DNA polyplexes formed using PEGylated biodegradable hyperbranched polymers

Article abstract of DOI:10.1002/mabi.200900378

A novel PEGylated biodegradable hyperbranched PEG-b-PDMAEMA has been synthesized. The low toxicity, small molecular weight PDMAEMA chains were crosslinked using a biodegradable disulfide-based dimethacrylate (DSDMA) agent to yield higher molecular weight hyperbranched polymers. PEG chains were linked onto the polymer surface, masking the positive charge (as shown by Zeta potential measurements) and reducing the toxicity of the polymer. The hyperbranched structures were also cleaved under reducing conditions and analyzed, confirming the expected component structures. The hyperbranched polymer was mixed with DNA and efficient binding was shown to occur through electrostatic interactions. The hyperbranched structures could be reduced easily, generating lower toxicity oligomer chains. (Chemical Presented).

Full text of DOI:10.1002/mabi.200900378

Full Paper
DNA Polyplexes Formed Using PEGylated
Biodegradable Hyperbranched Polymers
Lei Tao, William C. Chou, Beng H. Tan, Thomas P. Davis*
A novel PEGylated biodegradable hyperbranched PEG-b-PDMAEMA has been synthesized. The
low toxicity, small molecular weight PDMAEMA chains were crosslinked using a biodegrad-
able disulfide-based dimethacrylate (DSDMA) agent to yield higher molecular weight hyper-
branched polymers. PEG chains were linked onto the polymer surface, masking the positive
charge (as shown by Zeta potential measurements) and reducing the toxicity of the polymer.
The hyperbranched structures were also cleaved under reducing conditions and analyzed,
confirming the expected component structures.
The hyperbranched polymer was mixed with
DNA and efficient binding was shown to occur
through electrostatic interactions. The hyper-
branched structures could be reduced easily,
generating lower toxicity oligomer chains.
Introduction
have the potential to complex with negative charged DNA
or RNA, generating neutral or positive charged polyplexes,
Many human diseases, such as cancer and cystic fibrosis,
with the ability to cross the negative charged cell
[12]
[
1–3]
have genetic origins.
‘‘Gene therapy,’’ insertion of a
membrane.
There are two main obstacles to the use of
targeting gene into cells to replace or override defective
genes, may well be a promising therapeutic approach.
Direct administration of naked deoxyribonucleic acid
cationic polymers, polyplex unpacking, and cytotoxicity. It
is known that increase in the positive charge on a polymer
improves cellular uptake and transfection efficiency, but
the accompanying detrimental toxicity effect originating
from the destabilization and loss of integrity of the cell
(
DNA) is unviable as poor transfer efficiency and enzymatic
degradation negate effective therapeutic implementation.
Viral (recombinant viruses) and non-viral (synthetic
membrane is also enhanced, leading to a narrow operating
[13,14]
[
4–8]
materials) have been proposed as vehicles for DNA.
window between efficiency and severe toxicity.
To
Despite their efficient delivery ability, viral vectors have
many drawbacks, such as cargo capacity, resistance to
repeat infection and safety concerns, limiting their
reduce the cytotoxicity stemming from positive charge,
neutral, non-toxic, and biocompatible polymers, such as
poly(ethylene glycol) (PEG) and dextran, have been
conjugated with DNA polyplexes to mask the positive
charge, reducing their non-specific binding capacity,
improving solubility and stability, and minimizing toxicity
[
9–11]
application.
Cationic polymers have attracted a lot
of research interest as potential non-viral vectors as they
[
15–17]
in vivo and in systemic delivery.
approach to improve synthetic polymer gene delivery is by
Another promising
T. P. Davis, L. Tao, W. C. Chou
Centre for Advanced Macromolecular Design (CAMD), School of
Chemical Sciences and Engineering, The University of New South
Wales, Sydney, NSW 2052, Australia
[18–22]
exploiting biodegradable cationic polymers.
Many
low molecular weight polymers possess low toxicity
compared to their higher molecular weight counterparts,
butare notsuitable forformingstable polyplexes forin vivo
Fax: (þ612) 9385 6250; E-mail: t.davis@unsw.edu.au
B. H. Tan
[
23–25]
ꢀ
administration.
Therefore cross-linking low molecu-
Institute of Materials Research and Engineering (IMRE), A STAR
(
Agency for Science, Technology and Research), 3 Research Link,
lar weight polymers through biodegradable linkages might
prove to be an efficient route to effective gene delivery
Singapore 117602
Macromol. Biosci. 2010, 10, 632–637
632
ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
DOI: 10.1002/mabi.200900378

DNA Polyplexes Formed Using PEGylated Biodegradable . . .
vectors, via high molecular weight cationic polymers. After
gene delivery, the polymers can undergo degradation,
resulting in the reduction of positive charges per molecular
and unspecific interactions. Additionally, the degradation
products are also more amenable to unpack and release free
DNA in the cell. For example, Forrest et al. crosslinked PEI
Abbreviations: deoxyribonucleic acid (DNA); polyethylene
glycol (PEG); chain transfer agent (CTA); dichloromethane (DCM);
0
molecular weight (MW); polydispersity index (PDI); 2,2 -azoisobu-
tyronitrile (AIBN); N,N-dimethylacetamide (DMAc); 4-(dimethyla-
mino)pyridine (DMAP); gel permeation chromatography (GPC);
molecular weight cut-off (MWCO); phosphate buffer (PB); phos-
phate buffered saline (PBS).
(
ꢁ800, essentially non-toxic but inefficient for gene
delivery), withdiacrylates togeneratepoly(b-aminoesters).
Compared to commercial 25 K PEI, the biodegradable
polymers were essentially non-toxic (at 14–30 K), and could
deliver DNA, mediating gene expression 2–16-fold more
Measurement
[
26]
efficiently.
dimethylamino)ethyl methacrylate) (PDMAEMA) was
evaluated as a gene delivery agent and displaying
Recently, biodegradable linear poly(2-
Gel permeation chromatography (GPC) analyses of polymers were
performed in N,N-dimethylacetamide (DMAc) (0.03% w/v LiBr,
(
ꢃ1
0
.05% BHT stabilizer) at 50 8C (flow rate: 0.85 mL ꢂ min ) using a
[
27]
Shimadzu modular system comprising a DGU-12A solvent degasser,
anLC-10ATpump,aCTO-10Acolumnoven,andanRID-10Arefractive
index detector. The system was equipped with a Polymer
significantly reduced cytotoxicity in a range of cell lines.
Centre for Advanced Macromolecular Design (CAMD)
recently developed biodegradable hyperbranched
PDMAEMA as a potential gene delivery agent,
2
Laboratories 5.0 mm bead-size guard column (50 ꢄ 7.8 mm )
[
28]
as part
2
5
4
3
followed by four 300 ꢄ 7.8 mm linear PL columns (10 , 10 , 10 ,
of a synthetic research program into producing novel
approaches to biodegradable, well-defined polymeric
and 500). Calibration was performed with narrow polydisperse
6
ꢃ1
polystyrene standards ranging from 500 to 10 g ꢂ mol
.
[
29–32]
1
structures.
In this paper, we describe the synthesis
H NMRspectrawereobtained usinga Bruker AC300F(300 MHz)
Spectrometer or a Bruker DPX300 (300 MHz) Spectrometer. Multi-
plicities were reported as singlet (s), broad singlet (bs), doublet (d),
triplet (t), quad (q), and multiplet (m). Agarose gel electrophoresis
was carried out with ReadyAgrose mini Gel (BioRad).
of PEGylated biodegradable hyperbranched PDMAEMAand
itsuseforDNAbinding. Thisworkrepresentsanadvanceon
our previous work as PEGylation has been used to shield the
charge of the hyperbranched polymer as demonstrated
through masking of positive charge via Zeta potential
measurements. In previous work we have shown that the
masking of charge by PEGylation also leads to reduced
Methods
[
33–35]
protein fouling of metal nanoparticles,
and therefore
PEG Trithiocarbonate (CTA, 1)
improved biocompatibility. Reversible addition–fragmen-
tation chain transfer (RAFT) technology allows control of
the polymerization process to yield low molecular weight
oligomers with narrow dispersity chain lengths and low
toxicity. We describe the use of a cross-linking agent,
disulfide-based dimethacrylate (DSDMA), 1,2-bis(2-(3-
methylbuta-1,3-dien-2-yloxy)ethyl) disulfane, tolinkPEGy-
lated-oligomers into biodegradable hyperbranched struc-
tures. To our knowledge, this is the disclosure of a synthesis
strategy to create biodegradable PEG-PDMAEMA hyper-
branched copolymers for DNA polyplex formation.
4-Cyano-4-(ethylthiocarbonothioylthio) pentanoic acid
(1.00 g, 3.8 mmol), PEG 1 100 (3.84 g, 3.2 mmol), and DMAP
(0.10 g) were dissolved in dry DCM (50 mL), DCC (0.99 g,
4.8 mmol) was added under nitrogen atmosphere. The
mixture was stirred at 22 8C for 16 h, and filtered to remove
4
white solid. The solvent was dried over MgSO and distilled
under vacuum to yield a yellow residue. The crude was
purified bycolumn chromatographyon silicagel [methanol
in DCM (1.0–8.0%)] to yield the product as a yellow waxy
1
solid (3.31 g, 76.9%). H NMR (300.18 MHz, CDCl
3
): d
), 3.89–3.52 (m, 48.7H,
),
), 1.35 (t, 3H,
(
ppm) ¼ 4.27–4.24 (m, 2H, COOCH
2
PEG unit), 3.37 (s, 3H, OCH
3
), 3.34 (q, 2H, J ¼ 7.4 Hz, SCH
2
Experimental Part
2.68–2.32 (m, 4H, CCH
J ¼ 7.4 Hz, SCH CH
280, 1 111, 843.
2
CH
2
), 1.87 (s, 3H, CCH
3
ꢃ1
2
3
). IR (cm ): 2 880, 1 736, 1 466, 1 343,
Materials
1
0
N,N -dicyclohexylcarbodiimide (DCC, 99%, Sigma), 4-(dimethyla-
mino) pyridine (DMAP, 99%, Aldrich), PEG 1 100 monomethyl ether
(
Fluka), and DNA sodium salt from salmon testes (DNA, Sigma)
Synthesis of Hyperbranched PEG-b-PDMAEMA
were used as purchased. 4-Cyano-4-(ethylthiocarbonothioylthio)
[36]
A typical polymerization is described as follows:
pentanoic acid was prepared as previously described,
1,2-bis(2-
DMAEMA (316 mg, 2.0 mmol), CTA (135 mg, 0.1 mmol),
DSDMA (87 mg, 0.3 mmol) and AIBN (4.9 mg, 0.03 mmol)
were dissolved in DMAc (3.0 mL). Aliquots were transferred
to four different vials that were then sealed with rubber
septa. Each vial was deoxygenated by purging with
(
3-methylbuta-1,3-dien-2-yloxy)ethyl) disulfane was prepared as
[37]
0
2,2 -Azoisobutyronitrile (AIBN, 98%,
previously described.
Sigma–Aldrich) was recrystalized twice from acetone, dichloro-
methane (DCM, 99%, Ajax) was stored over calcium hydride and
distilled before using.
Macromol. Biosci. 2010, 10, 632–637
ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.mbs-journal.de
633

L. Tao, W. C. Chou, B. H. Tan, T. P. Davis
nitrogen for 30 min prior to placement in a preheated water
bath at 70 8C. The vials were taken out at 4, 8, 22 and 29 h.
Immediate cooling with ice-water bath and exposure to air
quenched the polymerizations. The polymer solutions were
subjected to direct GPC analyses. The polymers were
collected after precipitation from DCM to hexane and then
dried under vacuum.
Deoxyribonucleic Acid (DNA) Binding by the
Protonated Hyperbranched Polymer
ꢃ1
Deoxyribonucleic acid (DNA, 0.2–1.0 mg ꢂ mL ) was dis-
solved in 5% glucose aqueous solution. A series of
ꢃ1
protonated PEG-b-PDMAEMA solutions (0.1–1.0 mg ꢂ mL
)
in 5% glucose aqueous were added slowly to the DNA
solution. The amount of the PEG-b-PDMAEMA added was
calculated based on the chosen positive/negative (P/N)
ratio of the polymer to DNA. The solution was incubated at
Static Light Scattering (SLS) Analysis of the
2
5 8C for 30 min with gentle stirring, and then the solution
Hyperbranched PEG-b-PDMAEMA
was used directly for gel electrophoresis.
A Brookhaven BI-9000AT Digital Autocorrelator was used
for SLS measurements which applies vertically polarized
laser light of wavelength 632.8 nm. In SLS, measurements
were carried out at a temperature of 25 8C (unless otherwise
stated) to determine the weight-average molecular weight
Analysis of Particle Size and Zeta Potential of
Polyplexes
The determination of hydrodynamic diameters and zeta
(
Mw) of the hyperbranched copolymer in DMAc solution
ꢃ1
potentials of the DNA polyplexes (10 mg ꢂ mL DNA and 2–
[
38,39]
from Zimm plots according to the relation;
ꢃ1
5
00 mg ꢂ mL protonated PEG-b-PDMAEMA in 5% glucose
aqueous solution) was performed at 25 8C by a Zetasizer
Nano dynamic light scattering detector (Malvern Instru-
ments, He–Ne laser 633 nm). The mean diameter was
obtained from the arithmetic mean using the relative
intensity of each particle size.
"
#
2
2
Kc
1
q Rg
¼
1 þ
þ 2A2c
(1)
DRu Mw
3
where K is the optical constant, which depends on the
refractive index increment of the polymer solution
2
2
2
4
(
K ¼ 4p n (dn/dc) /N l ). Here, c is the concentration of
A
the polymer solution, n the refractive index of the solvent,
u the angle of measurement, l the wavelength of laser
Results and Discussion
light, DR
solvent)], dn/dc the refractive index increment of the
copolymer solution, and N is the Avogadro’s constant. The
u
the excess Rayleigh ratio [DR
u
¼ R
u
(solution) – R
u
Preparation of Biodegradable Hyperbranched
PDMAEMA
(
A
PEGylated chain transfer agent (1) (Scheme 1) was prepared
scattering angles ranged from 508 to 1308 at 108 intervals
while the copolymer concentration ranged from 1 to
using the reaction between commercial methyl ether PEG
(
ꢁ1 100) and 4-cyano-4-(ethylthiocarbonothioylthio) pen-
ꢃ1
2
1
0 mg ꢂ mL . A plot of Kc/R versus [sin (u/2) þ kc] (where
u
tanoic acid in the presence of DCC using DMAP as a
k is a plotting constant) can be used to determine the
molecular parameters. A simultaneous extrapolation to
zero angle and concentration yields an intercept, that is the
inverse of the Mw.
1
catalyst. The H NMR spectrum (Figure 1a) of the CTA
exhibited signals at 4.25 and 3.37 ppm corresponding to
the ester group and the methyl ether moiety, respectively.
The integration ratio of these two peaks is 2/3.08,
The refractive indices of copolymers in DMAc were
measured by using a BI-DNDC differential refractometer at
a wavelength of 620 nm to determine the refractive index
increment (dn/dc) of each solution. The instrument was
primarily calibrated with potassium chloride (KCl) in
aqueous solution and the dn/dc value was found to be
0
.150 ꢅ 0.011 mL ꢂ gꢃ1 for PEG-b-PDMAEMA in DMAc.
Protonation of the Hyperbranched Polymer
Hyperbranched copolymer (30 mg) was dissolved in HCl
aqueous solution (5.0 mL of 0.01 M). After removal of excess
acid by centrifugation filtration [molecular weight cut-off
(
MWCO): 10 000], water was removed by a freeze drying
Scheme 1. Synthesis of biodegradable hyperbranched PEG-b-
process to yield polymer as a yellow powder.
PDMAEMA and the subsequent cleavage reaction.
Macromol. Biosci. 2010, 10, 632–637
634
ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
DOI: 10.1002/mabi.200900378

DNA Polyplexes Formed Using PEGylated Biodegradable . . .
could not be used to quantitatively
analyze the polymers. Consequently,
SLS was employed to characterize the
‘
‘true’’ molecular weight yielding a
weight average molecular weight of
Mw ꢁ 342 K.
Cleavage of the Biodegradable
Hyperbranched Polymer
As every branched oligomer is linked
through a disulfide bond, the polymer
should be cleavable in the presence of
reductants. Consequently, the polymer
was treated with DTT (0.1 M) in DMAc
1
Figure 1. H NMR spectra of (a) CTA, (b) hyperbranched PEG-PDMAEMA copolymer.
close to the predicted 2/3, indicative of complete esterifica-
tion. This PEGylated CTA was employed to synthesize
polymer from DMAEMA, adding DSDMA as crosslink
agent to increase the degree of polymer branching
solution for 16 h. The GPC chromatogram (Figure 3)
indicated that the post-reduction polymer possessed a
much lower molecular weight [Mn (GPC) ꢁ11 600] than the
original polymer. The narrow polydispersity of the cleaved
polymer (PDI ꢁ 1.18) also indicated uniformity, consistent
with RAFT polymerization control, demonstrating a design
control capacity over the final degraded polymer via the
original synthesis protocol.
[
37,40–42]
whilst avoiding undissolvable gel formation.
final polymer was prepared with a ratio, [CTA]/[DSDMA]/
The
1
[
monomer] ¼ 1:3:20. The
H NMR spectrum of the
final copolymer (Figure 1b) displayed signals at 4.06
and 3.37 ppm corresponding to the ester group of
PDMAEMA and methyl ether group from the CTA moiety.
The integration ratio of these two peaks was ꢁ42/3.0,
close to the predicted value of 40/3, confirming that
the oligomer chains have predetermined chain lengths.
The signal at 2.86–3.00 ppm was attributed to the
methylene group adjacent to the disulfide, confirming that
the cross-linking agent was incorporated into the polymer
structure.
Deoxyribonucleic Acid (DNA) Binding to the
Biodegradable Hyperbranched PDMAEMA
Deoxyribonucleic acid (DNA) sodium salt from salmon
testes was used as model DNA. The hyperbranched
degradable PEG-PDMAEMA was protonated prior to DNA
binding. The protonated polymer was mixed with DNA
having different P/N ratios, and the binding was monitored
by agarose gel electrophoresis. The negative signal from
DNA decreased with an increase in P/N ratio, as shown in
Figure 4a. When the ratio reached 1:1, the DNA was
neutralized perfectly and there was no DNA complex
Thepolymerizationsweremonitoredovertimeasshown
in Figure 2. As expected both the molecular weight and
branched polymer ratio increased with increase in reaction
times. As GPC was calibrated with polystyrene standards it
Figure 2. Gel permeation chromatography (GPC) traces of hyper-
branched polymers generated from [Monomer]/[CTA]/[DSDMA]/
[
initiator] ratio of 20:1:3:0.3 at 70 8C.
Figure 3. Polymers (a) before and (b) after incubating with DTT.
Macromol. Biosci. 2010, 10, 632–637
ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.mbs-journal.de
635

L. Tao, W. C. Chou, B. H. Tan, T. P. Davis
Keywords: biodegradable; hyperbranched;
oligomers; RAFT
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DOI: 10.1002/mabi.200900378
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