied and in
general, three parameters constitute complex formation;
19,20
hydrogen bonding, hydrophobic effects, and charge. Poly-
(ethylene glycol) (PEG) has been shown to form stable com-
plexes with proteins at low pH and up to pH 8, and such com-
plexes are formed strictly through hydrogen bonding.
21,22
Hydrophobic interactions are more difficult to ascertain, and
the effects of hydrophobicity are generally manifested in a
cooperative fashion.
23,24
Xia et al. demonstrated enhanced
binding to lysozyme by introducing a pyrene label to poly(2-
acrylamido methylpropylsulfate).
25
The enhanced binding
resulted from interactions between the probe and lysozyme’s
hydrophobic cleft. Interestingly, molecular dynamics simu-
lations have demonstrated oligonucleotide strand separation
when complexed with weakly interacting charge associations
in conjunction with hydrophobic moieties such as gold nano-
particles.
26
IPECs based on charge are most common and are
routinely employed for nucleotide delivery. IPECs prepared via
electrostatic associations consistently maintain a 1 : 1 stoichio-
metry, and deviations from 1: 1 binding result from drastic
changes in architecture (e.g. branching). Furthermore, regard-
less of a strong or weak polyelectrolyte, polyion exchange and
substitution readily occur when a small molecular salt is
present.
27,28
Ammonium-based polymers (e.g. PEI,
29,30
DMAPMA,
10,31,32
DMAEMA
33–35
) are typically utilized for the
cationic block, but polymers possessing phosphonium groups
have also been reported.
36–40
To promote efficacious delivery, polymeric vehicles incor-
porate multiple modes of interaction. Reineke and co-
workers
41,42
investigated IPEC formation between plasmid
DNA (pDNA) and poly(glycoamidoamines) (PGAAs). It was
determined that while long-range charge-charge interactions
drive IPEC assembly, hydrogen bonding plays a vital role in
stabilizing the complexes. Furthermore, the less dense amine-
containing polymers have stronger electrostatic associations
with pDNA.
Our research group has focused on the development of con-
trolled, tailored (co)polymers afforded via RAFT polymeriz-
ation, and more specifically, the synthesis of these constructs
directly in aqueous media. Recent efforts have centred on a
rational design for preparing polymeric drug/gene delivery
systems, thus allowing construction of responsive
micelles,
43–45
theranostics,
32
peptide mimics,
46
modular co-
polymers,
47,48
and vehicles for endosomal escape.
49
We have
previously demonstrated targeted delivery of siRNA via BICs as
well as with disulfide-bound amphiphilic copolymers.
31,50
In
our experience, we have noted a block-length dependence for
efficacious siRNA delivery.
10
Although mechanisms for IPEC
and BIC formation have been proposed, information regarding
complex dissociation and the role of block length on IPEC
stability is lacking. To our knowledge, this is the first study
directed toward elucidating the nature of the cationic block
length with regard to gene suppression utilizing well-defined
block copolymers which form stable, monodisperse BICs with
oligonucleotides. specifically when maintaining N : P =
1. Herein, we report the synthesis of hydrophilic-block-cationic
copolymers prepared by aRAFT, and through the use of a com-
bination of circular dichroism (CD), analytical ultracentrifuga-
tion (AUC), and solution differential scanning calorimetry
(DSC), structural and binding effects on gene suppression with
regard to cationic block length (DMAPMA) were ascertained.
Increasing DMAPMA length promotes complex stability and
increases the binding constant. Due to large binding con-
stants, the thermodynamics of BIC dissociation could not be
determined; however, the kinetics of gene suppression are
indicative of an ion exchange/substitution mechanism, provid-
ing evidence for a pathway for siRNA and/or oligonucleotide
release in vitro.
Experimental
Materials
All reagents were purchased from Sigma and used as received
unless otherwise noted. 4,4′-Azobiscyanovaleric acid (V-501)
was purchased from Wako and was recrystallized twice from
methanol. Azobisisobutyronitrile (AIBN) was recrystallized
from methanol. N-(3-Aminopropyl)methacrylamide hydrochlo-
ride (APMA) was purchased from Polysciences. N,N-(3-
Dimethylaminopropyl)methacrylamide (DMAPMA) and tri-
ethylamine (TEA) were distilled prior to use. 4-Cyanopentanoic
acid dithiobenzoate (CTP), di-N-hydroxy succinimide activated
folic acid (diNHS-FA), and N-(2-hydroxypropyl)methacrylamide
(HPMA) were prepared according to literature pro-
cedures.
31,51,52
HPLC purified oligonucleotides (siRNA against
Gaussia Luciferase and the dsDNA analogue of siRNA) were
purchased from Integrated DNA Technologies, Inc. The siRNA
sequences targeting Guassia Luciferase are as follows: Sense
strand 5′-AGAUGUGCAACUUUUGCUACCGCAUCU-3′and the
antisense strand 5′-AGGAGAUGCGGUAGCAAAAGUUGCACA
UCUUU-3′. The DNA analogue sequences of siRNA are as
follows: Sense strand 5′-AGATGTGCAATTTTGCTACCGCATCT-3′
and the antisense strand 5′-AGGAGATGCGGTAGCAAAAGTT
GCACATCTTT-3′. Oligonucleotides (siRNA and dsDNA) were
heated at 95 °C for 10 min and were allowed to slowly cool to
room temperature prior to use. Concentrations of oligonucleo-
tide (siRNA and dsDNA) are reported as duplex concentrations
unless otherwise noted. The Biolux® Gaussia Luciferase assay
kit used for the determination of gene suppression was pur-
chased from New England Biolabs, Inc. Gibco® RPMI 1640
cell culture media (with and without folic acid) were purchased
from Life Technologies Corporation. KB cells (human epider-
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mal cancer cells) expressing the Gaussia Luciferase gene
(KB-GLuc) were prepared as previously reported.
32
For reac-
tions requiring nitrogen, ultrahigh purity nitrogen (purity
≥99.998%) was used. Spectra/Por® regenerated cellulose dia-
lysis membranes (Spectrum Laboratories, Inc) with a molecular
weight cut-offof 12–14 kDa were used for dialysis, and the dia-
lysis conditions were maintained for 3 days at 4 °C, pH 4.
Polymer synthesis
Synthesis of poly(HPMA-stat-APMA) macroCTA (P1). The
macro chain transfer agent (CTA) was prepared employing
V-501 as the primary radical source and CTP as the chain
transfer agent at 70 °C. HPMA (6.80 g, 47.5 mmol) and APMA
(405 mg, 2.28 mmol) were added to a 100 mL round-bottomed
flask, dissolved in 1 M acetate buffer (pH 5.2, 0.27 M acetic
acid and 0.73 M sodium acetate), and diluted to a final volume
of 50 mL. ([M]
o
= 1 M). The initial feed composition was
95 mol% HPMA and 5 mol% APMA. The round-bottomed
flask was septum-sealed and purged with nitrogen for 1 h
prior to polymerization. The HPMA-stat-APMA macroCTA was
prepared with a [M]
o
/[CTA] ratio = 800/1, while the [CTA]/[I]
ratio was kept at 5/1; the reaction was allowed to proceed for
3.5 h. The polymerization was quenched by rapid cooling in
liquid nitrogen followed by exposure to air. The macroCTA was
isolated by dialysis (pH 3–4) at 4 °C and recovered by lyophili-
zation with a yield of 93%.
Synthesis of poly[(HPMA-stat-APMA)-block-DMAPMA] co-
polymers (P2–P4). The poly(HPMA-stat-APMA) macroCTA was
chain extended with DMAPMA also using V-501 as the radical
source at 70 °C. DMAPMA and poly(HPMA-stat-APMA) were
added to a round-bottomed flask, dissolved in acetate buffer to
give [M]
o
= 1 M. The round-bottomed flask was septum-sealed
and subsequently purged with nitrogen for 1 h. Block copoly-
mers were prepared with a [M]
o
/[CTA] = 200, while the [CTA]/[I]
was kept at 5/1. Each polymerization was terminated at prede-
termined time intervals by rapid cooling in liquid nitrogen
and subsequent exposure to air. The poly[(HPMA-stat-APMA)-
block-DMAPMA] (P2–P4) copolymers were purified by dialysis
(pH 3–4, 4 °C) and recovered by lyophilization with yields of
92–97%.
Since thiocarbonylthio moieties have been demonstrated to
be cytotoxic,
53
these end-groups were removed from poly-
[(HPMA-stat-APMA)-block-DMAPMA] (P2–P4) copolymers fol-
lowing a standard literature procedure.
54
A typical reaction is
as follows: poly[(HPMA
171
-stat-APMA
13
)-block-DMAPMA
27
](P2)
(500 mg, 11.8 μmol) was added to a 25 mL round-bottomed
flask and dissolved with 6.0 mL of DMF. Azobisisobutyronitrile
(AIBN; 49.1 mg, 0.300 mmol) was then added to the flask
giving an AIBN/copolymer ratio of 25 : 1. The following solu-
tion was then septum-sealed and purged with nitrogen for 1 h
and allowed to react at 70 °C for 4 h. The resulting copolymer
was precipitated from DMF in cold, anhydrous diethyl ether
and washed repeatedly. This step was repeated three times,
and the copolymer was dried in vacuo overnight. The recovered
yields were 85–89%.
Copolymer functionalization with folic acid. DiNHS-FA was
prepared following a standard literature procedure.
31
Follow-
ing isolation of the chain terminated block copolymer, the
pendent, functional, primary amine groups from the incorpor-
ated APMA were labelled with NHS-activated folic acid. A
typical reaction is as follows: 50 mg (1.18 μmol) of poly-
[(HPMA
171
-stat-APMA
13
)-block-DMAPMA
27
](P2) copolymer was
dissolved in 390 μL of DMSO to give a final concentration of
3.00 mM. A total of 150 times excess of diNHS-FA (116 mg) was
then dissolved in 350 μL of DMSO, and P2 copolymer solution
was added to the diNHS-FA solution at a rate of 20 μL every
20 min. Triethylamine (TEA) was added to a final concen-
tration of 39 mM to serve as a catalyst. The resulting solution
was shielded from light and allowed to react for 48 h at room
temperature. Following reaction, excess ammonium hydroxide
(100% by volume) was added to quench the remaining acti-
vated esters from activated folic acid. Quenching was carried
out for 24 h. The resulting reaction mixture was then directly
placed in dialysis tubing and was first dialyzed against 0.6 M
NaCl solution for 24 h followed by dialysis against DI water for
3 days. The folate-conjugated poly[(HPMA-stat-APMA)-block-
DMAPMA] copolymers were recovered by lyophilization with
yields of 96–98%.
Formation of hydrophilic-block-cationic/oligonucleotide
complexes
Preparation of copolymer/GLuc DNA complexes for solution
differential scanning calorimetry. Folic acid labelled poly-
[(HPMA-stat-APMA)-block-DMAPMA]/GLuc DNA complexes
were prepared with a N: P ratio = 1 (i.e. neutral complexes).
The GLuc DNA duplex concentration was maintained at 75 μM
for all complexes. A typical preparation is as follows: 128 μLof
a 600 μM GLuc DNA stock solution was combined with
417.7 μL of sodium cacadylate buffer (10 mM NaAs). Next,
454.3 μL of a 370.4 μM poly[(HPMA
171
-stat-APMA
13
)-block-
DMAPMA
27
](P2) stock solution was added. The GLuc DNA/
copolymer complex solution was vortexed and equilibrated for
30 min. After equilibration, the solution was degassed for
30 min prior to DSC measurements. The DNA and polymer
stock solutions were prepared in 10 mM sodium cacadylate
buffer at pH 7.2.
Preparation of copolymer/siRNA complexes for gene sup-
pression. Folic acid labelled poly[(HPMA-stat-APMA)-block-
DMAPMA]/siRNA complexes were prepared with a N : P ratio =
1; the siRNA concentration was maintained at 100 nM. A
typical preparation is as follows: 1.30 μL of a 185.2 μM poly-
[(HPMA
171
-stat-APMA
13
)-block-DMAPMA
27
](P2) stock solution
was combined with 3.30 μLofa20μM siRNA stock solution.
This copolymer/siRNA complex solution was then diluted with
127.4 μL of RPMI 1640 medium containing no FBS. The solu-
tion was mixed by vortexing and equilibrated for 30 min. After
equilibrating, the solution was further diluted with 528 μLof
RPMI 1640 medium (supplemented with FBS) and vortexed to
ensure a homogenous solution. The RNA and polymer stock
solutions were prepared in 10 mM phosphate buffer at pH 7.4.
Polymer Chemistry Paper
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Gene suppression of Gaussia Luciferase in KB cells
Cell culture. KB-GLuc cells were maintained and prolifer-
ated in RPMI 1640 (with folic acid) medium supplemented
with 10% fetal bovine serum (FBS), 100 units mL
−1
penicillin,
and 100 μgmL
−1
streptomycin at 37 °C in 95% air humidified
atmosphere and 5% CO
2
.
Gene suppression. Twenty four hours prior to treatment,
the KB-GLuc cell medium was replaced with RPMI
1640 medium containing no folic acid and was supplemented
with 10% FBS. After this, the cells (12 000 cells per well) were
seeded in a 48 well plate (Corning Inc.). Cells were treated with
200 μL of a hydrophilic-block-cationic copolymer/siRNA
complex solution. Dharmafect was utilized as the positive
control, and the preparation of Dharmafect/siRNA complexes
followed the manufacturer protocols. The delivered siRNA con-
centration was maintained at 100 nM for all complexes, and
the KB-GLuc cells were treated for 24–48 h prior to measure-
ment. The extent of Gaussia Luciferase suppression was deter-
mined using a Biolux® Gaussia Luciferase assay kit (New
England Biolabs, Inc.). After incubation, 10 μL of medium was
transferred to a 96 well plate and combined with 10 μLof
assay buffer. The luminescence was immediately determined
utilizing a Biotek Synergy2 MultiMode Microplate Reader. All
gene suppression studies were performed in triplicate. The
passage number for KB-GLuc cells was 11.
Characterization
All polymers were characterized by aqueous size exclusion
chromatography (ASEC) using an eluent of 1 wt% acetic acid–
0.1 M Na
2
SO
4
(aq) at a flow rate of 0.250 mL min
−1
at 25 °C,
Eprogen, Inc. CATSEC columns (100, 300, and 1000 Å), a
Polymer Laboratories LC1200 UV/Vis detector, a Wyatt Optilab
DSP interferometric refractometer (λ= 690 nm), and a Wyatt
DAWN-DSP multi-angle laser light scattering (MALLS) detector
(λ= 633 nm). Absolute molecular weights and polydispersities
were calculated using the Wyatt Astra (version 4) software.
ASEC chromatograms obtained for poly(HPMA-stat-APMA) (P1)
and poly[(HPMA-stat-APMA)-block-DMAPMA] (P2–P4; before
and after chain extensions) are presented in Fig. 1. The dn/dc
measurements for poly(HPMA-stat-APMA) and (co)polymers
were performed with a Wyatt Optilab DSP interferometric
refractometer (λ= 690 nm) at 35 °C and determined using
Wyatt DNDC (version 5.90.03). Conversions for the macroCTA
and the chain extension with DMAPMA were determined by
comparing the area of the monomeric UV signal detected at
274 nm at t
0
to the area at t
x
using a Polymer Laboratories
LC1200 UV/vis detector.
Copolymer compositions were determined with a Varian
Mercury
PLUS
300 MHz spectrometer in D
2
O, and spectra were
recorded with a delay time of 2 s.
1
H NMR was used to deter-
mine the copolymer composition of poly(HPMA-stat-APMA)
and poly[(HPMA-stat-APMA)-block-DMAPMA] copolymers by
integration of the relative intensities of the methyne-proton
resonances of HPMA at 3.75 ppm and the dimethyl-proton
resonances of DMAPMA at 2.75 ppm.
Conjugation of folic acid to poly[(HPMA-stat-APMA)-block-
DMAPMA] copolymers was verified by UV/vis spectroscopy (see
ESI†).
1
H NMR was performed on a Varian Mercury
PLUS
300 MHz spectrometer in DMSO-d
6
with delay times of 2 s.
The amount of conjugated folic acid was estimated by inte-
gration of the methyne-proton resonance of HPMA at
3.75 ppm and the proton resonance of folic acid at 8.64 ppm
(s, PtC
7
H, 1H). These values were estimated by employing a
Lorentzian/Gaussian line fit using MestReNova (version 6.0.2-
5475). UV/vis spectroscopy was carried out using a Perkin-
Elmer Lambda 35 spectrophotometer for folic acid conjugated
poly[(HPMA-stat-APMA)-block-DMAPMA] copolymers. An
average extinction coefficient of 8000 M
−1
cm
−1
for free folic
acid in phosphate buffer (10 mM Pi, 100 mM NaCl, pH 7.4)
was used to determine the number of folic acid units.
Variable-angle dynamic light scattering (DLS) measurements
of hydrophilic-block-cationic copolymer/siRNA complexes under
aqueous conditions were performed using incident light of
633 nm from a Spectra Physics Model 127 He–Ne laser operat-
ing at 40 mW. The angular dependence (60°–120° in 10° incre-
ments) of the autocorrelator functions was determined with a
Brookhaven Instruments BI-200SM goniometer with an Ava-
lanche photodiode detector and TurboCorr correlator. DLS
measurements were carried out at a complex concentration
(siRNA + FA-block copolymer) of 1.0 mg mL
−1
in phosphate
buffer (10 mM Pi, pH 7.4) at 25 °C. The mutual diffusion coeffi-
cients (D
m
) were determined from the relation
Γ¼Dmq2
in which Γand q
2
represent the decay rate of the autocorrela-
tion function and the square of the scalar magnitude of the
scattering vector, respectively. The hydrodynamic radius (R
h
)
was then calculated from the Stokes–Einstein equation
DmDO¼ðkbTÞ=ð6πηRhÞ
in which ηis the solution viscosity, k
b
is Boltzmann’sconstant,
and Tis the temperature in Kelvin. Zeta-potential measure-
ments were carried out at a complex concentration of 0.5 mg
mL
−1
in 20 mM NaCl solution at pH 7.4 using a Malvern Zetasi-
zer Nano Series Instrument. Samples were vortexed to ensure a
Fig. 1 ASEC-MALLS of poly(HPMA-stat-APMA) macroCTA (P1) and sub-
sequent chain extensions with DMAPMA (P2–P4).
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homogeneous solution and equilibrated for 30 min at 25 °C
prior to measurement. To remove dust, samples were centri-
fuged at 14 000 RPM for 10 min. Both DLS and zeta-potential
measurements were performed in triplicate.
The ellipticity of the oligonucleotides as well as the
polymer/oligonucleotide complexes was determined utilizing a
Jasco J-815 circular dichroism spectropolarimeter. Oligo-
nucleotides were prepared in phosphate buffer (10 mM Pi,
pH 7.4) at a concentration of 1.0 μM. Oligonucleotide/copolymer
complexes were also prepared in phosphate buffer at an oligo-
nucleotide concentration of 1.0 μM; the copolymer concen-
tration was adjusted to maintain a N : P = 1. Spectra were
recorded with a scan rate of 10 nm min
−1
, a 0.5 nm bandwidth,
and a time constant of 2 s. The signal-to-noise was doubled for
all spectra by averaging four scans. To determine defects of the
secondary structure, the characteristic peaks of B-form oligonu-
cleotide (right-handed helices) at 280 nm (maximum), 250 nm
(minimum), and 260 nm crossover (from positive to negative)
were monitored for discrepancies after complexation.
55
All calorimetric experiments were carried out using a
Calorimetric Sciences Corporation Nano DSC-II solution differ-
ential scanning calorimeter (DSC). Sodium cacadylate buffer
(10 mM, pH 7.2) was used for the running buffer. The GLuc
DNA (analogue of Gaussia Luciferase siRNA) concentration was
maintained at 75 μM while the concentrations of poly[(HPMA-
stat-APMA)-block-DMAPMA] (P2–P4) copolymers were adjusted
to maintain a nitrogen-to-phosphate (N : P) ratio equal to 1 (i.e.
neutral complexes). CpCalc (Version 2.1, Calorimetric Sciences
Corp.) was used to subtract buffer-buffer scans from buffer-
sample scans. Linear-polynomial baselines were applied to
each scan for the determination of the molar heat capacity.
Sedimentation-velocity experiments were performed in a
Beckman XLA Analytical Ultracentrifuge at 20 °C at 50 K rpm.
Data were collected at 260 nm and processed in DCDT+ to
produce g(s) sedimentation coefficient distributions.
56
A fixed
concentration of ssDNA was titrated with increasing concen-
trations of polymer P2 or P3. Data were converted to S
w
and
analyzed according to a 1 : 1 binding model in Scientist 3.
57
Results and discussion
Synthesis of poly(HPMA-stat-APMA) macroCTA, chain
extensions with DMAPMA, and folic acid conjugation
A statistical macroCTA consisting of a theoretical feed ratio of
95 mol% HPMA and 5 mol% APMA was prepared via aqueous
RAFT polymerization (Scheme 1). The polymerization was per-
formed in 1 M acetate buffer (0.27 M acetic acid, 0.73 M
sodium acetate, pH = 5.2) at 70 °C using 4-cyanopentanoic
acid dithiobenzoate (CTP) and 4,4′-azobiscyanovaleric acid
(V-501) as the CTA and initiator, respectively. Experimentally
determined compositions for the macroCTA by
1
H NMR
closely resemble initial feed ratios which were calculated to be
93 mol% HPMA and 7 mol% APMA. The resulting poly-
(HPMA
171
-stat-APMA
13
)(P1) macroCTA was subjected to a
series of chain extensions with DMAPMA under similar con-
ditions (Scheme 1). Fig. 1 illustrates ASEC-MALLS chromato-
grams for the macroCTA and these subsequent chain
extensions indicating shifts to lower elution volume, narrow
polydispersities (PDI) (<1.10), and unimodal peaks. Copolymer
composition, copolymer molecular weights (M
n
and M
w
),
PDI, and dn/dcvalues are presented in Table 1 for P1 and
Scheme 1 Synthetic pathway for the preparation of poly[(HPMA-stat-APMA)-block-DMAPMA] copolymers and subsequent complexation with
oligonucleotides.
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poly[(HPMA-stat-APMA)-block-DMAPMA] (P2–P4) copolymers.
Copolymer compositions were determined via
1
H NMR by inte-
gration of the relative intensities of the methyne-proton reson-
ance of HPMA at 3.75 ppm to that of the dimethyl proton
resonances of DMAPMA at 2.75 ppm in D
2
O. The compositions
obtained from
1
H NMR correlate well with molecular weights
determined via ASEC-MALLS (Fig. 1).
The pendent, primary amine of APMA conveniently allows
for facile conjugation of electrophilic compounds, and in our
case, the cellular targeting moiety, folic acid (Scheme 1). Con-
jugation of folic acid was conducted in DMSO at room temp-
erature for 48 h.
1
H NMR and UV/vis spectroscopy were used to
quantify the folic acid conjugation for each hydrophilic-block-
cationic copolymer (see ESI, Fig. S2†), and the content
exceeded 92% (∼12 out of 13 amines). We are most concerned
with the delivery of neutral complexes (i.e. nitrogen-to-phos-
phate (N : P) ratio = 1) as excessive cationic charges (N : P > 1)
would encourage universal transfection as well as increase
cytotoxicity. With folic acid pendently conjugated to APMA,
allowing for targeted cellular delivery, only the tertiary amine
containing block of DMAPMA will form an electrostatic associ-
ation with the chosen oligonucleotide, while the HPMA seg-
ments promote water stability, biocompatibility, and non-
immunogenicity. We have chosen aRAFT as it allows for facile
synthesis of tailored architectures with predetermined mole-
cular weights and low PDIs under mild conditions. Our
narrowly dispersed, well-defined, folic acid-conjugated hydro-
philic-block-cationic copolymers are especially suited for for-
mation of homogeneous block ionomer complexes (BIC) with
oligonucleotides, therefore allowing for the correlation of BIC
dissociation to the extent of gene suppression in vitro.
Characterization of hydrophilic-block-cationic/oligonucleotide
complexes
We chose to use a double-stranded DNA (dsDNA) which is ana-
logous to siRNA (vide infra), so complexes formed with DNA
should resemble those prepared with RNA for comparative
purposes. Table 2 presents the hydrodynamic radius (R
h
),
ζ-potential, and PDI for GLuc/copolymer and siRNA/copolymer
complexes. Not surprisingly, both GLuc DNA and siRNA
complexes increase in size (R
h
) with increasing DMAPMA
block length (18–30 nm); interestingly, these systems become
more monodisperse with increasing size (PDIs < 0.2). Typically,
intercomplex bridging is observed for larger cationic block
lengths, and we have observed similar phenomena utilizing an
analytical ultracentrifuge. Charge neutrality was confirmed by
ζ-potential and gel electrophoresis (Fig. 2). Lanes 2–4 and 6–8
(Fig. 2) represent the BICs prepared with either siRNA or GLuc
DNA, respectively; lanes 1 and 5 serve as the siRNA and GLuc
DNA controls, respectively. As can be seen, both DNA and RNA
condensation occur while maintaining charge neutrality, an
important requirement which prevents off-target effects and
cytotoxicity. The complexes prepared with GLuc DNA or siRNA
are comparable, monodisperse, and charge-neutral.
Nucleic acids possess secondary structure alterations of
which can be monitored via circular dichroism (CD).
58,59
Typi-
cally, B-form (standard, right-handed helix) DNA possess three
CD spectral characteristics: a 280 nm maximum, a 250 nm
minimum, and a 260 nm crossover (from positive to nega-
tive).
55
However, these trends are sequence dependent. The CD
spectrum for GLuc DNA is presented in Fig. 3A (black curve).
Table 3 presents the minimum, maximum, and the crossover
for GLuc DNA, siRNA, and oligonucleotide-hydrophilic-block-
Table 1 Molecular weight (M
n
,M
w
), polydispersity (PDI), composition,
conversion (ρ), and dn/dcvalues for P1 macroCTA and P2–P4
copolymers
Sample M
n,Tha
(kDa) M
n,expb
(kDa) M
wb
(kDa) PDI
b
Comp
c
(mol%)
ρ
d
(%) dn/dc
e
P1 22.2 24.0 26.4 1.10 93 : 7 20 0.168
P2 27.7 29.3 31.4 1.07 80 : 6 : 14 14 0.167
P3 32.4 33.0 35.3 1.07 71 : 5 : 24 25 0.167
P4 41.4 41.5 44.4 1.07 59 : 5 : 36 47 0.165
a
Theoretical M
n
,(M
n,Th
), calculated from conversion (ρ) using M
n,Th
=
([M]
o
/[CTA] × M
w,monomer
×ρ+M
w,CTA
.
b
Experimental M
n
(M
n,Exp
)was
determined by aqueous SEC-MALLS.
c
As determined by
1
H NMR.
d
Conversions were determined by comparison of the UV signal at
274 nm of the monomer at t
0
to that at t
x
.
e
Determined by Wyatt
Optilab DSP interferometric refractometer (λ= 690 nm).
Table 2 The hydrodynamic radii (R
h
), polydispersity (PDI), and ζ-poten-
tial for siRNA and GLuc DNA-hydrophilic-block-cationic copolymer
complexes
Sample R
h
(nm) PDI
a
ζ-potential (mV)
siRNA N/A
b
N/A
b
−35.1
siRNA-P2 18.7 0.206 −2.16
siRNA-P3 26.3 0.058 1.35
siRNA-P4 31.7 0.046 2.38
GLuc DNA N/A
b
N/A
b
−36.2
DNA-P2 18.9 0.214 −2.36
DNA-P3 26.8 0.064 −0.099
DNA-P4 31.8 0.042 1.87
a
Determined at 90°.
b
The excess scattering compared to solvent was
too low for an accurate determination.
Fig. 2 Gel electrophoresis of siRNA (Lane 1) and GLuc DNA (Lane 5) as
well as oligonucleotide complexes prepared with P2 (Lanes 2 and 6), P3
(Lanes 3 and 7), and P4 (Lanes 4 and 8).
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cationic copolymer complexes. The GLuc DNA possesses a
272 nm maximum, a 250 nm minimum, and a 260 nm cross-
over. These spectral characteristics drastically shift when com-
plexed with hydrophilic-block-cationic copolymers, and when
complexed with P2,P3,andP4 to form BICs with GLuc DNA, a
red shift in the maximum, crossover, and minimum is
observed. Additionally, a decrease in ellipticity for the
maximum and a more negative increase in the ellipticity for the
minimum are observed; these trends exist for all polymers uti-
lized. These changes in structure are similar to the melt
(thermal denaturation) spectra (see ESI, Fig. S3†)ofGLucDNA.
Alternatively, RNA helices typically exist in the A-form (also
right-handed helix), which possesses a 270 nm maximum,
240 nm minimum, and a 250 nm crossover. Again, these
characteristics are sequence dependent. Fig. 3B depicts the
molar ellipticity of siRNA (Black curve) and siRNA/hydrophilic-
block-cationic copolymer complexes (Red, Blue, and Green
curves). The siRNA possesses a 262 nm maximum, a 235 nm
minimum, and a 253 nm crossover. These characteristics
remain when siRNA is complexed with P2,P3, and P4, except a
decrease in ellipticity for the maximum and a more negative
increase in ellipticity for the minimum are observed. The
changes to siRNA’s spectral characteristics when bound to P2,
P3, and P4 also resemble melting spectra (see ESI, Fig. S4†).
We hypothesize that, for both siRNA and GLuc DNA, complexa-
tion slightly denatures the helix to minimize charge repulsion
thus stabilizing the block ionomer complex.
Binding characteristics of hydrophilic-block-cationic/GLuc
DNA complexes as determined via analytical
ultracentrifugation and solution differential scanning
calorimetry
Analytical ultracentrifugation was conducted to determine
characteristics of complex formation. Utilizing double-
stranded DNA (dsDNA), samples were too polydisperse to accu-
rately determine the binding parameters, therefore the single
strands (sense strand and antisense strand) were utilized for
AUC (Fig. 4). P2 (Fig. 4A) and P3 (Fig. 4B) were titrated into a
fixed amount of ssDNA. From these titrations, complexation
readily occurs as evidenced by shifts to increased apparent
Fig. 3 Molar ellipticity of (A) GLuc DNA and DNA-hydrophilic-block-
cationic copolymer complexes, and (B) siRNA and siRNA-hydrophilic-
block-cationic copolymer complexes.
Table 3 The maximum, cross-over, and minimum for oligonucleotide
and oligonucleotide-hydrophilic-block-cationic copolymer complexes
Sample λ
Max
(nm) λ
Cross-over
(nm) λ
Min
(nm)
siRNA 262 253 235
siRNA-P2 263 253 234
siRNA-P3 263 253 234
siRNA-P4 266 251 233
GLuc DNA 272 260 250
DNA-P2 287 269 254
DNA-P3 293 271 253
DNA-P4 299 282 257
Fig. 4 Monitoring the association of single-stranded DNA with hydro-
philic-block-cationic copolymers. g(s*) sedimentation coefficient distri-
bution of a titration of ssDNA with P2 (A) and P3 (B). The black arrows
indicate complexation (i.e. shifts toward increased S
apparent
) when titrat-
ing siRNA with hydrophilic-block-cationic copolymers.
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sedimentation (S
Apparent
) values. Furthermore, an association
constant may be obtained via a global fit to a 1 : 1 stoichio-
metric model.
57
The binding constants related to complex for-
mation utilizing P2 and P3 were in the range of 10
4
and 10
7
,
respectively, values typical for electrostatic association.
Although complexation was observed for P2 and P3, ssDNA
titrated with P4 produced polydisperse complexes (data not
shown), and a binding constant was not determined.
DSC measurements of the excess heat capacity relative to a
reference cell allow thermodynamic determinations of dis-
sociation (folded-to-unfolded).
60
From these melting profiles,
complex stability can be elucidated. Due to the large amounts
of material required for DSC, utilizing siRNA becomes cost
prohibitive. Therefore, a dsDNA analogue of siRNA was used in
all of our DSC studies.
Fig. 5 depicts the molar heat capacity (MHC) thermograms,
for GLuc DNA and GLuc DNA/copolymer complexes formed
with P2,P3 and P4. The GLuc DNA duplex dissociation temp-
erature, or melting temperature (T
m
), was determined to be
54.4 °C for uncomplexed dsDNA (Fig. 5A). Furthermore, T
m
values increased with DMAPMA block length (P2, 88.4 °C, P3
90.2 °C, P4, 91.8 °C, respectively (Fig. 5B–D)). This trend of
increasing T
m
indicates that polymer complexation signifi-
cantly stabilizes the DNA duplex, greater cationic block length
conferring greater duplex stability. These findings are also
corroborated by the changes in secondary structure as
observed with CD (Fig. 3).
Since the magnitude of the melting endotherm, represent-
ing the breaking of hydrogen bonds between the sense and
antisense strands, may mask changes corresponding to dis-
sociation, ssDNA was separately complexed with hydrophilic-
block-cationic copolymers P2–P4. Fig. 6 presents the excess
heat capacity (ΔC
p,XS
) thermograms for copolymer complexes
prepared with the sense and antisense strands. For the
scanned temperature range (30 °C–125 °C), no dissociation is
observed (i.e. no exotherm or endotherm) regardless of hydro-
philic-block-cationic polymer utilized in complexation. The
absence of dissociation is not surprising, since the binding
constants are so large. A binding constant of 10
4
would indi-
cate 1/10 000 dissociated molecules. With the concentration
used, the dissociated species would be at or below the detec-
tion limit for the calorimeter.
Gene down-regulation
To determine the role of cationic block length on gene sup-
pression, gene down-regulation was monitored in KB cells
which stably express Gaussia Luciferase. Fig. 7 demonstrates
the kinetics of gene knockdown as a function of DMAPMA
block length. Increasing the cationic block slightly increases
gene suppression; however, increasing the cationic block also
increases the cytotoxicity (see ESI, Fig. S7†). Appreciable cell
death occurred with P4 while P2 maintained negligible cyto-
toxicity over a 48 h period. Interestingly, the observed gene
knockdown maximum is shifted toward longer times when
increasing DMAPMA block length, from 32 h for P2 to 40 h for
P4. These shifts in the maxima agree with literature reports for
polyion exchange/substitution rates, since increasing mole-
cular weight increases the time required for complete ion
exchange.
27,28,61
Regardless of the nature of a dissociation or
ion exchange/substitution mechanism, the trend is clear –an
Fig. 6 Excess heat capacity thermograms of hydrophilic-block-cationic
copolymer binding to GLuc antisense and sense strands.
Fig. 7 Gene knockdown kinetics for complexes consisting of siRNA and
P2 (blue), P3 (red), and P4 (green). Dharmafect was utilized as the posi-
tive control (Black). Error bars represent the standard deviation of tripli-
cate experiments.
Fig. 5 Molar heat capacity thermograms for (A) GLuc DNA, (B) DNA-P2
complexes, (C) DNA-P3 complexes, and (D) DNA-P4 complexes.
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increase in binding/stability and the expected increase in time
to reach maximum gene suppression (Scheme 2).
Conclusions
Aqueous RAFT was utilized to prepare poly[(HPMA-stat-APMA)-
block-DMAPMA] copolymers with controlled lengths of
DMAPMA. Well-defined BICs were prepared with siRNA and its
dsDNA analogue, and the complexes formed with siRNA and
GLuc DNA were comparable. AUC demonstrated increased
binding constants with increasing cationic (DMAPMA) block
length. Solution differential scanning calorimetry was con-
ducted to determine BIC stability. The melting temperature of
GLuc DNA, the siRNA analogue, significantly shifted to higher
temperatures when complexed with hydrophilic-block-cationic
copolymers. Furthermore, increasing DMAPMA block length
increased the T
m
of each block ionomer complex (P4 >P3 >
P2). Since thermal denaturation of the DNA provided a large
endotherm, single-stranded DNA was complexed with hydro-
philic-block-cationic copolymers to determine thermodynamics
of dissociation. Since no complex dissociation was observed
under our experimental conditions, we believe that the
binding constants are so high that any species dissociation
would be below the instruments detection capability. While
the precise nature of BIC complexation/decomplexation is not
fully understood, these studies clearly demonstrate an increase
in gene suppression with increasing DMAPMA block length,
and more importantly, longer DMAPMA block lengths pro-
duced a time-delay in achieving a gene knockdown maximum.
Additionally, the kinetics for achieving these maxima are con-
sistent with ion exchange/substitution rates for IPECs and
BICs. While we have demonstrated the effect of block length
on gene suppression, the effect of the cationic-anionic registry
(i.e. arrangement of cationic charges along the phosphate-
helix backbone) on binding strength, and subsequently, gene
suppression is still under investigation.
Acknowledgements
The authors would like to thank Dr Rangachari and Dr Savin
for the use of the CD spectropolarimeter and DLS instruments,
respectively. This work was funded through NSF EPSCoR
EPS-0903727.
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