Solid-phase synthesis of a lysine-capped bis-dendron with remarkable DNA delivery abilities

Article abstract of DOI:10.1039/b804771e

Solid-phase synthesis of a generation 3.0 polyamidourea 1→3 C-branched bis-dendron followed by capping of the peripheral amino groups with l-lysine gave an efficient transfection reagent.

Full text of DOI:10.1039/b804771e

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Solid-phase synthesis of a lysine-capped bis-dendron with remarkable DNA
delivery abilities†
Siew-Eng How,‡ Asier Unciti-Broceta, Rosario M. Sa´nchez-Mart´ın and Mark Bradley*
Received 20th March 2008, Accepted 30th April 2008
First published as an Advance Article on the web 12th May 2008
DOI: 10.1039/b804771e
Solid-phase synthesis of a generation 3.0 polyamidourea 1→3
C-branched bis-dendron followed by capping of the peri-
pheral amino groups with L-lysine gave an efficient trans-
fection reagent.
A number of investigations have demonstrated that the high-
generation polycationic dendrimers (e.g. PAMAM G >5, con-
taining 128 primary amines + 126 tertiary amines) show efficient
transfection properties.13 However, as the dendrimer generation
increases, so does cytotoxicity, while difficulties in obtaining pure
compounds increases proportionately.1,13 What would be highly
desirable would be a synthetically accessible, low-generation, yet
highly efficiently transfecting dendrimer.
Polymers have emerged as a vital component of many medical
technologies and as a result demands have arisen for the adaptation
of the size, topology, chemistry and properties of a multitude of
polymeric materials.1,2 In this arena, dendrimers have acquired a
privileged status, firstly because of their unique structural features
as highly branched, symmetrical, monodisperse polymers, and sec-
ondly due to their tunable chemistry, which allows the production
of compositionally and structurally controlled macromolecules.3
Polycationic dendrimers have been widely investigated as vec-
tors for drug delivery and release,1,4–6 and also as devices for
efficiently transfecting cells (e.g. SuperFect, PAMAM derivatives,
etc).7–11 However, despite all these efforts, the mechanisms and
structural requirements which enable DNA–dendrimer complexes
(also called dendriplexes) to cross the cellular membrane,12 enter
the cytoplasm and then release their cargo are still unclear.
In this manuscript,14 the solid-phase synthesis and remarkable
transfection abilities of a G 3.0 polyamidourea 1→3 C-branched
bis-dendron are reported, where the peripheral amino groups
of the structure have been capped with L-lysine.15 The den-
dritic structures were synthesized using a divergent, microwave-
assisted, solid-phase approach with the dendrimer assembled on
polystyrene resin via an acid labile linker.16 The acid-cleavable
polyamine scaffold 4 was used as a core for assembling the
dendrons (prepared as previously reported16,17) and attached onto
the solid support to give 5 (see Scheme 1). The dendrimer was
constructed (up to G 3.0) by the sequential addition of the
AB3 isocyanate-type monomer 619 under microwave irradiation
conditions, followed by the displacement of the methyl ester with
propane-1,3-diamine (see Scheme 2). The use of tris-branched
monomer 6 leads to a rapid increase in terminal functionality
(2 → 6 → 18 → 54),16 with acid treatment of resin-bound
dendrimer 7 giving G 3.0 bis-dendron 8 (see Fig. 1). Treatment
of 7 with Fmoc-Lys(Boc)-OH/DIC/HOBt followed by Fmoc-
deprotection and resin cleavage gave dendrimer 9, with 1H-
NMR analysis (see characterization in the ESI†) suggesting that
approximately 50 out of the 54 terminal amino groups had been
School of Chemistry, King’s Buildings, West Mains Road, Edinburgh, EH9
3JJ, UK. E-mail: Mark.Bradley@ed.ac.uk
† Electronic supplementary information (ESI) available: General chem-
istry procedures, experimental details for the synthesis, characterization
of the novel compounds, and biological assay protocols. See DOI:
10.1039/b804771e
‡ Current address: School of Science and Technology, University Malaysia
Sabah, Kota Kinabalu 88999, Sabah, Malaysia.
Scheme 1 Reagents and conditions: (a) NEt3, DMAP, DMF, 81%; (b) polymer-supported Pd,18 pyrrolidine, THF, reflux, 94%; (c) aminomethyl resin,
DIC, HOBt, DCM; (d) 5% N2H4·H2O in DMF.
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Scheme 2 Reagents and conditions: (a) monomer 6, DMAP, DIPEA, DMF–DCM 1 : 1, lW, 100 ◦C, 60 min; (b) propane-1,3-diamine, MeOH, 72 h;
(c) TFA–DCM–H2O (9.5 : 0.25 : 0.25), 3 h; (d) Fmoc-Lys(Boc)-OH, DIC, HOBt, DCM–DMF 1 : 1, overnight; (e) 20% piperidine in DMF, 2 × 30 min.
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Fig. 1 Structures of G 3.0 polyamidourea 1→3 C-branched bis-dendrons 8 and 9.
coupled (attempts to force the reaction further were not successful
despite the use of prolonged microwave heating).
The ability of the dendrimers to complex DNA was studied
by conventional electrophoretic DNA retardation assays. Den-
driplexes were formulated as a function of dendrimer/DNA
weight ratios, and representative electrophoretic gel images are
shown in Fig. 2, showing that dendrimers 8 and 9 completely
suppressed the electrophoretic mobility of plasmid DNA (this was
also observed for the positive controls SuperFect and Effectene).
The transfection properties of dendrimers 8 and 9 were evalu-
ated with four different mammalian cell lines (HEK293T, HeLa,
ND7, and B16F10) employing pEGFP-N1 as a GFP-reporter.
Dendriplexes were formulated at two weight ratios (10 : 1 and
20 : 1) and tested in triplicate using SuperFect (a dendrimeric
material) and Effectene (a lipid formulation) as controls, with
GFP expression evaluated by flow cytometry.20
Fig. 2 Electrophoretic DNA retardation assays. Dendrimers 8 and 9 were
complexed with pEGFP-N1 at weight ratios of 10 : 1 and 20 : 1, loaded
onto an agarose gel (1% agarose, 1 lg mL−1 ethidium bromide) and run at
100 V for 1 h. Positive controls (Sup = SuperFect; Eff = Effectene) were
formulated as suggested by the supplier. Control = naked pEGFP-N1
(containing both supercoiled and nicked circular forms).
was especially high in B16F10 cells at a weight ratio of 20 :
1, with 83% of the cells having fluorescence above background
As observed in Fig. 3, dendrimer 9 showed high gene delivery
efficacy with HEK293T, ND7 and B16F10 cells. GFP expression
2268 | Org. Biomol. Chem., 2008, 6, 2266–2269
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and R.M.S.-M. thanks the Royal Society for a Dorothy Hodgkin
Fellowship.
Notes and references
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Fig. 3 Percentage of GFP-expressing cell population.
(see ESI† for the flow cytometry data), better than either SuperFect
(68%) or Effectene (21%). Since B16F10 (melanoma) cells are
considered to be a hard-to-transfect cell line,21 this result highlights
the potential of this reagent. Flow cytometry analysis of neuron-
derived ND7 cells showed that dendrimer 9 efficiently delivered
pEGFP-N1 into this cell line at a 10 : 1 weight ratio with
71% efficiency. Poor transfection efficiency was found in HeLa
cells, consistent with previous work,12,22 which demonstrated that
dendriplexes use a caveolin-dependent pathway as a preferential
internalisation route12 and that HeLa cells are poor at caveolin-
dependent endocytosis.22
It was interesting to observe that the transfection properties of 8
(displaying 54 amino groups) was insignificant compared to the L-
lysine derivative 9 (with 108 amino groups on the surface), clearly
indicating that charge density and size are important parameters in
transfection with dendriplexes (8 and 9 were found to be non-toxic
at the weight ratios used in all the cell lines tested as determined
by MTT assay (>90% cell viability)).
In conclusion, solid-phase methodology in association with
microwave heating was used to efficiently construct a G 3.0
polyamidourea 1→3 C-branched bis-dendron that, following
capping with L-lysine, led to a dendrimer with approximately 108
primary amines. This dendrimer showed remarkable transfection
abilities in various mammalian cell lines, comparable or better than
SuperFect, a commercially available dendrimer-type transfection
reagent.
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20 All experiments were carried out in serum-containing media.
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Acknowledgements
S.E.-H. thanks the Ministry of Science, Technology and Inno-
vation, Malaysia (MOSTI) and the University Malaysia Sabah
for a scholarship. A.U.-B./M.B. thank the MRC for funding
This journal is
The Royal Society of Chemistry 2008
Org. Biomol. Chem., 2008, 6, 2266–2269 | 2269
©