Degradable PEG-folate coated poly(DMAEA-co-BA)phosphazene-based polyplexes exhibit receptor-specific gene expression

Article abstract of DOI:10.1016/j.ejps.2007.12.003

A new cationic biodegradable polyphosphazene was developed, bearing both pendant primary and tertiary amine side groups, poly(2-dimethylaminoethylamine-co-diaminobutane)phosphazene (poly(DMAEA-co-BA)phosphazene). PEG and PEG-folate were coupled to polyple

Full text of DOI:10.1016/j.ejps.2007.12.003

european journal of pharmaceutical sciences 3 3 ( 2 0 0 8 ) 241–251
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/ejps
Degradable PEG-folate coated
poly(DMAEA-co-BA)phosphazene-based
polyplexes exhibit receptor-specific gene expression
a
a
a
b
J. Luten , M.J. van Steenbergen , M.C. Lok , A.M. de Graaff ,
a
a
a,∗
C.F. van Nostrum , H. Talsma , W.E. Hennink
a
Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Faculty of Pharmaceutical Sciences,
Utrecht University, P.O. Box 80.082, 3508 TB Utrecht, The Netherlands
Department of Biochemistry & Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1,
b
3
584 CL Utrecht, The Netherlands
a r t i c l e i n f o
a b s t r a c t
Article history:
A new cationic biodegradable polyphosphazene was developed, bearing both pen-
Received 6 October 2007
Received in revised form
dant primary and tertiary amine side groups, poly(2-dimethylaminoethylamine-co-
diaminobutane)phosphazene (poly(DMAEA-co-BA)phosphazene). PEG and PEG-folate were
coupled to polyplexes based on this poly(DMAEA-co-BA)phosphazene, leading to small (size
100 and 120 nm, respectively) and almost neutral particles. In vitro tissue culture experi-
ments showed a low cytotoxicity of both uncoated and coated polyplexes. However, the
PEG coated polyplexes showed a 2-fold lower transfection activity in OVCAR 3 cells as com-
pared to the uncoated polyplexes. On the other hand, the PEG-folate coated polyplexes had
a 3-fold higher transfection than the PEGylated polyplexes. When free folate was added
to the transfection medium, only the transfection activity of the targeted polyplexes was
reduced, indicating internalization of the targeted PEG polyplexes via the folate receptor.
Confocal laser scanning microscopy confirmed a lower binding and uptake of the PEGylated
polyplexes by OVCAR-3 cells when compared to uncoated and folate-PEGylated polyplexes.
While uncoated polyplexes induced aggregation of erythrocytes at polymer concentrations
of 0.09 ␮g/mL, the PEGylated systems could be incubated at ten times higher concentra-
tion before aggregation occurred indicating excellent shielding of the surface charge of
the polyplexes by grafting of PEG. In conclusion, the targeted delivery of poly(DMAEA-
co-BA)phosphazene bases polyplexes and their improved compatibility with erythrocytes
makes them interesting for in vivo applications.
4
December 2007
Accepted 4 December 2007
Published on line 8 December 2007
Keywords:
Gene delivery
Polyplex
Polyphosphazene
Folate
Biodegradable
©
2008 Published by Elsevier B.V.
1
.
Introduction
get tissues with minimal toxicity and immunogenicity, and
deliver the intact gene into the nucleus of target cell to get
high levels of gene expression. Viral vectors are the most
efficient gene delivery systems known so far, however, they
also have some severe drawbacks, like the induction of an
A carrier system needs to fulfill the following requirements
to be a promising candidate for in vivo gene delivery. The
carrier should be able to efficiently accumulate in specific tar-
∗
Corresponding author. Tel.: +31 30 2536964; fax: +31 30 2517839.
E-mail address: W.E.Hennink@uu.nl (W.E. Hennink).
0928-0987/$ – see front matter © 2008 Published by Elsevier B.V.
doi:10.1016/j.ejps.2007.12.003

2
42
european journal of pharmaceutical sciences 3 3 ( 2 0 0 8 ) 241–251
immune response, in particular after repeated administration,
possible recombination with wild-type viruses, limitations
in the size of inserted DNA and oncogeniticity (McTaggart
and Al-Rubeai, 2002; Gorecki, 2001; Schreier, 1994; Byrnes
et al., 1995). Synthetic carriers such as polymers, therefore,
have become an attractive alternative due to their relative
safety and their lack of restraints on the size of the plas-
mid DNA to be delivered (Felgner and Rhodes, 1991; Curiel
et al., 1991). Among the most studied polymers are pEI,
pLL and pDMAEMA (Asayama et al., 1997; Boussif et al.,
PEGylation in the case of polyphosphazene-based polyplexes
requires a polymer with a functional group that is able to cova-
lently bind the PEG chain with or without a targeting ligand.
In this paper, we report the synthesis, characterization of a
polyphosphazene capable of post-PEGylation and the in vitro
evaluation of the corresponding PEGylated polyplexes with
and without folate targeting groups in OVCAR-3 cells.
2
.
Materials and methods
1
995; van de Wetering et al., 1997). However, these poly-
2
.1.
Materials
mers are either non-biodegradable (pEI and pDMAEMA) or
show low transfection activity (pLL). In recent years there
has been an increasing interest in biodegradable polymers,
like polyesters, polyphosphazenes, degradable pEI, polyphos-
phoesters (Luten et al., 2003, 2006, 2007; Kim et al., 2005;
Zhong et al., 2005; Christensen et al., 2006) as nonviral gene
delivery vectors. Recently, the biodistribution and in vivo trans-
fection efficiency of polyplexes composed of plasmid DNA
and p(DMAEA)phosphazene were investigated after intra-
venous administration in tumour bearing mice (de Wolf et
al., 2005). Polyplexes based on poly(DMAEA)phosphazene were
shown to have a preferential tumor gene expression (organ
gene expression <1/100 of tumour gene expression). This
observed selectivity mediated by the p(DMAEA)phosphazene
polyplexes could enable the application of this polymer to
deliver therapeutic genes to tumours. However, the polyplexes
were rapidly cleared from the circulation (<7% ID, at 60 min
after administration). One way of improving the circulation
kinetics of colloidal gene delivery particles is by PEGylation
Hexachlorocyclotriphosphazene (NPCl2)3 (>99.99%), calcium
ꢀ
sulphate
dihydrate,
folic
acid,
N-ethyl-N -(3-
dimethylaminopropyl)-carbodiimid hydrochloride (EDC),
N-hydroxysuccinimide (NHS), benzyl chloroformate (Z-Cl),
di-tert-butyl dicarbonate (BOC2O), 1,4-diaminobutane (BA)
and 2-dimethylaminoethylamine (DMAEA) were purchased
from Aldrich (Zwijndrecht, The Netherlands) and were used
as received. COOH-PEG5000-NH2·HCl and PEG5000-NHS were
purchased from Nektar (Huntsville, AL, USA). 1,4-Dioxane
(
Biosolve, Valkenswaard, The Netherlands) was dried over
and distilled from sodium metal using benzophenone as
an indicator. INF-7, a 24 amino acid containing peptide
with fusogenic activity derived from the influenza virus,
was synthesized via standard Fmoc solid-phase synthesis
(
Plank et al., 1994). PEI (branched, 25 kDa) was purchased
from Sigma (Zwijndrecht, The Netherlands). PDMAEMA
Mn = 92 kDa) was synthesized by a radical polymerization of
-dimethylaminoethyl methacrylate in an aqueous solution
Cherng et al., 1996). The plasmid pCMVLacZ, containing a
(
2
(
(Verbaan et al., 2004; de Wolf et al., 2007b). However, a major
drawback of PEGylation of polyplexes is their lower transfec-
tion activity due to the loss of non-specific binding to the target
cell. Introduction of targeting moieties on their surfaces is
an interesting option to restore the transfection potential of
poly(DMAEA)phosphazene-DNA polyplexes.
bacterial LacZ gene preceded by a nuclear localization signal
under control of a CMV promoter, was purchased from San-
vertech (Heerhugowaard, The Netherlands). The plasmid DNA
was labeled with Cy5 dye using the Mirus Label It nucleic acid
labeling kit (Sopachem, Wageningen, The Netherlands) and
the cells were embedded in FluorSave Reagent (Calbiochem,
San Diego, USA) for confocal laser scanning microscopy
Targeting of the folate receptor, a glycopolypeptide with a
−9
high affinity (K < 10 M) for folic acid and the physiologic
d
circulating form of the vitamin, N5-methyltetrahydrofolate
(
CLSM) experiments.
(Antony, 1996; Guo et al., 2006; Wang and Hsiu, 2005) had
received much attention in recent years, since the folate recep-
tor has been shown to be over expressed in human cancer
cells and in addition, folic acid is a relatively small molecule
2.2. Synthesis of poly(DMAEMA-co-BA)phosphazene
(
MW 441 Da) which does consequently have only limited
Mono-Z-diaminobutane was prepared as shown in Fig. 1,
according to literature procedures (Atwell and Denny, 1984).
In detail, a solution of di-tert-butyl dicarbonate (100 mmol,
21.8 g) in dioxane (400 mL) was added over a period of 5 h to a
solution of 1,4-diaminobutane (700 mmol, 70.4 mL) in dioxane
(400 mL) The mixture was stirred overnight at room temper-
ature and the solvent was removed in vacuo. Water (400 mL)
was added to the residue and stirred for 20 min. The insoluble
bis-substituted product was removed by filtration. Next, the
filtrate was extracted with DCM (3× 200 mL) and the organic
layers were washed with brine (200 mL), dried with MgSO4, and
concentrated in vacuo to afford 17 g N-BOC-1,4-diaminobutane
(88%). 1H NMR (CDCl3, ı in ppm) 4.94 (BOC-NH, 1H, br), 3.03
(CH2, 2H, br), 2.64 (CH2, 2H, br), 1.98 (NH2, 2H, br), 1.42 (CH2CH2,
4H, br), 1.38 (CH3, 9H, s).
effects on the dimensions of the carrier system (Corona et
al., 1998; Elnakat and Ratnam, 2004; Sudimack and Lee, 2000).
To improve the availability of folate for receptor-binding,
attachment to a poly(ethylene glycol) (PEG) spacer was found
effective for the targeted delivery liposomes (Lee and Low,
1
994; Reddy and Low, 2000; Lee and Kim, 2005) and similar
findings were reported for polyplexes (Seymour et al., 1991).
As aforementioned, PEGylation of polyplexes has been shown
to significantly improve their pharmacokinetics after intra-
venous administration (Verbaan et al., 2004; Cho et al., 1998;
Ogris et al., 1999). On the other hand, PEGylated cationic
polymers have been shown to exhibit less satisfactory DNA-
condensing properties (Erbacher et al., 1999; Zuidam et al.,
2
000) which can be overcome by conjugating PEG to pre-
formed polyplexes (post-PEGylation) (Verbaan et al., 2004;
van Steenis et al., 2003; Blessing et al., 2001). To use post-
Next, N-BOC-1,4-diaminobutane (13.9 mmol, 3.5 g) was
reacted with Z-Cl (1.1 equiv., 3.2 g) under Schotten–Baumann

european journal of pharmaceutical sciences 3 3 ( 2 0 0 8 ) 241–251
243
Fig. 1 – Synthesis of mono-Z-diaminobutane.
◦
conditions (1 M aq. NaOH, 1.1 equiv., 0–20 C, 16 h) to give
then sealed under high vacuum. The reaction mixture was
heated to 250 C for 6 h. The polymer/trimer mixture was then
1
◦
the required unsymmetrical dicarbamate (6.0 g, 82%). H NMR
(
2
CDCl3, ı in ppm) 7.35 (CH, 5H, s), 5.09 (CH2, 2H, s), 4.69 (NH,
H, br), 3.21 (CH2, 2H, br), 3.12 (CH2, 2H, br), 1.51 (CH2CH2, 4H,
cooled to room temperature and diluted with 1,4-dioxane
(200 mL), containing mono-Z-diaminobutane (1.6 g, 7.0 mmol,
NH2/P-Cl ratio 0.1) and triethylamine (20 mL, in excess). The
reaction mixture was stirred for 2 days at room tempera-
ture followed by the addition of 2-dimethylaminoethylamine
(DMAEA) (19 mL, 0.17 mol). The mixture was stirred for 6 days
followed by removal of the formed triethylamine-HCl salt by
filtration and the filtrate containing the polymer was con-
centrated under reduced pressure. Next, the polymer was
dissolved in 100 mL MeOH and the Z-group was removed using
Pd/C (1.0 g, 10%) and H2 gas overnight at room temperature.
Subsequently, the reaction mixture was concentrated and
the polymer was dialyzed (cellulose acetate with a molecular
weight cut off of 12,000–14,000 Da) against water for 3 days.
The polyphosphazene was collected by lyophilization (yield:
300 mg, 4%).
br), 1.43 (CH3, 9H, s).
The dicarbamate was subsequently selectively depro-
◦
tected with TFA in DCM (1:1) (0 C, 1 h) to yield mono-Z-
diaminobutane in 76%. 1H NMR (CDCl3, ı in ppm) 7.34 (CH,
5
br), 2.73 (CH2, 2H, br), 1.98 (NH2, 2H, br), 1.51 (CH2CH2, 4H,
br).
A polyphosphazene co-polymer with pendant primary
and tertiary amines was synthesized as shown in Fig. 2.
Poly(dichloro)phosphazene was synthesized via bulk polymer-
ization. A glass ampoule was treated with TMS-Cl, cleaned and
dried in a vacuum oven prior to use. Next, the ampoule was
filled with hexachlorocyclotriphosphazene (4.0 g, 70 mmol P-
Cl), and CaSO4·2H2O (10 mg, 0.06 mmol) and the ampoule was
H, s), 5.08 (CH2, 2H, s), 4.95 (NH, 1H, br), 3.18 (CH2, 2H,
Fig. 2 – Synthesis of poly(DMAEA-co-BA)phosphazene.

2
44
european journal of pharmaceutical sciences 3 3 ( 2 0 0 8 ) 241–251
Fig. 3 – Synthesis of PEG-folate-NHS and coupling to poly(DMAEA-co-BA)phosphazene.
2
.3.
Synthesis of NHS-activated folate-conjugated PEG
2.4.
Characterization of
poly(DMAEMA-co-BA)phosphazene
Folate N-hydroxysuccinimidyl ester (folate-NHS) was pre-
pared according to a literature procedure (Fig. 3) (Cho et al.,
1H NMR spectra were recorded on a Varian G-300 300 MHz
spectrometer (Varian, Palo Alto CA, USA) and 31P NMR spectra
on a Varian Inova 500 MHz spectrometer (Varian, Palo Alto, CA,
USA). Chemical shifts are given relative to tetramethylsilane or
phosphoric acid (85%) as an external reference; measurements
were performed in CD3OD.
Weight average molecular weight (Mw) and number aver-
age molecular weight (Mn) of the synthesized polymers were
determined by gel permeation chromatography (GPC), using a
Viscotek VE 2001 system (de Wolf et al., 2007a). Two Shodex SB-
804 M columns with a pre-column (Shodex OH-pak SB-G) were
connected to a Triple Detector Array 302, equipped with a low
and a right angle light scattering detector, a viscometer detec-
tor and a refractive index detector. The eluent was 0.3 M NaAc
(pH 4.4) with a flow-rate of 1.0 mL/min (Jiang et al., 2006). The
refractive index increment (dn/dc) of p(DMAEA)phosphazene
was determined by injecting p(DMAEA) phosphazene solu-
tions with different concentrations (1–10 mg/mL) directly into
2
005). In brief, folic acid (65 mg) was dissolved in DMSO (2.6 mL)
to which triethylamine (0.05 mL) was added. After addition of
N-hydroxysuccinimide (NHS) (38 mg, 2.2 equiv.), and N-ethyl-
ꢀ
N -(3-dimethylaminopropyl)-carbodiimid hydrochloride (EDC)
(
1
30 mg, 1.1 equiv.), the mixture was stirred in the dark for
8 h. Folate-NHS was coupled to NH2-PEG-COOH as follows:
the folate-NHS solution was added to PEG (350 mg, 70.0 ␮mol)
dissolved in a mixture of DMSO (2.5 mL and triethylamine
(
0.1 mL), after which the mixture was stirred overnight. The
folate-PEG-COOH was dialyzed extensively against deionized
H2O. Insoluble products including unconjugated folic acid
were removed by filtration through a 0.2-␮m filter (Millipore,
Bedford, MA). The soluble product was collected and freeze-
dried. Next, folate-PEG-COOH was reacted with EDC/NHS (10
equiv. to COOH) in DMSO to yield folate-PEG-NHS. The con-
jugate was purified by precipitation in diethylether, collected
◦
and stored at −20 C.

european journal of pharmaceutical sciences 3 3 ( 2 0 0 8 ) 241–251
245
Fig. 4 – 1H NMR spectra of Z-protected p(DMAEA-co-BA)phosphazene (A) and p(DMAEA-co-BA)phosphazene (B).
the refractive index detector and was determined to be
.159 mL/g. Molecular weight data analysis was performed by
to 0.5 ␮mol glycine/mL. As a control p(DMAEA)phosphazene,
lacking primary amines, was used.
0
OmniSEC 4.1 software.
For the detection of the PEG conjugates a mesopor column
was used; detection was done with a UV detector (363 nm).
DMF/LiCl (10 mM) was used as the eluent with a flow rate of
2.5.
PEGylation of polyplexes
0
.7 mL/min.
PEGylation of polyplexes was performed by adding DMSO solu-
tions of either NHS-PEG-folate (53.9 mg/mL stock solution) or
NHS-PEG (49.1 mg/mL stock solution) to the polyplexes, and
incubating these for 15 min at room temperature. The poly-
plexes were prepared by adding 200 ␮L of 4-(2-hydroxyethyl)-
1-piperazine-ethanesulfonic acid (HEPES) buffer (5.0 mM, pH
7.4), containing 15 ␮g of plasmid DNA to 800 ␮L of HEPES buffer
(5.0 mM, pH 7.4), with 90 ␮g of polymer (6:1, w/w, N/P ratio
18). The resulting dispersion was vortexed for 5 s and after
30 min incubation time at room temperature the size of the
The amount of primary amines in the polymer was
determined spectrophotometrically by the ninhydrin assay
Romberg et al., 2006). Samples were prepared in 1 M sodium
(
acetate buffer, pH 5.5, containing approximately 0.2 ␮mol
NH2/mL. Next, 1 mL of this polymer solution was taken and
freshly prepared ninhydrin solution (2.0 g ninhydrin and 0.3 g
hydrindantin dissolved in 75 mL of 2-methoxyethanol and
2
5 mL of 4 M sodium acetate buffer, pH 5.5) was added.
The mixtures were vortexed and incubated for 15 min at
1
◦
◦
00 C. After cooling to room temperature, the samples were
polyplexes was determined using DLS at 25 C. To this dis-
diluted with 5 mL of 50% ethanol in water and the absorbance
was measured at 570 nm with a PerkinElmer Lambda 2
UV/vis spectrophotometer. Glycine in a 1 M acetate buffer
was used for calibration. The calibration curve was linear up
persion 50 ␮L of either NHS-PEG-folate or NHS-PEG solution
was added. The concentration of the PEG solutions was var-
ied by diluting the stock solutions mentioned above to obtain
different PEG/primary amine ratios.

2
46
european journal of pharmaceutical sciences 3 3 ( 2 0 0 8 ) 241–251
2
.6.
Size and charge measurements of the different
studies with the following modifications. A 16-well glass plate
polyplexes
was used instead of a 96-well plate and after one hour incuba-
tion time, the cells were rinsed with phosphate buffered saline
(PBS), fixed with 2.0% paraformaldehyde solution in PBS for
Z-average diameter and polydispersity of the poly-
mer/plasmid DNA complexes were measured by dynamic light
scattering (DLS) using a Malvern ALV CGS-3 system equipped
with an argon-ion laser (488 nm, 10.4 mW) (uniphase). The
ALV correlator software in combination with DTS (Nano)
and ALVFilereader software was used. The instrument was
calibrated with 100 nm poly(styrene) particles dispersed in
water. In a typical experiment, 200 ␮L of HEPES buffer (5.0 mM,
pH 7.4), containing 15 ␮g of plasmid DNA was added to 800 ␮L
of HEPES buffer (5.0 mM, pH 7.4), with various amounts of
polymer. The resulting solution was vortexed for 5 s and after
◦
1 h at 4 C and rinsed again with PBS. Cells were embedded in
FluorSave Reagent and covered with a cover glass. Fluorescent
and transmitted light microscope images of cells were taken
simultaneously using a Leica TCS-SP microscope and analyzed
using Leica TCS-SP Power Scan software (Leica Microsystems,
Rijswijk, The Netherlands).
2
.9.
Polyplex-induced erythrocyte aggregation
Blood was collected from the vena cava inferior of female
BALB/c mice. Heparin (approximately 50 Units per sample) was
added to prevent blood coagulation. The blood samples were
3
0 min incubation time at room temperature the size of the
◦
polyplexes was determined using DLS at 25 C.
◦
The ꢀ-potential of the polyplexes was measured at 25 C
using a Zetasizer Nano-Z (Malvern). The instrument was
calibrated using a poly(styrene) dispersion with a known ꢀ-
potential. In a typical experiment, 200 ␮L of HEPES buffer
◦
centrifuged for 12 min at 4 C at 1200 × g and the pelleted ery-
throcytes were washed three times with HBS (5.0 mM Hepes,
150 mM NaCl, pH 7.4). Polyplexes were prepared as described
before at a DNA concentration of 15 ␮g/mL. In a V-shaped 96-
well plate 100 ␮L of polyplex dispersion were pipetted by serial
dilution of the polyplex solution, leading to DNA concentra-
tions ranging from 3.7 ng/mL to 15 ␮g/mL.
(
5.0 mM, pH 7.4), containing 15 ␮g of plasmid DNA was added
to 800 ␮L of HEPES buffer (5.0 mM, pH 7.4) with various
amounts of polymer. The resulting solution was vortexed for
5
s and after 30 min incubation time at room temperature the
Subsequently, 50 ␮L of a 450 mM NaCl solution was added,
followed by the addition of 50 ␮L of a 1% erythrocyte sus-
pension in HBS. The erythrocytes were allowed to sediment
overnight. Erythrocyte aggregation was scored visually.
surface charge properties of the polyplexes was determined.
2
.7.
Transfection studies
Transfection studies were done in OVCAR-3 cells as described
previously using pCMVLacZ as reporter gene (van Steenis et
al., 2003; Cherng et al., 1996). In brief, 96-well plates were
3.
Results and discussion
4
−2
seeded with cells at a density of 3 × 10 cm
24 h before
3.1.
Synthesis of poly(DMAEMA-co-BA)phosphazene
transfection. Polyplexes were prepared as follows: 150 ␮L of
polymer solution in HBS was added to 50 ␮L of plasmid solu-
tion (50 ␮g/mL) in HBS, and after incubation for 30 min at room
temperature, 5 ␮L of either PEG-NHS (9 mg/mL) or PEG-folate
solution (10 mg/mL) in DMSO was added, and the polyplexes
were incubated for another 15 min. Finally, 45 ␮L of INF-7 pep-
tide (170 ␮g/mL) was added to the dispersion before transfer
to the cells. After rinsing the cells with HBS, 100 ␮L of poly-
plex dispersion and 100 ␮L of culture 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 deter-
mine the number of viable cells using an XTT colorimetric
assay. Transfection efficiencies were normalized to that of
pDMAEMA polyplexes (polymer/DNA ratio of 3/1) in the pres-
ence of serum. Also, the transfection activity of polyplexes
of PEI (branched, 25 kDa) in the presence of 5% serum was
measured.
A poly(DMAEMA-co-BA)phosphazene with pendant primary
and tertiary amines was synthesized as shown in Fig. 2. The
synthesis of poly(DMAEMA-co-BA)phosphazene was explored
via several routes. The use of mono-BOC-diaminobutane and
mono-BOC amino ethanol instead of mono-Z-diaminobutane
was unsuccessful, because deprotection of the primary amine
in CH2Cl2/trifluoro acetic acid 1:1 led to extensive chain
scission. Therefore, the Z-group was investigated as protect-
ing group to synthesize the aimed polymer. Direct mono-Z
protection of 1,4-diaminobutane was practically impossible
(yields were typically below 3% using Z-Cl in aq. NaOH/THF
◦
at 0 C) while the di-Z protected diamine was formed pre-
dominately (Atwell and Denny, 1984). Mono-Z-diaminobutane
was therefore synthesized following a known method that
consisted of a route via mono-BOC-mono-Z-diaminobutane
(Biagbrough et al., 1996). The mono-Z-protected diaminobu-
tane was subsequently used to synthesize the Z-protected
poly(DMAEMA-co-BA)phosphazene. First, mono-Z-protected
diaminobutane was reacted with poly(dichloro)phosphazene
in a 1:10 NH2/P-Cl ratio. Then, an excess of DMAEA was used
to substitute the remaining chloride atoms. Other strategies,
like adding both substituents simultaneously or reversing the
2
.8.
Confocal laser scanning microscopy studies
order of addition of the substituents were unsuccessful. 1
H
The uptake and intracellular presence of polyplexes with
Cy5-labeled plasmid DNA was visualized via confocal laser
scanning microscopy. This experiment with labeled DNA was
essentially performed as described above for the transfections
NMR analysis showed (Fig. 4) that 12% of the side groups was Z-
protected BA. This was also confirmed with a ninhydrin assay
after the Z-group was successfully removed by Pd/C and H2-
gas (Fig. 4) (Romberg et al., 2006). The 31P NMR spectrum of

european journal of pharmaceutical sciences 3 3 ( 2 0 0 8 ) 241–251
247
Fig. 6 – Particle diameter and ꢀ-potential of the
poly(DMAEA-co-BA)phosphazene polyplexes at different
polymer/DNA ratios (w/w) in HEPES buffer (5.0 mM, pH 7.4).
for poly(DMAE)phosphazene (Luten et al., 2003). The addition
of NaCl (final concentration 150 mM) to the polyplexes caused
a gradual increase in size (from 80 to 170 nm in 10 h). This has
been observed before for poly(DMAE)phosphazene and also
for other polymers (Luten et al., 2006; Ogris et al., 1998). The
surface grafting of PEG to the polyplexes affected both size
and surface charge (Fig. 7). The polyplexes were made at a
polymer/DNA ratio of 6 (w/w) and after 30 min PEG-NHS or
folate-PEG-NHS is added followed by 15 min of incubation. In
contrast to naked polyplexes, the PEGylated polyplexes were
stable in 150 mM NaCl for more than 4 h. In line with obser-
vation for othe pegylated polyplexes (Verbaan et al., 2004; van
Steenis et al., 2003), a small increase in size was observed (from
to 80 nm (no PEG) to 140 nm at a PEG/primary amine ratio of
6, Fig. 7) with increasing extent of PEG grafting. Zeta potential
measurements (Fig. 7) showed that PEG grafting resulted in a
drop of the surface charge from +22 to 6 mV, indicating effec-
tive shielding of the surface charge of the polyplexes by the
PEG chains.
Fig. 5 – GPC results of the PEG-folate construct (A) and folic
acid (B) at 363 nm. A physical mixture of folic acid and PEG
did not cause a shift of the folate signal (data not shown).
the deprotected polymer showed only one signal at 1.7 ppm,
indicating complete substitution of the chloride atoms and
overlap of P-NH-BA and P-NH-DMAEA signal.
GPC analysis showed that the weight average molecu-
lar weight and the number average molecular weight of
poly(DMAEA-co-BA)phosphazene were 20 and 8.5 kDa, respec-
tively.
Conjugation of PEG-folate to the polyplexes caused even
more pronounced effects (Fig. 8). The size of the targeted
polyplexes increased from 80 nm (no PEG) to 160 nm at
PEG/primary amine ratio of 6. At the PEG/primary amine ratio
of 1.5 and higher, the surface charge of these polyplexes
was neutral to slightly negative as has been observed before
for folate targeted systems (van Steenis et al., 2003) and is
attributed to negatively charged carboxylic acid group of the
folate ligand.
3
.2.
Synthesis of NHS-activated folate-conjugated PEG
To PEGylate preformed polyphosphazene-based polyplexes
so called post-PEGylation), an NHS-activated PEG folate con-
(
jugate was prepared according to a described method (Cho et
al., 2005). GPC analysis was used to analyze the synthesized
conjugate. GPC analysis showed that PEG-folate had a much
shorter retention time than the free folic acid (Fig. 5A and B),
whereas a physical mixture of folate and PEG (not shown) did
not show this shift in retention time. This demonstrates that
folate was indeed covalently bound to the PEG chains.
3
.3.
Size and charge measurements of the (un)coated
polyplexes
Dynamic light scattering experiments showed that the
poly(DMAEMA-co-BA)phosphazene is capable to condense
plasmid DNA, yielding polyplexes with a size of around 80 nm
and a ꢀ-potential of +22 mV (Fig. 6) at polymer/plasmid ratios
(
w/w) higher than 6 (N/P = 18). This figure also shows that
neutral aggregates were formed between polymer/plasmid
ratio of 1.5:1 and 3:1 whereas negatively charged polyplexes
with a size of 90 nm were detected at a polymer/plasmid
ratio of 0.75:1. This behavior has been observed before for
other polymer-based polyplexes (Jiang et al., 2006) and also
Fig. 7 – Particle diameter and ꢀ-potential of the
post-PEGylated poly(DMAEA-co-BA)phosphazene
polyplexes at a polymer/DNA ratio of 6 (w/w) and at
different PEG/NH2 ratios in HEPES buffer (5.0 mM, pH 7.4).

2
48
european journal of pharmaceutical sciences 3 3 ( 2 0 0 8 ) 241–251
can be seen in Fig. 9. The activities of polyplexes based on
p(DMAEMA) (N/P 5, 42 mU/well) and BPEI (N/P 6, 62 mU/well)
were used as a reference. The non-PEGylated polyplexes
gave an expression of 45 mU of ␤-galactosidase per well and
were twice as active as the PEG shielded polyplexes. This
is expected since shielding of the polyplexes by PEGylation
reduces the non-specific binding of the polyplexes and sub-
sequent internalization by the OVCAR-3 cells (Verbaan et al.,
2004; van Steenis et al., 2003).
The transfection levels of the folate targeted system were
0 mU of ␤-galactosidase per well and were even slightly
6
Fig. 8 – Particle diameter and ꢀ-potential of the targeted
poly(DMAEA-co-BA)phosphazene polyplexes at a
polymer/DNA ratio of 6 (w/w) and at different PEG/NH2
ratios in HEPES buffer (5.0 mM, pH 7.4).
higher than the transfection levels obtained for the uncoated
polyplexes. The transfection activity of the polyplexes in the
presence of an excess of folic acid (1.0 mM) was also stud-
ied (Fig. 9). It was found that the transfection levels of the
uncoated polyplexes and for the PEGylated polyplexes were
unaffected and remained at the levels obtained without folic
acid incubation. Importantly, the transfection activity of the
folate targeted polyplexes decreased from 60 to 25 mU ␤-
galactosidase per well, a level comparable to that of the
PEGylated polyplexes. These results indicate that the increase
in transfection activity of the folate polyplexes is caused by
binding of the polyplexes to the folate receptor followed by
internalization (folate receptor endocytosis) and eventually to
gene expression (Sabharanjak and Mayor, 2004; Lee and Low,
994).
The relative cell viability of cells incubated with the
un)coated polyplexes is shown in Fig. 10. This figure shows
that the uncoated polyplexes displayed a high cell viability
0.8) at a polymer/DNA ratio of 6 and conjugation with PEG
or PEG-folate does not alter the cell viability of the gene car-
rier. For other polymers an increase of cell viability has been
observed when polyplexes were coated, but probably because
of the low toxicity of the uncoated polyphosphazene poly-
plexes we do not observe this effect (Lee and Kim, 2005; van
Steenis et al., 2003).
3
.4.
In vitro transfection studies with (un)coated
poly(DMAEA-co-BA)phosphazene-based polyplexes
The effect of PEGylation with and without folate on the trans-
fection activity of poly(DMAEA-co-BA)phosphazene-based
polyplexes was studied using OVCAR-3 cells. Ovarium car-
cinoma cells are known to have a high expression of folate
receptors (Corona et al., 1998).
1
The addition of the endosomal membrane disrupting INF-7
peptide to the polyplex dispersion, increased the transfection
activity with a factor 10–15 for the non-coated as well as the
coated poly(DMAEA-co-BA)phosphazene-based polyplexes.
Previously we found that, poly(DMAEA)phosphazene-based
polyplexes did not benefit from the presence of INF-7 in
the transfection of COS-7 cells (Luten et al., 2003). Perhaps
either polyplexes are taken up via different routes by COS-7
and OVCAR-3 cells or poly(DMAEA-co-BA)phosphazene- and
poly(DMAEA)phosphazene-polyplexes are taken up via differ-
ent pathways (Wong et al., 2007). It is also possible that the
molecular weight of the polymer plays an important role on
the transfection properties of poly(DMAEA)phosphazene poly-
plexes. A recent study (de Wolf et al., 2007a) revealed that the
polymer molecular weight not only affects the transfection
efficiency of the polyplexes, but also polyplex formation and
cytotoxicity. The use of poly(DMAEA-co-BA)phosphazene with
a higher molecular weight could lead to better transfection
properties, but this needs further investigation.
(
(
3.5.
Confocal laser scanning microscopy
The cellular association and internalization of the poly-
plexes was studied using CLSM and fluorescently labeled
plasmid. OVCAR-3 cells were incubated with different poly-
plexes, either uncoated, PEGylated or folate targeted, with
◦
a polyphosphazene/plasmid ratio of 6:1 for 1 h at 37 C. The
The effect of PEGylation of the poly(DMAEA-co-
BA)phosphazene polyplexes on their transfection activity
results are shown in Fig. 11. Fluorescence is observed at the
cell membrane as well as in the cytosol of the cells when
Fig. 9 – Transfection efficiencies of polyplexes, post-PEGylated with NHS-PEG or NHS-PEG-folate at 1.5 PEG:NH2 ratio, at a
polymer/DNA ratio of 6:1 (w/w), in medium, in folate-rich medium (1 mM). Efficiencies were determined by measuring the
␤-galactosidase activity. Polyplexes based on p(DMAEMA) (N/P 5) and BPEI (N/P 6) were used as a reference.

european journal of pharmaceutical sciences 3 3 ( 2 0 0 8 ) 241–251
249
Fig. 10 – Cell viabilities after treatment with polyplexes, post-PEGylated with NHS-PEG or NHS-PEG-folate at 1.5 PEG:NH2
ratio, at a polymer/DNA ratio of 6:1 (w/w), in medium, in folate-rich medium (1 mM). Polyplexes based on p(DMAEMA) (N/P
5
) and BPEI (N/P 6) were used as a reference.
they were incubated with uncoated polyplexes, indicating
cell association and cell internalization (Fig. 11A). This is in
line with previous papers in which it is shown that posi-
tively charged polyplexes are able to bind to and be taken
up by cells (Zuidam et al., 2000). For the PEGylated phos-
phazene polyplexes less intense fluorescence was observed
both at the cell membrane and in the cytosol (Fig. 11B)
which points to lower non-specific interaction between the
PEGylated polyplexes and cells as compared to non-coated
particles. Lower binding of the polyplexes to the cell lead
to decrease in cellular uptake of the polyplexes and a lower
transfection activity, in line with the observations reported
in Fig. 9. Incubation of cells with folate-PEG polyplexes
(
Fig. 11C) caused a more intense fluorescence both at the
cell membrane and in the cell as compared to the PEGylated
polyplexes, suggesting more cell association and internaliza-
tion of the targeted polyplexes. PEGylation with folate-PEG
restores the cellular uptake of the polyplexes, leading to higher
transfection levels, as observed in the transfection experi-
ments.
3
.6.
Polyplex-induced aggregation of erythrocytes
Upon intravenous injection of polyplexes, interactions
between polyplexes and blood components will occur
(
2
Verbaan et al., 2004; Dash et al., 1999; Moreau et al.,
002). To study whether PEGylation decreases adverse
interactions between the polyplexes and erythrocytes, the
different polyplexes (polymer/plasmid ratio was 6), either
uncoated, PEGylated, or with PEG-folate, were incubated
with erythrocytes and the aggregation was monitored visu-
ally. Fig. 12 shows the haemaglutinating properties of the
polyplexes when they were incubated with erythrocytes.
As
a control pDMAEMA-based polyplexes (polymer/DNA
ratio 3:1) were used. The pDMAEMA-based polyplexes
possessed the strongest erythrocyte aggregating activ-
ity as reflected by the lowest polymer concentration at
which erythrocyte aggregation did not occur (0.04 ␮g/mL).
Also the uncoated polyphosphazene-based polyplexes dis-
Fig. 11 – CLSM-study of OVCAR-3 cells upon incubation
played
0.09 ␮g/mL). When the polyphosphazene-based polyplexes
were post-PEGylated with PEG or with PEG-folate (1.5 equiv-
alents to N) erythrocyte aggregation occurred at 15
a relatively high erythrocyte aggregating activity
with poly(DMAEA-co-BA)phosphazene/DNA 6:1 polyplexes
(
◦
for one hour at 37 C (A), post-PEGylated
poly(DMAEA-co-BA)phosphazene/DNA 6:1 polyplexes (B)
and post-PEGylated with folate-PEG
poly(DMAEA-co-BA)phosphazene/DNA 6:1 polyplexes (C).
The pictures to the right are the direct transmitted light
pictures taken simultaneously with the CLSM-pictures. The
plasmid DNA was labeled with Cy5.
a
times higher polymer concentration as compared to the
uncoated polyphosphazene-based polyplexes. This shows
that PEG indeed effectively shields the positive charge of
the polyplexes thereby reducing a specific cellular interac-
tions.

2
50
european journal of pharmaceutical sciences 3 3 ( 2 0 0 8 ) 241–251
Fig. 12 – Erythrocyte aggregation by different polyplexes. Haemaglutination is expressed as the highest polymer
concentration at which aggregation was not observed (n = 2).
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4
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