Effects of structural modification on gene transfection and self-assembling properties of amphiphilic dendrimers

Article abstract of DOI:10.1039/b518174g

A library of novel amphiphilic, self-assembling dendrimers was designed and synthesised to evaluate the effects of structural changes on transfection efficiency. The Royal Society of Chemistry 2006.

Full text of DOI:10.1039/b518174g

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Effects of structural modification on gene transfection and self-assembling
properties of amphiphilic dendrimers†
Marine Guillot,a Sara Eisler,a Kathrin Weller,b Hans P. Merkle,b Jean-Louis Gallanic and Franc¸ois Diederich*a
Received 21st December 2005, Accepted 13th January 2006
First published as an Advance Article on the web 26th January 2006
DOI: 10.1039/b518174g
A library of novel amphiphilic, self-assembling dendrimers
was designed and synthesised to evaluate the effects of
structural changes on transfection efficiency.
A number of strategies are being employed to make the efficient
delivery of foreign genetic material into a cell possible, i.e. gene
transfection. While synthetic vectors are generally less efficient
than their viral counterparts,1 facile structural modifications allow
for a systematic study of the factors governing transfection
efficiency.2 Cationic lipids, polymers and dendrimers are among
the most promising synthetic vectors so far.3,4 Combination of
their essential features led to the realisation of small amphiphilic
dendrimers for use as gene carriers.5 Preliminary in vitro cell
culture studies have validated this structural motif and identified
compound 1 as the best candidate (Fig. 1). With this lead in hand,
we examined the effects of systematically altering its chemical
structure. We present here the first results in a comprehensive
study of structure–activity relationships (SARs). Due to the
diversity of the structural and biological parameters involved in the
cellular delivery of plasmid DNA, an optimisation of transfection
activity requires, in addition to exhaustive cell biological studies,
correlations to physicochemical properties. For this reason, the
basic ability of the vectors to self-assemble at the air–water
interface was explored in Langmuir films.
Amphiphile 1 consists of two first generation dendrons, one
lipophilic and one hydrophilic, connected by a rigid tolane
(diphenylacetylene) core. The first variable of interest is the size
and constitution of this linear spacer. Surprisingly, in many vector
systems the spacer length has been found to exert a large impact on
gene delivery efficacy.6 Therefore, the tolane moiety was extended
by one phenylene-ethynylene unit to provide structure 2.
A common feature among both viral and cationic lipid gene
carriers is their ability to self-assemble.7 The lipophilic dendrons
in our molecules are crucial as they direct this process, and the
structures were thus varied as follows. Cholesterol has strong
intermolecular packing ability8 and is bio-compatable.9 Hence, a
aLaboratorium fu¨r Organische Chemie, ETH-Zu¨rich, 8093, Zu¨rich, Switzer-
land. E-mail: diederich@org.chem.ethz.ch; Fax: +41 44 63 21109; Tel: +41
44 63 22992
Fig. 1 Library of amphiphilic dendrimers for gene transfection. Counter-
ions to the protonated amines are trifluoroacetates.
bInstitut fu¨r Pharmazeutische Wissenschaften, ETH-Zu¨rich, 8093, Zu¨rich,
Switzerland. E-mail: hmerkle@pharma.ethz.ch; Fax: +41 44 63 31314;
Tel: +41 44 63 37310
cInstitut de Physique et Chimie des Mate´riaux de Strasbourg, 67034,
Strasbourg Cedex, France. E-mail: gallani@ipcms.u-strasbg.fr; Fax: +33
388 107246; Tel: +33 388 107164
† Electronic supplementary information (ESI) available: Full synthetic
protocol for vector 8, spectral characterization of all vectors, complete cell
biological methods and data including error estimations and Langmuir
methods. See DOI: 10.1039/b518174g
cholesteryl ester was introduced to afford compound 3. In another
approach, the degree of lipophilicity in 1 was approximately
maintained by the incorporation of N,N-dioctylamido branches
to give vector 4.10 The main change involves an increase in the
volume of the resulting dendron. The further exchange of the
linear dodecylamido chains in lead compound 1 with rather exotic,
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large cyclododecylamido residues should lower the lipophilicity in
derivative 5.11,12
Careful design of the hydrophilic dendron is also required as
the protonated ammonium centres must bind to plasmid DNA
strongly enough for compaction and protection, allow interaction
with the negatively charged extracellular matrix, elicit cellular
uptake and then allow release into the cytosol and access to the
nucleus prior to transfection.13 In the case of cationic lipids, surface
charge density has been identified as a determinant factor for
the control of this process.14 Structural motifs from commonly
used poly(ethyleneimine) (PEI), poly(propyleneimine) (PPI), and
poly(aminoamine) (PAMAM) dendrimers were therefore incor-
porated, providing first generation vectors 6–8.15
Vector 9 incorporating a second generation PAMAM dendron
was also synthesised. The DNA–cationic liposome complexes are
usually taken up into the cell by an endocytotic pathway after
which endosomal escape must occur. While the exact mechanism
of the latter process is unknown for our amphiphilic dendrimers, a
few theories in the field of gene transfection have been presented.16
The internal, protonatable tertiary amine centres of 9 may result
in swelling of the complex upon acidification of the endosome,
invoking the so-called proton sponge effect. This mechanism may
facilitate the release of DNA into the cytoplasm.
With this library in hand, we determined the biological activity
and the self-assembling behaviour of the amphiphilic vectors.
In vitro transfection assays were carried out using human cer-
vical carcinoma cells (HeLa).‡ For compaction, plasmid DNA
encoding for green fluorescent protein (GFP) was mixed with
compounds 1–9 at various charge excess ratios (CEs)§ in the
absence of serum, and was incubated with HeLa cell cultures.
After 24 hours of incubation, the transfection efficiency (TE) was
evaluated by fluorescence activated cell sorting (FACS). Toxicity
of the compounds was determined in HeLa cells using a cell
proliferation assay. As for Langmuir films, chloroform solutions
of the vectors were deposited on the water surface and pressure–
area (p–A) isotherms were then measured. The morphology of
the amphiphilic monolayers was visualised using Brewster angle
microscopy (BAM).
Results of the transfection experiments are shown in Fig. 2a
for compounds 2–5 and in Fig. 2b for the hydrophilic library,
in comparison to reference vector 1. Generally, changes to the
cationic dendron improve gene expression slightly, whereas modi-
fications to the core and the lipophilic dendron have a negative
effect on transfection. Connecting the dendrons of compound
1 to a slightly elongated core unexpectedly results in a marked
decrease in the activity of compound 2 at CE = 2–4. A similar
outcome was observed in preliminary studies when a much
larger central scaffold was utilized and attempts at transfection
failed.17
Fig. 2 Transfection efficiency of new dendrimers in HeLa cells in
comparison with reference compound 1, measured as percentage of living
cells transfected and normalized to LipofectamineTM 2000 (LPF): (a)
Compounds 2–5, core extension and lipophilic library. (b) Compounds
6–9, hydrophilic library.
extendable to our dendrimeric system, the lipophilicity of the
vectors should be as follows, 1 ≈ 4 > 5. However, this ranking is not
reflected by the transfection results. Direct correlations between
increased lipophilicity in solution and increased gene transfer
efficiency in a biological system cannot be necessarily postulated,
with each specific carrier motif having its own rationale.20 Given
that the new dendrons in 4 and 5 have in common shorter, bulkier
branches than 1, it is possible that steric hindrance could disturb
the self-assembling process.11
All vectors 6–8 bearing two positive charges have a similar to
slightly better activity than the triply charged reference compound
1 at CE ≥ 4 (Fig. 2b). Therefore, within the first generation,
changes to the number, length and constitution of the branches
have little to no effect at these concentrations. The only difference
in activity is observed at CE = 2 between the PPI derivative 6
and the PEI derivative 7. The branches of 6 and 7 differ only by a
single methylene unit, but the activity of the shorter molecule 7 is
completely abolished at this concentration.
In spite of the expected proton sponge effect, a large decrease
in activity is observed between the first and the second generation
PAMAM derivatives 8 and 9. Different generations of globular
PAMAM dendrimers have the same charge density although
in vector 9 only one dendron has been incorporated, allowing
spreading of the charge due to electrostatic repulsion between
the protonated amines. The resultant decrease in surface charge
density may in turn hinder escape from the endosome and explain
the low activity of 9.
Equally unexpectedly, dendrimer 3 has almost no activity at
low to medium charge excess ratios (CE = 2–8). Cationic lipids
incorporating cholesterol units have been widely exploited for gene
delivery but a helper lipid is usually required to destabilize the
endosomal membrane and allow cytosolic access.3,18,19 In our case,
DNA condensation may be very tight and the resultant complex
may have too low fluidity.
Vectors 4 and 5 have similar transfection capabilities but overall
are less efficient than 1. From literature studies, a ranking of
lipophilicity in surfactants may be made.10–12 If this ranking were
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The toxicity of synthetic gene carriers is a major concern. Cell
death was evaluated at the same concentrations as were used in
the transfection assays for compounds 2–9 (Fig. 3). Structural
changes have a high impact on cell viability. The modification of
the core as in compound 2, results in a dramatic increase in toxicity
compared to reference vector 1 (Fig. 3a). On the contrary, variation
of the lipophilic dendron generally leads to a decrease in cellular
toxicity. Vectors 4 to 5 are slightly less toxic than 1. Compound
3 is harmless to the cells even at the concentrations where some
activity is detectable, which may be linked to the biodegradability
of the cholesterol sub-unit.9,18
Fig. 3 Toxicity of new dendrimers in comparison with reference com-
pound 1 and normalized to LipofectamineTM 2000 (LPF): (a) Compounds
2–5, core extension and lipophilic library. (b) Compounds 6–9, hydrophilic
library.
Fig. 4 p–A Isotherms recorded on pure water subphase and Brew-
ster-angle microscopy images: (a) Isotherms of vectors 6–9 in comparison
with reference compound 1 and BAM images of the film of 6. (b) Isotherm
of vectors 2–5 in comparison with reference compound 1 and BAM images
of the film of 2.
The most efficient gene carriers, compounds 6 to 8, are also
found to be the most toxic ones in the whole library (Fig. 3b).
Contrasting these results is the lower toxicity of second gener-
ation vector 9. Generally, an increase in dendrimer generation
corresponds to increased toxicity.21 In this case, the low cell death
is most likely due to the low transfection activity of 9.22
(protonated ammonium groups). The cross-section of the tolane
2
˚
(ca. 25 A ) is somewhat smaller than the cross-section of the
While study of the Langmuir films offers some insight into
the biological results, strict correlations are naturally difficult to
establish. One may nevertheless observe some trends. The most
active vectors in this library (namely 6 to 8) are the compounds that
behave similarly to reference molecule 1: their isotherms reveal a
chains or polar heads and hence creates a void space. In order
to compensate for this void space, the cores are either strongly
tilted (70◦) or, more probably, the molecules dimerise through
core–core interaction (p–p interactions). These dimers then behave
as a single “supermolecule” with a critical packing factor of
∼1 (as defined in ref. 10). Brewster-angle microscopy (BAM)
observations confirm that these compounds (and also 4 and 9)
indeed form homogeneous monolayers. Molecules with such a
packing factor are usually capable of forming bilayers or vesicles,
hence the ability to mimic organic structures such as membranes
or liposomes.
liquid-like behaviour, with no first-order phase transition and final
2
˚
molecular areas ranging between 85 A (molecule 1) (Fig. 4a) and
2
˚
95 A (molecule 6). These observations and the value of the final
molecular areas indicate that the molecules most probably form
monomolecular films within which the intermolecular interactions
are dominantly van der Waals (alkyl chains) and/or electrostatic
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On the contrary, the compounds with lower transfection
efficiency show markedly different behaviours on the Langmuir
trough, e.g. solid-like behaviour (2 and 5) and first-order phase
transitions (2 and 4) (Fig. 4b).23 The longer core of 2 and the larger
lipophilic heads of 4 and 5 make it impossible for a dimerisation
to occur. These molecules therefore have a critical packing factor
greater than 1, which does not favour the formation of films or
membranes. Indeed, BAM observations reveal the presence of
three-dimensional supramolecular architectures in the “films” of
compounds 2, 3 and 5. Compound 4 has a transition to some
undetermined supramolecular organisation that is not a bilayer.
BAM can only hint at such supramolecular architectures through
the observation of uneven or optically heterogeneous films and
getting a definitive proof would require additional experiments
such as cryo-transmission electron microscopy (TEM) or small
angle X-ray scattering. Still, a reasonable hypothesis based on
our observations and on the molecular architecture would be that
compounds 2–5 form micelles.
In conclusion, we have presented the effect of rational structural
modifications on the transfection activity of small amphiphilic
dendrimers. A clear sensitivity to steric requirements of the
lipophilic dendron was observed. Within this first library, every
change that has an effect on the packing ability clearly lowers the
transfection efficiency. On the other hand, small alterations to the
cationic dendron structure have limited impact on the biological
activity in comparison to lead compound 1.
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Acknowledgements
We gratefully acknowledge financial support from the NCCR
“Nanoscale Science”, NSERC “National Science and Engineering
Research Council of Canada” and the German “Fonds der
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‡ In comparison to the original studies,5 the HeLa cell line was used
instead of HEK293 cells and the transfection efficiency was evaluated
using FACS. Consequently, the absolute values for transfection efficiency
of reference vector 1 are higher in the present study.
§ The unitless CE ratio is defined as the number of positive charges on
the dendrimers divided by the number of negative charges present on the
plasmid DNA. Based on an average molecular weight of 666 g mol−1 per
base pair (bp), 1 lg of DNA is assumed to carry 3 nmol of negative charges.
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