Synthesis of a library of polycationic lipid core dendrimers and their evaluation in the delivery of an oligonucleotide with hVEGF inhibition

Article abstract of DOI:10.1016/j.bmc.2006.03.029

This article follows on from our previous work in the area of non-viral gene delivery using polycationic dendrimers (PCDs). Herein we report on the synthesis and efficacy of a new library of lipid core PCDs in the delivery of the anti-angiogenic oligonucleotide (ODN-1) to retinal pigment epithelial cells. ELISA was used to monitor hVEGF levels in cells transfected with dendriplexes, Cytofectin GSV and control (non-transfected). At 48 h, hVEGF titres had returned to that of the untransfected control for Cytofectin GSV however, a number of dendriplexes continued to exhibit a marked reduction in hVEGF titres.

Full text of DOI:10.1016/j.bmc.2006.03.029

Bioorganic & Medicinal Chemistry 14 (2006) 4775–4780
Synthesis of a library of polycationic lipid core dendrimers and
their evaluation in the delivery of an oligonucleotide
with hVEGF inhibition
Harendra S. Parekh,a,* Robert J. Marano,b Elizabeth P. Rakoczy,b
Joanne Blanchfieldc and Istvan Tothc
aSchool of Pharmacy, The University of Queensland, St Lucia, Qld 4072, Australia
bCentre for Opthalmology and Visual Science and Lion’s Eye Institute, University of Western Australia,
2 Verdun Street, Nedlands, WA 6009, Australia
cSchool of Molecular and Microbial Sciences, The University of Queensland, St Lucia, Qld 4072, Australia
Received 6 December 2005; revised 15 February 2006; accepted 15 March 2006
Available online 5 April 2006
Abstract—This article follows on from our previous work in the area of non-viral gene delivery using polycationic dendrimers
(PCDs). Herein we report on the synthesis and efficacy of a new library of lipid core PCDs in the delivery of the anti-angiogenic
oligonucleotide (ODN-1) to retinal pigment epithelial cells. ELISA was used to monitor hVEGF levels in cells transfected with den-
driplexes, Cytofectin GSVTM and control (non-transfected). At 48 h, hVEGF titres had returned to that of the untransfected control
for Cytofectin GSVTM however, a number of dendriplexes continued to exhibit a marked reduction in hVEGF titres.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
limited success. Electroporation is one such technique
that uses short electrical pulses to create pores in the
cell membrane so facilitating uptake of naked DNA/
ODNs.5 Major drawbacks with the use of electropor-
ation are that the technique is laborious with large
amounts of DNA/ODN required to achieve transfec-
tion, coupled with high cell mortality.6 Viral vectors
are to-date the most efficient gene delivery systems,
however, concerns are emerging about both the short-
and long-term risks they pose. Concerns exist over
potentially lethal immune reactions and limitations
on the size of DNA that can be introduced into cells
using viruses.7 A major obstacle in the delivery of oli-
gonucleotides using a non-viral approach is exposure
to extra- and intracellular barriers, for example,
nucleases.8,9 Several chemically synthesized cationic
delivery systems such as cationic liposomes, polymers
and dendrimers have been developed to overcome
the hurdles associated with gene delivery.10–12 A den-
drimer generally comprises of multiply branched
generations of a polycationic functionality to allow
for electrostatic complexation with DNA/ODN. In
recent years, the delivery of ODNs using dendrimers
and studies to determine uptake into cells have
been reported primarily using polyethyleneimine-
(PEI), polyamidoamine-(PAMAM) and more recently
The transfer of DNA/RNA into eukaryotic cells using
safer alternatives to viral vectors is an area that con-
tinues to receive a great deal of research interest.1–3
Antisense therapy aims to replace defective DNA by
transfer of corrected exogenous genes or portions
thereof to target cells, so transferring replication to
the rectified DNA sequences.4 Attempts to deliver
naked DNA/oligonucleotides (ODNs) have met with
Abbreviations: Boc, tert-Butyloxycarbonyl; Dde, N-(1-(4,4-dimethyl-
2,6-dioxocyclohexylidene)ethyl); CH3CN, acetonitrile; DCC, di-
cyclohexyl carbodiimide; DCM, dichloromethane; DIPEA, N,N-diiso-
propylethylamine; DMF, N,N-dimethylformamide; ES-MS, electro-
spray mass spectroscopy; HBTU, O-(1H-benzotriazol-1-yl)-1,1,3,
3-tetramethyluroniumhexafluorophosphate; HF, hydrogen fluoride;
HOBt, 1-hydroxybenzotriazole; ITC, isothermal titration microcalori-
metry; LAA, lipoamino acid; MBHA, 4-methyl benzhydryl amine;
ODN-1, Oligodeoxynucleotide; RP-HPLC, reverse phase-high perfor-
mance liquid chromatography; RPE, retinal pigment epithelium; TFA,
trifluoroacetic acid; THF, tetrahydrofuran; hVEGF, human vascular
epithelial growth factor; TMS, trimethyl silane; TIPS, triisopropyl
silane.
Keywords: Dendrimer; Oligonucleotide; hVEGF; Antisense therapy.
*
Corresponding author. Tel.: +61 7 3365 7430; fax: +617 3365 1688;
e-mail: hparekh@pharmacy.uq.edu.au
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.03.029

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H. S. Parekh et al. / Bioorg. Med. Chem. 14 (2006) 4775–4780
polypropylenimine(PPI)starburst
dendrimers.13–16
2. Chemical synthesis
These systems have delivered promising results and
have been elaborated further to increase transfection
efficiency by complexation of ODNs with liposomes
to form lipoplexes.17
2.1. Lipoamino acids
Lipoamino acids of varied chain length (C8-, C10-, C12-,
C14- and C18-enantiomeric mixtures) were prepared
following literature methods by reaction of diethyl ace-
toamidomalonate (1) with the appropriate bromoalkane
under reflux (Scheme 1).28 The resulting intermediate
was refluxed with 90% v/v HCl acid and neutralized to
afford the desired lipoamino acid (2). To prepare the
lipoamino acid for solid-phase synthesis (SPS), the
N-terminus was protected using Boc2O (3).
To this end PCDs incorporating lipoamino acids
(LAAs) show much promise as non-viral gene delivery
systems in the transport of oligonucleotides to target
sites.1,2,18 PCDs confer stability against digestive
nucleases and aid transport of oligonucleotides across
biological membranes.19,20 We investigated the efficien-
cy of delivering an anti-angiogenic oligonucleotide by
complexing with
a library of poly-lysine/arginine
PCDs.2,21 ODN-1 (GAGCCGGAGAGGGAGCGC
GA; MW = 6282) has a sequence homologous to a
portion of the 50 untranslated region (50 UTR) of
hVEGF—the main factor associated with uncontrolled
intraocular revascularization. ODN-1 has been shown
to inhibit expression of hVEGF.22 The overexpression
of hVEGF and resulting macular degeneration/diabet-
ic retinopathy is the major cause of blindness in pa-
tients aged over 60 years in the developed world.23,24
Here we describe the synthesis of a library of lipid
core poly-lysine/arginine dendrimers using solid-phase
Boc synthetic techniques. We envisaged that the
amphiphilic nature of the lipid core PCDs would raise
solubility issues, however, this was not realized and by
introducing a carbohydrate moiety into the synthetic
procedure a number of glycodendrimers (40–45) were
afforded.25 Complexation of the PCDs with ODN-1
was carried out in deionized water using a 6:1 charge
ratio (+:ꢀ) as determined previously by isothermal
microcalorimetric titrations.26 ODN-1/dendrimer com-
plexes were applied to retinal pigment epithelial cells
(RPE51) and media collected at 24 and 48 h with
transfection efficiency measured indirectly by hVEGF
reduction due to the presence of ODN-1. Results from
these findings were compared to those from the
Cytofectin GSVTM/ODN-1 control complex.27
2.2. Galactosyl glutarate
To enable incorporation of a carbohydrate group onto
the dendrimer using solid-phase synthesis, tert-butyl
glutarate was coupled via an amide linkage to D-galac-
tose (Scheme 2).
Peracetylated galactose was stirred with 30% HBr/AcOH
to afford peracetyl galactosyl bromide (4b, a-anomer) fol-
lowed by reaction with NaN3 to yield the corresponding
azide (5a, b-anomer). Reduction of the azide allowed
direct condensation with mono-tert-butyl glutarate to
afford 6a. Finally, acidolytic cleavage of the ester using
TFA yielded the desired building block (6b) ready for
dendrimer synthesis (see Fig. 1 for general structure).29
2.3. Dendrimers and glycodendrimers
Dendrimers were synthesized using stepwise solid-phase
Boc-chemistry (see Tables 1 and 2). Glycodendrimers
40–45 incorporated building block 6b coupled via the
acid to the e-NH2 termini of L-lysine (Fig. 2). To prevent
aggregation of the polar heads and lipidic tails within
each dendrimer, a spacer (C6) was introduced. Boc-lipo-
amino acids were used in C8, C10, C12, C14 and C18 chain
Scheme 1. Synthesis of Lipoamino acids.28 Reagents and conditions: (i) Br–(CH2)n–CH3, NaOEt, reflux (ii) H+ reflux, then NaOH to pH 7
t
(iii) Boc2O, BuOH/H2O, NaOH (pH 13), then citric acid to pH 7.
Scheme 2. Synthetic strategy towards galactosyl-glutarate.29 Reagents: (i) 30% HBr/AcOH (ii) NaN3, n-Bu4NHSO4, NaHCO3 in DCM (iii) 10% Pd/
C in EtOH, H2 (iv) HOOC(CH2)3COOtBu, DCC, DIPEA, THF (v) 90% TFA(aq), TIPS/DCM.

H. S. Parekh et al. / Bioorg. Med. Chem. 14 (2006) 4775–4780
4777
3. Results and discussion
3.1. Microscopic examination
Initial microscopic analysis was performed on RPE cells
treated with dendrimers or dendriplexes to assess degra-
dation of cells (Fig. 3).
It is evident that microscopic analysis of RPE cells
transfected with Cytofectin GSVTM/ODN and dendri-
mer/ODN complexes appears to be healthy (Figs.
3b and c) and viable for further in vitro studies.
An effect of the cationic dendrimers on RPE cell
morphology becomes apparent when administered
alone, which can be attributed to their surfactant-like
properties (Fig. 3d). For a number of dendrimer/
ODN-1 complexes a similar morphological change
was observed 48 h post-transfection. This change
was observed via microscopic analysis (as per
Fig. 3d) and the detail of cell morphology change ob-
served for each complex is available in the supporting
information.
Figure 1. General structure of dendrimers 16–35.
Table 1. C8, C10, C12, C14 and C18, lipoaminoacids; C6, –OC(CH2)5NH–
(spacer); G, glycine; K, lysine; R, arginine
Dendrimer
R3
R2
R1
W1
X1
Y1
Z1
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
—
—
—
—
—
—
—
—
—
K
—
K
K
—
K
—
K
—
K
K
R
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
C6
C6
C6
C6
C6
C6
C6
C6
C6
C6
C6
C6
C6
C6
C6
C6
C6
C6
C6
C6
C8
C8
C8
C8
C8
C10
C10
—
—
—
—
—
—
—
—
C10
—
—
—
—
—
—
C8
C8
C8
C10
C10
—
(C8)2
C10
C10
(C8)2
(C8)2
—
—
(C10 2
(C10 2
(C10 2
C10
(C10 2
(C10 2
(C10 2
)
3.2. ELISA
)
—
—
)
—
—
—
—
—
—
—
—
—
—
—
—
ELISA was performed on media collected from dendri-
mer/ODN-1 conjugates 16–35 and 40–45 in addition to
Cytofectin GSVTM/ODN-1 (Cyt) and non-transfected
control (Cytofectin GSVTM alone, Con) at 24 and 48 h
post-transfection (Fig. 4).
R
)
R
)
C10
C10
—
R
)
—
K
—
K
—
K
C12
C12
—
—
(C12 2
(C12 2
(C12 2
(C12 2
)
All but one (20) of the dendrimers resulted in a mod-
erate reduction in hVEGF expression compared to the
commercially available transfection agent Cytofectin
GSVTM in the initial 24 h period. This clearly demon-
strates release of ODN-1 from the dendriplex and
delivery to the target site within the nucleus of the
cell. Cells transfected using Cytofectin GSVTM exhibit-
ed the expected profile of 40–50% reduction in the ini-
tial 24 h, followed by no significant reduction in the
subsequent 24 h. The profile of 25 was very similar
to Cytofectin GSVTM and it may be interesting to ex-
plore modifications of 25 to attempt prolonging its
efficacy.
)
—
)
C12
C12
)
Table 2. Abbreviations as in Table 1; GD, glycodendrimer; Gal-Lys,
6b coupled to e-NH2 termini of L-lysine
GD
R1
U
W
X
Y
Z
40
41
42
43
44
45
K
K
K
K
K
—
—
—
C6
C6
—
—
C6
C6
C14
C18
—
C14
C18
Gal-Lys-
Gal-Lys-
—
C14
C18
—
Gal-Lys-
Gal-Lys-
—
—
Gal-Lys-
Gal-Lys-
—
(C14 2
(C18)2
)
—
—
Of the library studied, dendrimers 16, 40 and 41 dis-
played a sustained reduction of hVEGF concentration
between 40% and 50% after 48 h, however, cell mor-
phology resembled that in Figure 3d after this time
period. The reduction in hVEGF titre (ꢁ20%) in the
second 24 h period of study for dendrimer 16 is encour-
aging. The structure of 16 consists of three short C8-
LAAs with a net 4+ charge. Previous studies in this
area identified dendrimers consisting of two C14-LAAs
with an 8+ charge or two C18-LAAs with a 4+ charge
as being very efficient at transfecting RPE cells with
hVEGF concentrations remaining at between 50%
and 60% of control after 48 h.1 It is interesting that
16 although comprising of much shorter LAAs has a
transfection efficiency comparable to those previous
dendrimers. Furthermore, the introduction of more
LAAs (i.e., 17 and 18) did not correlate to better activ-
ity by way of hVEGF reduction.
Figure 2. General structure of glycodendrimers 40–45.
lengths. Upon completion of the synthesis, all dendri-
mers were cleaved from the resin using the high HF pro-
tocol, purified using RP-HPLC and characterized by
mass spectrometry.

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H. S. Parekh et al. / Bioorg. Med. Chem. 14 (2006) 4775–4780
Figure 3. Microscopic analysis of RPE cells 24 h post-transfection with: (a) Control cells (untransfected); (b) Cytofectin GSVTM/ODN-1 complex; (c)
Dendrimer/ODN-1 complex; (d) Unconjugated dendrimers (1 mg/mL).
Figure 4. Expression of VEGF (% of untransfected control).
A number of dendrimers were functionalized with galac-
tose—chosen at random as the carbohydrate moiety,
primarily to enhance hydrophilicity. The introduction
of galactose was also undertaken to explore the possibil-
ity of exploiting any active transport mechanisms, how-
ever, a larger glycodendrimer library comprising of
numerous carbohydrates would be required to fully
address this issue. Of the glycodendrimers constructed
40 and 41 displayed similar hVEGF titres at 24 and
48 h (ꢁ60% of control) and each comprises of three
C14 and C18 LAAs, respectively, with both dendrimers
carrying a net 8+ charge. Once again, decreasing (42
and 43) or increasing (44 and 45) the number of LAAs
did not further reduce hVEGF titres.
4. Conclusion
Synthesis and evaluation of a library of polycationic
dendrimers and glycodendrimers towards transfection
of RPE cells has been demonstrated. From the library
of dendrimers synthesized there does not seem to be
an obvious trend relating dendrimer structure to
hVEGF titre, although a number of complexes were
moderately efficient in transfecting human cells
(RPE51). Dendrimers 16, 40 and 41 displayed a marked
reduction in hVEGF titres after 48 h compared to the
commercially available standard Cytofectin GSVTM,
however, a significant change in cellular morphology
was also present 48 h post-transfection for these and a

H. S. Parekh et al. / Bioorg. Med. Chem. 14 (2006) 4775–4780
4779
number of other dendrimers (see supporting informa-
tion). To enhance the hydrophilicity of dendrimers with
relatively long LAAs (C14/C18), a carbohydrate moiety
was introduced (40–45), although in hindsight insolubil-
ity in aqueous systems was not an issue. Although glyco-
dendrimers 40 and 41 displayed a marked reduction in
hVEGF at 48 h, this was also accompanied by a marked
change in cell morphology. Dendrimers 42–45 were effi-
cient in transfecting cells at 24 h, however, this returned
to control levels after a further 24 h with moderate-no
change in cellular morphology. Here we have shown
that incorporating three LAAs and galactose as a carbo-
hydrate moiety provides moderate hVEGF reduction in
the initial 24 h post-transfection, however, it returns to
control levels in the subsequent 24 h. There is evidence
of altered cellular morphology from extended exposure
of the RPE cells to the dendriplexes and further studies
need to be carried out to determine the significance of
this. Further work is underway to address the delivery
of oligonucleotides to cell lines using glycodendrimers
derived from various carbohydrates whilst maintaining
healthy viable cells over an extended time course. The ef-
fects of complex size, shape and surface charge on trans-
fection efficiency and cell viability in various cell lines
are also underway.
using the procedure described above. The resin was
dried over KOH under vacuum. The dendrimers were
cleaved from the resin using HF (10 mL) and p-cresol
(1 mL) at 0 ꢁC for 1 h. The cleaved dendrimers were pre-
cipitated using diethyl ether, then re-dissolved in 50%(aq)
CH3CN and lyophilized to afford an amorphous
powder.
5.2. Purification
Analytical RP-HPLC was performed on a Shimadzu
instrument (LC-10AT liquid chromatograph, SCL-10A
system controller, SPD-6A UV detector, a SIL-6B auto
injector with a SCL-6B system controller and columns
C4 (Vydac, 5 lm pore size, id = 4.6, 250 mm), C18 (Zor-
bax, 3.5 lm pore size, id = 4.6, 150 mm)) to identify the
synthesized dendrimers and ascertain the appropriate
gradient conditions for preparative HPLC. Preparative
HPLC was undertaken with ꢁ100 mg of crude dendrimer
separated on a Waters HPLC system (Model 600 control-
ler, 490E UV detector, F pump and TSK Gel C4/C18 col-
umns with 10lm pore size and 22 mm id) using an
acetonitrile/water gradient and characterized by ES-MS
(Perkin Elmer API 3000 instrument). The resulting den-
drimers were used as diastereomeric mixtures.
5.3. Complexation
5. Experimental
Oligodeoxynucleotide, ODN-1 (1.0 mg/mL in deionized
water), was added to each dendrimer (1.0 mg/ mL in
deionized water), mixed for 15 min, diluted with
200 lL sterile water and lyophilized. Complexes were
reconstituted in deionized water (100 lL) prior to trans-
fection. The molar ratio of 6:1 (+:ꢀ) dendrimer/ODN-1
was used for complexation as previously determined by
ITC experiments.32,33 Cytofectin GSVTM/ODN complex-
es were prepared according to the manufacturer’s
instructions (Glen Research, Stirling, VA, USA) using
Optimemꢂ media.
MBHA resin was purchased from Reanal (Hungary)
and protected amino acids were obtained from Nova-
Biochem (Australia). DMF and TFA of peptide synthe-
sis grade were purchased from Auspep (Parkville,
Australia). HPLC grade acetonitrile was purchased
from Labscan Asia Co. Ltd (Bangkok, Thailand). The
mobile phase was a mixture of solvent A (0.1% TFA
in water) and solvent B (0.1% TFA in 90%(aq) CH3CN)
at a flow rate of 1 mL/min.
5.1. General procedure for synthesis of dendrimers 16–35
and 40–45
5.4. In vitro experiment and ELISA
p-MBHA resin (100–200 mesh, 0.4 mmol/g loading) was
swelled in DMF in a fritted glass vessel for 90 min. An
activation mixture of HBTU (0.5 M in DMF, 4 equiv),
DIPEA (0.442 mL, 4 equiv) and amino acid (4 equiv)
was shaken with the resin for 15 min. Negative ninhy-
drin reaction (5 min) showed nearly quantitative cou-
pling (P99.6%) and the Boc group was removed using
100% TFA (2· 1 min) followed by in situ neutraliza-
tion.30,31 Dendrimers were synthesized on a 0.3 mmol
scale, and the following amino acid side-chain protec-
tion was used: Lys(Boc), Lys(Dde). Between all manip-
ulations the resin was washed exhaustively with DMF.
Upon completion of synthesis terminal Boc groups were
removed and in the case of glycodendrimers 40–45 the
resin was also shaken with freshly prepared 1 M
NaOMe (8 mL, 30 min), to remove the galactose-ace-
tyl-protecting groups prior to washing with DMF and
then methanol. Dde was removed using 2% v/v hydra-
zine monohydrate in DMF (2· 20 min washes) followed
by coupling of building block 6b (5-oxo-5-(2,3,4,6-tetra-
O-acetyl-b-D-galactosylamino)pentanoic acid, 3 equiv)
Cells of human RPE origin, RPE51, were grown to 80%
confluency in a 24-well plate and transfected with 1 lM
(final concentration) ODN-1 in quadruplet sets using
either Cytofectin GSVTM (as per the manufacturer’s
instructions) or the dendrimer/ODN-1 complexes. The
cells were grown under hypoxic conditions (5% CO2/
2% O2) for 48 h with media removed at both 24 and
48 h intervals. One hundred microlitres of media from
each sample at 24 and 48 h intervals was directly used
in the assay. The samples were then used in a sandwich
ELISA as per the manufacturer’s instructions (Cytel-
isaTM Human VEGF kit, CYTIMMUNE Sciences Inc.,
Maryland, USA). Statistical analysis was performed
on the data, which consisted of an ANOVA with post
hoc analysis using Tukey/Kramer and a Fishers LSD
procedure (p < 0.05).
5.5. NMR and MS
NMR measurements were performed on a Bruker
Avance 500 instrument using a 5 mm SEI probe at

4780
H. S. Parekh et al. / Bioorg. Med. Chem. 14 (2006) 4775–4780
25 ꢁC. Spectra were processed using Topspinꢂ 1.3 soft-
ware and TMS was used as the internal standard. Mass
spectrometric measurements were performed using a tri-
ple-quadrupole PE Sciex API 3000 mass spectrometer
with positive ion electrospray (ES). Accurate mass spec-
trometric measurements (HR-MS) were performed
using a Finnigan MAT 900 XL-Trap instrument with
a Finnigan API III electrospray source in positive ion
electrospray mode.
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5.5.1. tert-Butyl 5-oxo-5-(2,3,4,6-tetra-O-acetyl-b-D-ga-
lactosyl-1-amino)pentanoate (6a). H NMR (500 MHz,
1
CDCl3): d 6.23 (m, 1H, C1-Gal), 5.37 (dt, 1H, J = 1.1,
2.9, 14 Hz, C5-Gal), 5.17 (m, 1H, C2-Gal), 5.06 (m,
2H, C3,C4-Gal), 4.02 (m, 1H, C6-Gal), 2.19–1.90 (br
m, 18H, 4 · CH3, 3· CH2 (pent)), 1.38 (s, 9H,
C(CH3)3). 13C NMR (125 MHz, CDCl3): d 172.75,
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2.40–2.20 (br m, 4H, 2· CH2 (pent)), 2.11, 2.03, 2.00,
1.96 (s, 12H, 4· CH3), 1.91 (m, 2H, CH2 (pent)). 13C
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170.49, 170.04, 169.80 (C@O), 78.37 (C1-Gal), 72.30
(C5-Gal), 70.83 (C2-Gal), 68.36 (C4-Gal), 67.17 (C3-
Gal), 61.11 (C6-Gal), 35.11 (HNCOCH2), 32.64
(CH2COOH), 20.72, 20.66, 20.57, 20.52 (CH3), 19.96
(CH2CH2CH2). HR-MS m/z calcd for C19H27NO12Na
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Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmc.2006.03.029.
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