Polymer Side-Chain Degradation as a Tool to Control the Destabilization of Polyplexes

Article abstract of DOI:10.1023/B:PHAM.0000012165.68765.e6

Purpose. We purposed to design a cationic polymer that binds to pDNA to form polyplexes and that subsequently degrades within a few days at physiological pH and temperature, releasing the DNA in the cytosol of a cell. Methods. We synthesized a new monomer carbonic acid 2-dimethylamino-ethyl ester 1-methyl-2-(2-methacryloylamino)-ethyl ester (abbreviated HPMA-DMAE) and the corresponding polymer. Hydrolysis of the carbonate ester of both the monomer and the polymer was investigated at 37°C. The DNA condensing properties of the pHPMA-DMAE was studied using dynamic light scattering (DLS) and zeta potential measurements. Degradation of the polyplexes at 37°C and pH 7.4 was monitored with DLS and gel electrophoresis. In vitro transfections were performed in COS-7 cell line. Results. pHPMA-DMAE is able to condense DNA into small particles (110 nm) with a positive zeta potential. The half-life of the polymer and monomer at 37°C and pH 7.4 was around 10 h whereas at pH 5, the half-life was 380 h. In line with this, due to hydrolysis of the side groups, pHPMA-DMAE-based polyplexes dramatically increased in size at 37°C and pH 7.4 whereas at pH 5.0, only a very small increase was observed. Interestingly, intact DNA was released from the polyplexes after 48 h at pH 7.4 whereas all DNA remained bound to the polymer at pH 5.0. Polyplexes were able to transfect cells with minimal cytotoxicity if the endosomal membrane-disrupting peptide INF-7 was added to the polyplex formulation. Conclusions. Degradation of the cationic side-chains of a polymer is a new tool for time-controlled release of DNA from polyplexes, preferably within the cytosol and/or nucleus.

Full text of DOI:10.1023/B:PHAM.0000012165.68765.e6

Pharmaceutical Research, Vol. 21, No. 1, January 2004 (© 2004)
Research Paper
methylamino methacrylate) (pDMAEMA) or cationic lipids
such as DOTAP or DOPE (3–6). However, the current non-
viral carriers are less efficient in their transfection activity
than viral vectors. It is commonly accepted that polymer–
DNA complexes (also called polyplexes) are taken up by cells
by endocytosis (7). DNA has to be protected against degra-
dation inside the endosomes/lysosomes which is established
as long as the polyplexes stay intact in these cellular compart-
ments. Although some cationic polymers as such are thought
to be able to destabilize endosomes [e.g., pEI (8), polyami-
doamine dendrimers (9), or pDMAEMA (10)], endosomal
escape can be promoted using specific compounds [e.g., Gala-
(11) and INF-peptides (12,13) or poly(propylacrylic acid)
(14)]. Once escaped from the endosome, DNA has to disso-
ciate from the polymer and be transported into the nucleus
for transcription. Dissociation of polyplexes may occur by
anionic compounds (e.g., proteins or RNA) present in the
cytosol (5,15). Alternatively, dissociation of the DNA from
Polymer Side-Chain Degradation as a
Tool to Control the Destabilization
of Polyplexes
Arjen M. Funhoff,1 Cornelus F. van Nostrum,1
Adriënne P. C. A. Janssen,1 Marcel H. A. M. Fens,1
Daan J. A. Crommelin,1 and Wim E. Hennink1,2
Received July 30, 2003; accepted September 19, 2003
Purpose. We purposed to design a cationic polymer that binds to
pDNA to form polyplexes and that subsequently degrades within a
few days at physiological pH and temperature, releasing the DNA in
the cytosol of a cell.
Methods. We synthesized a new monomer carbonic acid 2-dimethyl-
amino-ethyl ester 1-methyl-2-(2-methacryloylamino)-ethyl ester (ab- the polymer can be achieved by polymer degradation. Poly(4-
breviated HPMA-DMAE) and the corresponding polymer. Hydro-
lysis of the carbonate ester of both the monomer and the polymer was
investigated at 37°C. The DNA condensing properties of the
pHPMA-DMAE was studied using dynamic light scattering (DLS)
and zeta potential measurements. Degradation of the polyplexes at
37°C and pH 7.4 was monitored with DLS and gel electrophoresis. In
vitro transfections were performed in COS-7 cell line.
hydroxy-L-proline ester) has been studied as one of the first
water-soluble, degrading gene delivery polymers (16,17). This
polymer showed a rapid degradation in the first 2 h at 37°C
and pH 7.0, after which degradation slowed down. Impor-
tantly, the degradation of the polymer was retarded when it
was complexed with DNA. A similar degradation behavior
was found for poly[␣-(4-aminobutyl)-L-glycolic acid]
Results. pHPMA-DMAE is able to condense DNA into small par-
ticles (110 nm) with a positive zeta potential. The half-life of the (PAGA) (18,19). Recently, degradable pEIs were designed
polymer and monomer at 37°C and pH 7.4 was around 10 h whereas
at pH 5, the half-life was 380 h. In line with this, due to hydrolysis of
the side groups, pHPMA-DMAE-based polyplexes dramatically in-
creased in size at 37°C and pH 7.4 whereas at pH 5.0, only a very
small increase was observed. Interestingly, intact DNA was released
from the polyplexes after 48 h at pH 7.4 whereas all DNA remained
bound to the polymer at pH 5.0. Polyplexes were able to transfect
cells with minimal cytotoxicity if the endosomal membrane-
disrupting peptide INF-7 was added to the polyplex formulation.
Conclusions. Degradation of the cationic side-chains of a polymer is
a new tool for time-controlled release of DNA from polyplexes, pref-
erably within the cytosol and/or nucleus.
by linking low-molecular-weight pEI blocks with oligo(L-
lactic acid-co-succinic acid) (20) or poly(ethylene glycol) (21)
via degradable bonds. However, the degradation time was
rather slow for the first polymer, taking weeks up to months,
and degradation of the PEG-containing copolymer is not re-
ported yet. Recently, Luten et al. reported on a new class of
cationic polymers for gene delivery: polyphosphazenes (22).
The half-lifes of the polymers were of the order weeks. Cel-
lular uptake of lipo/polyplexes and escape from the endosome
into the cytoplasm takes a number of hours (23). This means
that the polyplexes should be stable for a couple of hours
after administration and also at pH 5 (inside the endosome),
thereby protecting DNA against degradation by endosomal/
lysosomal DNases. Given the time between administration
and intracellular presence, the ideal degradable carrier is
stable at pH 5 but degrades after a few hours at pH 7.4. The
biodegradable polymers investigated so far rely on the (com-
plete) degradation of the polymer backbone, and the degra-
dation of these polymers is rather slow. The alternative ap-
proach we have chosen in this study is to design a polymer
with degradable cationic side groups, meaning that the back-
bone stays intact but the plasmid condensing side-chains are
removed in time. The condensing capacity of the polymer,
which relies on the multivalency of the interaction with the
plasmid, thereby decreases, and the plasmid will be released
and become available for intracellular transport. The polymer
that has been intensively studied for gene delivery in our
research group is pDMAEMA (10), which carries an ester
bond between the polymer backbone and each cationic side
group (Fig. 1). However, unlike the intrinsic hydrolytical sen-
sitivity of an ester group, it was demonstrated that this poly-
mer is completely stable at any tested pH, probably because
of the inability of water to access the ester bond, being too
KEY WORDS: biodegradation; gene delivery; polyplex.
INTRODUCTION
Gene therapy is considered to be a promising approach
to treat life-threatening diseases. In order to introduce suc-
cessfully foreign DNA into a cell, extracellular degradation of
DNA has to be prevented and cellular uptake has to occur.
To achieve this, both viral and nonviral carrier systems have
been developed over the years. Although viral systems (“vec-
tors”) are highly efficient in introducing DNA into cells (1,2),
they possess some serious disadvantages, such as the possible
induction of immune responses and problems with pharma-
ceutical-grade large-scale productions. Therefore, as an alter-
native for viral vectors, attention is focused on the design of
nonviral carriers. These include cationic polymers such as
poly-L-lysine (pLL), poly(ethylene imine) (pEI), and poly(di-
1 Department of Pharmaceutics, Utrecht Institute for Pharmaceutical
Sciences (UIPS), Utrecht University, Utrecht, The Netherlands.
2
To whom correspondence should be addressed. (e-mail:
W.E.Hennink@pharm.uu.nl)
0724-8741/04/0100-0170/0 © 2004 Plenum Publishing Corporation
170

Controlled Destabilization of Polyplexes
171
aminoethanol (2.22 g, 24.9 mmol, DMAE) was added drop-
wise. During addition, the CDI dissolved and the obtained
clear solution was stirred at room temperature for 1 h. Next,
this solution was washed with water (three times 20 ml), dried
over magnesium sulfate, and the solvent was evaporated un-
der reduced pressure, yielding DMAE-CI as a colorless liquid
(3.12 g, 68%).
NMR spectra were recorded on a Varian G-300 300 MHz
spectrometer (Varian, Palo Alto, CA, USA).
NMR (CDCl3, ␦ in ppm): 1H: 2.29 (s, 6H, N(CH3)2), 2.68
(t, 2H, CH2N), 4.48 (t, 2H, OCH2), 7.04 (s, 1H, NCHCHN),
7.40 (s, 1H, NCHCHN), 8.12 (s, 1H, NCHN). 13C: 45.7
(N(CH3)2), 57.5 (CH2N), 65.9 (OCH2), 117.1 (NCHCHN and
NCHCHN), 130.6 (NCHN).
DMAE-CI (3.12g, 17.0 mmol) was dissolved in 30 ml
dichloromethane and 1.83 g HPMA (12.8 mmol, 0.75 equiva-
lents) dissolved in 20 ml dichloromethane was added. Hy-
droquinone monomethyl ether (10 mg) was added to prevent
premature polymerization. This solution was put under a ni-
trogen atmosphere and stirred at room temperature for 5
days. The solvent was removed under reduced pressure, yield-
ing an oil (5.11 g, mixture of imidazole, unreacted DMAE-CI,
and HPMA-DMAE).
Fig. 1. Structural formulas of the polymers used in this study.
close to the hydrophobic polymer backbone (24). Therefore,
we designed a similar polymer, containing a degradable bond
between the cationic group and the polymer backbone (Fig.
1). We chose a carbonate ester, whose degradation rate is
higher than a normal ester and therefore favorable (25). The
backbone is derived from poly-2-hydroxypropyl methacryl-
amide (pHPMA), which is known to have no polymer-related
toxicity (26,27). The cationic group of the polymer is the same
as for pDMAEMA, the dimethyl amino group, which is par-
tially protonated at physiological pH, thus ensuring DNA
binding and condensation capability (10).
NMR (CDCl3, ␦ in ppm): 1H: 6.15 (bs, 1H, CONHCH2),
5.63 (s, 1H, H2CסC), 5.34 (s, 1H, H2CסC), 4.86 [m, 1H,
CH2CH(CH3)O], 4.20 (m, 2H, OCH2CH2), 3.7–3.3 [m, 2H,
CH2CH(CH3)O], 2.48 (t, 2H, OCH2CH2), 2.22 [s, 6H,
N(CH3)2], 1.97 [s, 3H, H2CסC(CH3)], 1.27 [d, 3H,
CH2CH(CH3)O]. 13C: 168.8 (CסO), 154.7 (CסO), 135.2
(H2CסC), 121.7 (H2CסC), 74.1 (CH), 65.1 (OCH2), 57.4
(CH2N), 45.3 [N(CH3)2], 43.8 (NHCH2), 18.5 (CHCH3), 17.3
(CCH3).
pHPMA-DMAE was synthesized by radical polymeriza-
tion under a nitrogen atmosphere as follows: approximately 1
g of the crude monomer was dissolved in 4 ml of 1 M aqueous
hydrochloric acid solution, and the pH was adjusted to 5.
Polymerization was carried out under shaking conditions at
70°C with ammonium peroxodisulfate as initiator (M/I ס 25).
After 20 h, the polymerization mixture was cooled down to
room temperature and transferred into a dialysis tube
(MWCO 3.5 kDa). After extensive dialysis against an NH4Ac
buffer of pH 5.0 (10 mM, last step 5 mM) at 4°C, the polymer
was collected after freeze drying. Molecular weight of the
polymer relative to dextran standards (Fluka) was deter-
mined by gel permeation chromatography (GPC) (10).
This study reports on the synthesis of the monomer and
polymer; the pH-dependent degradation of the monomer,
polymer, and polyplexes; and the transfection activity of poly-
plexes.
MATERIALS AND METHODS
Materials
The following materials were used as received: D,L-1-
amino-2-propanol 99+ % (Acros), N,N-dimethylaminoetha-
nol (DMAE, Aldrich, Zwijndrecht, The Netherlands),
1,1’-carbonyldiimidazole (CDI, Acros, Geel, Belgium). Meth-
acryloyl chloride of purity Ն 97% (Fluka, Zwijndrecht, The
Netherlands) was freshly distilled before use. pDMAEMA
(Mn ס 92 kg/mol) was synthesized via radical polymerization
(10). The plasmid pCMVLacZ, containing a bacterial LacZ
gene preceded by a nuclear localization signal under control
of a cytomegalovirus (CMV) promoter, was purchased from
Sanvertech (Heerhugowaard, The Netherlands). INF-7, a 24
amino acid containing peptide with fusogenic activity derived
from the influenza virus, was synthesized via standard Fmoc
solid-phase synthesis (12). HPMA was synthesized according
to Oupicky et al. (28).
Hydrolysis of the Monomer
Monomer (60 mg, crude mixture) was dissolved in 100 ml
of the appropriate buffer. Buffers used were acetic acid at pH
5.0 and HEPES at pH 7.4 (concentration buffer 10 mM, ionic
strength adjusted to 150 mM with NaCl). The solutions were
incubated at 37°C, and samples were periodically withdrawn
from these solutions and immediately analyzed with RP-
HPLC.
The RP-HPLC system consisted of a Waters pump
model 515 (Waters Associates, Milford, MA, USA), Spark
Marathon Basic+ injector (Spark, Emmen, The Netherlands)
and a LKB 2151 UV detector (LKB, Roosendaal, The Neth-
erlands) set at 210 nm. The columns used were a Merck
Synthesis of Carbonic Acid 2-Dimethylamino-Ethyl Ester
1-Methyl-2-(2-Methacryloylamino)-Ethyl Ester and
Corresponding Polymer [(p)HPMA-DMAE]
A dry round-bottom flask was loaded with 1,1’- LiChrosphere 100 (Merck, Darmstadt, Germany) RP-18 col-
carbonyldiimidazole (6.84 g, 45.0 mmol, CDI) and 50 ml di- umn (5 ␮m, 125 × 4 mm i.d.) and a RP-18 guard column (4 ×
chloromethane. To this suspension 2.50 ml N,N’-dimethyl- 4 mm). The mobile phase consisted of a water–acetonitrile

172
Funhoff et al.
95/5 (w/w) solution completed with 10 mM triethylamine and transfection, polyplexes were prepared as follows: 150 ␮l of
brought to pH 2 with perchloric acid. The flow rate was 1 polymer solution (various concentrations) was added to 50 ␮l
ml/min; injection volume was 20 ␮l.
of plasmid solution (50 ␮g/ml), and after incubation for 30
min at room temperature, 50 ␮l of either buffer or INF-7
solution (150 ␮g/ml) was added, and the polyplexes were in-
cubated for another 15 min before addition to the cells. After
rinsing the cells with HBS, 100 ␮l of polyplex dispersion and
100 ␮l of culture (with or without 10% serum) medium were
incubated with the cells for 1 h. After removal of the polyplex
medium, fresh culture medium was added, and the cells were
incubated for another 48 h. All transfection experiments were
performed in two identical series in separate 96-well plates.
One series was tested for reporter gene expression (␤-
galactosidase) by ONPG colorimetric assay; the other series
was used to determine the number of viable cells using an
XTT colorimetric assay. As a reference, pDMAEMA poly-
plexes prepared at the same DNA concentration and an N/P
ratio of 6 were used. The transfection activity of this polyplex
formulation was set at one.
Hydrolysis of the Polymer
Hydrolysis of the polymer was followed in time with 1H-
NMR in D2O buffered at pD 7.4 with NaH2PO4/Na2HPO4;
ionic strength adjusted to 150 mM with NaCl at 37°C in a
Varian Gemini 500 MHz NMR apparatus. Each hour a pro-
ton NMR spectrum was run, and the peak area of the
CH2CH(CH3)O (4 in Fig. 1) resonance was plotted in time.
Physical Characteristics of
pHPMA-DMAE-Based Polyplexes
The physical characteristics of pHPMA-DMAE-based
polyplexes were investigated as a function of the polymer-
nitrogen to DNA-phosphate (N/P) ratio. Plasmid DNA was
diluted in 20 mM HEPES buffer, pH 7.4, to a concentration of
75 ␮g/ml. A stock solution of the polymer (5 mg/ml) was
diluted in the same buffer to various concentrations (7–450 RESULTS AND DISCUSSION
␮g/ml). Polyplexes were made by the addition of 700 ␮l of a
polymer solution to 175 ␮l DNA solution, mixed thoroughly
and incubated for 30 min at room temperature. The Z-
average diameters of the formed polyplexes were determined
via dynamic light scattering (DLS) at 25°C, and zeta-potential
measurements were carried out as previously described (10).
Synthesis and Characterization of (p)HPMA-DMAE.
Synthesis of the monomer was performed in two steps. In
the first step N’N’-dimethylaminoethanol (DMAE) was acti-
vated by 1,1’-carbonyldiimidazole in a good yield (68%) and
good purity (>97% by NMR). In the second step, the acti-
vated alcohol was reacted with N-(2-hydroxypropyl) metha-
crylamide (HPMA). A kinetic study showed that when a
slight excess (∼30 mol%) of activated alcohol was used, no
unreacted HPMA could be detected by proton NMR after 5
days of reaction at room temperature. Removal of the solvent
resulted in a mixture of the desired product (HPMA-DMAE)
as well as unreacted activated DMAE and imidazole. Purifi-
cation of HPMA-DMAE from the reaction mixture was dif-
ficult to achieve (premature degradation of this compound;
low yields). The crude mixture was therefore directly poly-
merized because the impurities did not interfere with the radi-
cal polymerization and could easily be removed by the dialy-
sis procedure applied to purify and isolate the polymer.
Radical polymerization of the monomer was performed
in an aqueous 1 M HCl solution (with pH adjusted to pH 5 if
necessary to prevent degradation of the monomer) with good
yield (75%). The weight-average molecular weight (Mw) of
the polymer was 160 kDa and the number-average molecular
weight (Mn) was 20 kDa.
Polyplex Destabilization Studied with DLS
Polyplexes of pHPMA-DMAE and plasmid DNA were
made at a N/P ratio of 5.4, either in 10 mM HEPES pH 7.4 or
10 mM acetic acid pH 5, ionic strength adjusted to 150 mM
with NaCl. The size of these polyplexes was measured every
half hour with dynamic light scattering at 37°C for approxi-
mately 15 h. As a control, pDMAEMA-based polyplexes
(N/P ס 6) in HEPES buffer were measured.
Polyplex Destabilization Studied with Agarose
Gel Electrophoresis
Polyplexes of pHPMA-DMAE and plasmid DNA were
made by mixing 10 ␮l of plasmid solution, 200 ␮g/ml, with 40
␮l of pHPMA-DMAE solution, 200 ␮g/ml, corresponding
with a N/P ratio of 5.4 and incubated for 30 min at room
temperature. This was done in 10 mM HEPES pH 7.4 or 10
mM sodium acetate pH 5; ionic strength adjusted to 150 mM
with NaCl. As a control, pDMAEMA-based polyplexes at pH
7.4 were also investigated. These polyplex dispersions were
subsequently incubated at 37°C for 0, 2, 4, 7, 24, or 48 h. After
cooling down to room temperature, 10 ␮l of the correspond-
ing buffer and 3 ␮l of sample buffer (0.4% w/v bromophenol
blue, 10 mM EDTA, 50% v/v glycerol in water) were added
to 20 ␮l of each polyplex dispersion. Thirty microliters of
these solutions were applied to a 0.7% agarose gel containing
0.5 ␮g/ml ethidium bromide and were run at 100 V (15).
Hydrolysis of Monomer and Polymer
The hydrolysis of the monomer at 37°C was followed
with RP-HPLC. With the HPLC method used, both the re-
maining HPMA-DMAE as well as one of the hydrolysis prod-
ucts (HPMA) could easily be detected and quantified. The
relative peak areas of the HPMA-DMAE and HPMA as a
function of time and at pH 7.4 and 5 are depicted in Fig. 2
(note the different timescales of the x axes of the graphs). The
kobs for the hydrolysis of the monomer was calculated by
Transfection Studies
Transfection studies were done in COS-7 cells using the plotting the natural logarithm of the peak areas divided by the
plasmid pCMVLacZ as reporter gene as previously described peak area at time point zero vs. the time (see insets in the
(10,15). In brief, 96-well plates were seeded with cells at a figures; the calculated kobs at pH 5 and 7.4 are 5.0 × 10−7 s−1
density of 3 × 104 /cm2 24 h before transfection. At the day of [(half-life, 380 h) and 1.7 × 10−4 s−1 (half-life, 9.6 h), respec-

Controlled Destabilization of Polyplexes
173
Fig. 3. Hydrolysis of p(HPMA-DMAE) in time at pH 7.4 and 37°C.
Integral of the 1H-NMR peak at 4.8 ppm is shown on the y axis.
is comparable to polyplexes composed of DNA and other
cationic polymers (13,22,29).
Polyplex Destabilization Studied with DLS
The destabilization of p(HPMA-DMAE)-based poly-
plexes was investigated at pH 5.0 and pH 7.4 at 37°C by
dynamic light scattering. Figure 5 depicts the particle size of
the polyplexes as a function of time. While the size of poly-
plexes based on p(HPMA-DMAE) at pH 5.0 and of
pDMAEMA polyplexes at pH 7.4 was constant over the in-
vestigated time period, the size of p(HPMA-DMAE)-based
polyplexes at pH 7.4 increased dramatically. This indicates
that the side chains of p(HPMA-DMAE) indeed are removed
upon hydrolysis, even in the polyplexes. Due to the hydroly-
sis, the charge density of the polymer decreases in time, re-
sulting in decreasing binding/condensing capabilities of the
polymer. As the result, the polyplexes swell/aggregate in time
(Fig. 4). The time period in which aggregation of the poly-
plexes occurs is of the same order of magnitude as monomer/
polymer hydrolysis (compare Fig. 5 with Figs. 2 and 3).
Fig. 2. (ࡗ) Hydrolysis of the HPMA-DMAE monomer and (᭿) for-
mation of HPMA in time (37°C) at pH 7.4 (top) and at pH 5.0
(bottom). The insets show the natural logarithm of the peak areas
divided by the peak area at time point zero vs. the time.
Polyplex Destabilization Studied with Agarose
Gel Electrophoresis
tively]. The hydrolysis of pHPMA-DMAE was measured
with 1 H-NMR by following the integral of the
CH2CH(CH3)O peak from HPMA-DMAE (4 in Fig. 1),
which shifts from 4.8 to 3.9 ppm when the DMAE part is
removed by hydrolysis, in time (Fig. 3). The estimated half-
life of the polymer is ∼12 h, which corresponds well with the
half-life of the monomer under the same condition. This is
much faster than other reported polymers like pEI linked via
oligo(L-lactic acid-co-succinic acid) (20) or polyphosphazenes
(22).
Polyplexes incubated at 37°C for different time periods
were subjected to gel electrophoresis to investigate whether
Biophysical Characterization of
p(HPMA-DMAE)-Based Polyplexes
The DNA binding and condensation properties of
p(HPMA-DMAE) were investigated by DLS and zeta poten-
tial measurements as a function of the polymer to DNA ratio.
Figure 4 shows that when an excess of the polymer was added
(N/P > 4), the polymer was able to condense DNA into small
particles with a size of approximately 110 nm and a positive
zeta potential of approximately +19 mV. Neutral aggregates
were formed at an N/P ratio between 2 and 4, and negatively
Fig. 4. (ࡗ) Particle size and (᭿) zeta potential of p(HPMA-DMAE)-
charged polyplexes were formed at a ratio of 1. This behavior based polyplexes as a function of the N/P ratio.

174
Funhoff et al.
As DNA stays complexed with the polymer inside endo-
somes/lysosomes (pH 5), degradation by DNases is mini-
mized, while once inside the cytoplasm DNA will be released
via hydrolysis of the polymer cationic side-chains. However,
this hydrolysis is not so fast that DNA is released before
cellular uptake can take place. Both experiments indicate that
hydrolysis of the polymer is not or is hardly influenced by
c o m -
plexation with DNA. This is in contrast to other degradable
polymers like PAGA, where degradation was influenced by
complexation with DNA (16).
Transfection Efficiency and Cytotoxicity of
p(HPMA-DMAE)-Based Polyplexes
The transfection activity of pHPMA-DMAE-based poly-
plexes was investigated in COS-7 cells. When serum proteins
were present during transfection, no transfection activity was
found (data not shown). A low transfection activity of poly/
lipoplexes in the presence of serum proteins has been re-
Fig. 5. The size of polyplexes based on p(HPMA-DMAE) incubated
at (᭡) pH 7.4 and (ᮀ) pH 5.0 or pDMAEMA at pH 7.4 (छ).
the DNA was released from polyplexes by hydrolysis of ported before, and this was ascribed to aggregation and/or
p(HPMA-DMAE). Figure 6 shows that for pDMAEMA at destabilization of the poly/lipoplex due to proteins (32). This
pH 7.4 or p(HPMA-DMAE) at pH 5.0, all DNA remained in can be prevented by pegylation of the polymer or the poly-
the starting slots indicating that the DNA remained com- plexes, as has been shown for pDMAEMA (33). However,
plexed with the polymers. At pH 7.4 and 0 h and 4 h incuba- this was not the intention of this study. Therefore, serum
tion, all DNA was present in the starting slot, indicating that proteins were absent in further transfection experiments.
the polymer was still complexed with DNA. At 7 and 24 h of
Figure 7 shows the transfection of COS-7 cells and cyto-
incubation, no fluorescence signal could be detected. This can toxicity of p(HPMA-DMAE)-based polyplexes as a function
probably be attributed to the presence of aggregates (see Fig. of the polymer to DNA ratio in the absence or presence of
5), which are not accessible for ethidium bromide, as has also the membrane-disrupting peptide INF-7. As a reference,
been observed for pEI-DNA aggregates (30). Interestingly, pDMAEMA was used as a transfection agent, and its highest
after 48 h incubation, DNA was released in its intact form transfection efficiency was set to 1. p(HPMA-DMAE)-based
from the p(HPMA-DMAE)-based polyplexes, indicating that polyplexes in the absence of INF-7 are not toxic to cells at any
the polymer was hydrolyzed to such an extent that dissocia- of the tested concentrations and are able to transfect cells to
tion of the polyplexes had occurred. DNA release takes place some extent, but only at rather high polymer to DNA ratios.
between approximately 2–4 half-lifes; in that time, at least This behavior has been seen for other polymers and is prob-
75% of the side chains have degraded.
ably related to the low toxicity of the polymer (13,22). Inter-
Both the DLS and gel electrophoresis experiments indi- estingly, when the membrane-destabilizing peptide INF-7 was
cate that at physiological pH and temperature, the hydrolysis added to the polyplex dispersion, a drastic increase in the
of the polymer complexed with DNA is relatively fast transfection activity was observed. This likely indicates that
whereas at pH 5, the complexes are stable. In the literature, it p(HPMA-DMAE) polyplexes do not escape from the endo-
was shown that when DNA is complexed with a polymer, some, as previously reported for other polymers (12,13). Ad-
degradation by DNases is slowed down considerably (15,31). dition of the INF-7 peptide to pDMAEMA-based polyplexes
Fig. 6. Gel electrophoresis of polyplexes incubated at 37°C for 0, 2, 4, 7, 24, or 48 h. Lane 1, free DNA; lanes
2–7, pDMAEMA-based polyplexes, pH 7.4; lanes 8–13, p(HPMA-DMAE)-based polyplexes pH 7.4 [lane 9
(t ס 2 h) is empty]; lanes 14–19, p(HPMA-DMAE)-based polyplexes pH 5.0.

Controlled Destabilization of Polyplexes
175
ficking of the DNA toward the nucleus via nuclear localiza-
tion signal (NLS) peptides (34,35). Transfection with NLS
containing DNA is currently under investigation.
CONCLUSIONS
We have synthesized a new gene delivery polymer
(pHPMA-DMAE, Fig. 1) with hydrolyzable cationic side-
groups. It was shown that polyplexes based on this polymer
were stable at pH 5, but showed release of intact DNA within
48 h after incubation at pH 37°C and pH 7.4. Polyplexes based
on this polymer were able to transfect cells efficiently when a
membrane-destabilizing peptide was present while not being
toxic toward the cells. Thus, polymer side-chain degradation
is a new tool for controlled intracellular destabilization of
polyplexes. Future work will focus on nuclear targeting of the
intracellularly released DNA to further enhance transfection
efficiency and in vivo application of the new polymer.
ACKNOWLEDGMENTS
This research was supported by a grant from the Dutch
ministry of Economic Affairs (Project No. BTS-98150). The
authors thank Dr. R. Verrijk (OctoPlus Technologies,
Leiden, The Netherlands) for valuable discussions.
REFERENCES
1. M. G. Rots, D. T. Curiel, W. R. Gerritsen, and H. J. Haisma.
Targeted cancer gene therapy: the flexibility of adenoviral gene
therapy vectors. J. Controlled Release 87:159–165 (2003).
2. B. Blits, G. J. Boer, and J. Verhaagen. Pharmacological, cell, and
gene therapy strategies to promote spinal cord regeneration. Cell
Transplant. 11:593–613 (2002).
3. W. T. Godbey and A. G. Mikos. Recent progress in gene delivery
using non-viral transfer complexes. J. Controlled Release 72:115–
125 (2001).
Fig. 7. (A) Transfection efficiency and (B) cytotoxicity of polyplexes
based on p(HPMA-DMAE) (᭿) without and (ᮀ) with the mem-
brane-disrupting peptide INF-7. As a reference, pDMAEMA (᭹)
without and (᭺) with the peptide was used. Cells are grown for 48 h
after transfection.
4. T. Merdan, J. Kopecek, and T. Kissel. Prospects for cationic poly-
mers in gene and oligonucleotide therapy against cancer. Adv.
Drug Del. Rev 54:715–758 (2002).
also increased the transfection efficiency, however, the in-
crease was less dramatic than for p(HPMA-DMAE)-based
polyplexes [1.8-fold increase for pDMAEMA polyplexes vs.
6.2-fold increase for p(HPMA-DMAE) polyplexes]. This can
be ascribed to the superior intrinsic endosomal escape capa-
bility of pDMAEMA polyplexes. For pDMAEMA poly-
plexes, the capacity of INF to break down the endosomal
barrier adds little to the transfection efficiency. p(HPMA-
DMAE) polyplexes coated with the INF peptide showed
some minor cytotoxicity, but this toxicity is independent of
the polymer to DNA ratio (Fig. 7) and thus probably caused
by the peptide and not by the polymer. For pDMAEMA
polyplexes, coating with INF-7 does not influence the (sub-
stantial) toxicity of the polymer.
Although cytosolic delivery of released DNA is likely
enhanced for pHPMA-DMAE polyplexes (after endosomal
escape via the INF peptide) because of the hydrolysis of the
cationic side-chains and the subsequent disintegration of the
polyplexes, the transfection efficiency is comparable to poly-
plexes based on the nondegradable polymer pDMAEMA. It
is possible that one of the next barriers, like nuclear transport
or nuclear uptake, is limiting the transfection process, and
therefore protein expression is not enhanced for pHPMA-
DMAE-based polymers. Passage of these barriers may be
facilitated for nonviral vectors by improving the cellular traf-
5. S. C. De Smedt, J. Demeester, and W. E. Hennink. Cationic
polymer based gene delivery systems. Pharm. Res. 17:113–126
(2000).
6. M. D. Brown, A. G. Schatzlein, and I. F. Uchegbu. Gene delivery
with synthetic (non viral) carriers. Int. J. Pharm. 229:1–21 (2001).
7. C. M. Wiethoff and C. R. Middaugh. Barriers to nonviral gene
delivery. J. Pharm. Sci. 92:203–217 (2003).
8. O. Boussif, F. Lezoualch, M. A. Zanta, M. D. Mergny, D. Scher-
man, B. Demeneix, and J. P. Behr. A versatile vector for gene and
oligonucleotide transfer into cells in culture and in vivo – poly-
ethylenimine. Proc. Natl. Acad. Sci. U. S. A. 92:7297–7301 (1995).
9. J. Haensler and F. C. Szoka. Polyamidoamine cascade polymers
mediate efficient transfection of cells in culture. Bioconjug.
Chem. 4:372–379 (1993).
10. P. van de Wetering, E. E. Moret, N. M. E. Schuurmans-
Nieuwenbroek, M. J. van Steenbergen, and W. E. Hennink.
Structure-activity relationships of water-soluble cationic methac-
rylate/methacrylamide polymers for nonviral gene delivery. Bio-
conjug. Chem. 10:589–597 (1999).
11. N. K. Subbarao, R. A. Parente, F. C. Szoka, Jr., L. Nadasdi, and
K. Pongracz. pH-dependent bilayer destabilization by an amphi-
pathic peptide. Biochemistry 26:2964–2972 (1987).
12. C. Plank, B. Oberhauser, K. Mechtler, C. Koch, and E. Wagner.
The influence of endosome-disruptive peptides on gene-transfer
using synthetic virus-like gene-transfer systems. J. Biol. Chem.
269:12918–12924 (1994).
13. A. M. Funhoff, C. F. van Nostrum, G. A. Koning, N. M. E.
Schuurmans-Nieuwenbroek, D. J. A. Crommelin, and W. E. Hen-
nink. Endosomal escape of polymeric gene delivery complexes is

176
Funhoff et al.
not always enhanced by polymers buffering at low pH. Biomac-
romolecules (in press, 2004).
14. T. R. Kyriakides, C. Y. Cheung, N. Murthy, P. Bornstein, P. S.
Stayton, and A. S. Hoffman. pH-Sensitive polymers that enhance
intracellular drug delivery in vivo. J. Controlled Release 78:295–
303 (2002).
15. C. Arigita, N. J. Zuidam, D. J. A. Crommelin, and W. E. Hen-
nink. Association and dissociation characteristics of polymer/
DNA complexes used for gene delivery. Pharm. Res. 16:1534–
1541 (1999).
16. Y. B. Lim, Y. H. Choi, and J. S. Park. A self-destroying polycat-
ionic polymer: biodegradable poly(4-hydroxy-L-proline ester). J.
Am. Chem. Soc. 121:5633–5639 (1999).
17. D. Putnam and R. Langer. Poly(4-hydroxy-L-proline ester): low-
temperature polycondensation and plasmid DNA complexation.
Macromolecules 32:3658–3662 (1999).
18. Y-B. Lim, S-O. Han, H-U. Kong, Y. Lee, J-S. Park, B. Jeong, and
S. W. Kim. Biodegradable polyester, poly[alpha-(4 aminobutyl)-
L-glycolic acid], as a non-toxic gene carrier. Pharm. Res. 17:811–
816 (2000).
19. Y-B. Lim, C-H. Kim, K. Kim, S. W. Kim, and J-S. Park. Devel-
opment of a safe gene delivery system using biodegradable poly-
mer, poly[␣-(4-aminobutyl)-L-glycolic acid]. J. Am. Chem. Soc.
122:6524–6525 (2000).
20. H. Petersen, T. Merdan, K. Kunath, D. Fischer, and T. Kissel.
Poly(ethylenimine-co-L-lactamide-co-succinamide): a biodegrad-
able polyethylenimine derivative with an advantageous pH-
dependent hydrolytic degradation for gene delivery. Bioconjug.
Chem. 13:812–821 (2002).
21. C. H. Ahn, S. Y. Chae, Y. H. Bae, and S. W. Kim. Biodegradable
poly (ethylenimine) for plasmid DNA delivery. J. Controlled Re-
lease 80:273–282 (2002).
22. J. Luten, J. H. van Steenis, R. van Someren, J. Kemmink, N. M.
E. Schuurmans-Nieuwenbroek, G. A. Koning, D. J. A. Cromme-
lin, C. F. van Nostrum, and W. E. Hennink. Polyphosphazenes as
biodegradable non-viral vector systems. J. Controlled Release 89:
483–497 (2003).
23. C. M. Varga, K. Hong, and D. A. Lauffenburger. Quantitative
analysis of synthetic gene delivery vector design properties. Mol.
Therapy 4:438–446 (2001).
24. P. van de Wetering, N. J. Zuidam, M. J. van Steenbergen, O. A.
G. J. Houwen, W. J. M. Underberg, and W. E. Hennink. A
mechanistic study of the hydrolytic stability of poly(2-(dimethyl-
amino)ethyl methacrylate). Macromolecules 31:8063–8068 (1998).
25. W. N. van Dijk-Wolthuis, M. J. van Steenbergen, W. J. Under-
berg, and W. E. Hennink. Degradation kinetics of methacrylated
dextrans in aqueous solution. J. Pharm. Sci. 86:413–417 (1997).
26. J. Kopecek, P. Kopeckova, T. Minko, and Z. R. Lu. HPMA co-
polymer-anticancer drug conjugates: design, activity, and mecha-
nism of action. Eur. J. Pharm. Biopharm. 50:61–81 (2000).
27. D. Oupicky, C. Konak, K. Ulbrich, M. A. Wolfert, and L. W.
Seymour. DNA delivery systems based on complexes of DNA
with synthetic polycations and their copolymers. J. Controlled
Release 65:149–171 (2000).
28. D. Oupicky, C. Konak, and K. Ulbrich. DNA complexes with
block and graft copolymers of N-(2-hydroxypropyl) methacryl-
amide and 2-(trimethylammonio)ethyl methacrylate J. Biomater.
Sci. Polymer Ed. 10:573-590 (1999).
29. J. Y. Cherng, P. van de Wetering, H. Talsma, D. J. A. Crommelin,
and W. E. Hennink. Effect of size and serum proteins on trans-
fection efficiency of poly((2-dimethylamino)ethyl methacrylate)-
plasmid nanoparticles. Pharm. Res. 13:1038–1042 (1996).
30. T. Bieber, W. Meissner, S. Kostin, A. Niemann, and H. P. El-
sasser. Intracellular route and transcriptional competence of
polyethylenimine-DNA complexes. J. Controlled Release 82:441–
454 (2002).
31. W. T. Godbey, M. A. Barry, P. Saggau, K. K. Wu, and A. G.
Mikos. Poly(ethylenimine)-mediated transfection: a new para-
digm for gene delivery. J. Biomed. Mater. Res. 51:321–328 (2000).
32. J. P. Yang and L. Huang. Overcoming the inhibitory effect of
serum on lipofection by increasing the charge ratio cationic lipo-
some to DNA. Gene Ther. 4:950–960 (1997).
33. F. J. Verbaan, C. Oussoren, C. J. Snel, M. Verbeek, D. J. A.
Crommelin, W. E. Hennink, and G. Storm. Steric stabilization of
poly(2-(dimethylamino)ethyl methacrylate) based polyplexes
mediates prolonged circulation and tumor targeting in mice. J.
Gene Med.
34. V. Escriou, M. Carriere, D. Scherman, and P. Wils. NLS biocon-
jugates for targeting therapeutic genes to the nucleus. Adv. Drug
Deliv. Rev. 55:295–306 (2003).
35. E. Hebert. Improvement of exogenous DNA nuclear importation
by nuclear localization signal-bearing vectors: a promising way
for non-viral gene therapy? Biol. Cell 95:59–68 (2003).