Efficient gene transfection with functionalised multicalixarenes

Article abstract of DOI:10.1039/b712100h

Novel amino-functionalised multicalixarenes have been synthesised which show low cellular toxicity, effective DNA binding and, when featuring aliphatic amines, are efficient gene transfection agents. The Royal Society of Chemistry.

Full text of DOI:10.1039/b712100h

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COMMUNICATION
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Efficient gene transfection with functionalised multicalixarenes{
Ruth Lalor, Jessica L. DiGesso, Anja Mueller and Susan E. Matthews*
Received (in Cambridge, UK) 7th August 2007, Accepted 5th September 2007
First published as an Advance Article on the web 14th September 2007
DOI: 10.1039/b712100h
Novel amino-functionalised multicalixarenes have been synthe-
sised which show low cellular toxicity, effective DNA binding
and, when featuring aliphatic amines, are efficient gene
transfection agents.
The efficient introduction of genetic material into cells is an area of
considerable interest both to provide tools for biological research
and for the development of gene-based therapeutics.1,2 Synthetic
vectors, in particular linear polymers1 and dendrimers2 incorpor-
ating a high level of surface positive charge, or cationic lipids3 have
proved useful in promoting gene transfection. However, these are
often unable to afford transfection without the addition of either a
buffering agent such as chloroquine to reduce DNA degradation
in acidic lysosomes or an additional endosomolytic agent such as
dioleoyl-L-a-phosphatidyethanolamine (DOPE).4 One-component
Fig. 1 Calixarenes and multicalixarenes studied.
efficient gene transfection systems are thus an important target.
Calixarenes have recently been shown to have potential in a
A multi-step convergent synthetic route was designed for the
multicalixarenes (MCs) studied. The introduction of functionality
to the surface relied on the key bifunctional intermediate 9. This
was prepared from the known tripropyl derivative14 through
introduction of a masked amine as the phthalimide15 7 and
subsequent ipso nitration and reduction at the calixarene upper rim
(Scheme 1). Compound 9 was then either protected as the
orthogonal Boc derivative 10 to allow for the formation of
multicalixarenes featuring aromatic amines for DNA binding or
treated with a Boc-protected glycine derivative for the introduction
of surface aliphatic amines to yield 11. Coupling of these
functionalised outer surfaces to the central core was achieved
through initial deprotection (12, 13) and subsequent reaction with
activated pentafluorophenol esters 16 and 21 of the known
wide range of biological process and diseases including applica-
tions in drug delivery,5,6 as ion channel mimics7 and as anti-
tuberculosis agents.8 Single calixarenes,9 featuring guanidinium
functionality, have been demonstrated to promote DNA transfec-
tion and these studies indicate that architecture, size and
lipophilicity are important in achieving transfection with less
lipophilic derivatives requiring the addition of a DOPE adjuvent.
Equally the interaction of amino-calixarene dimers10 with DNA
has recently also been reported although their transfection ability
was not discussed.
The incorporation of calixarenes into dendrimeric or multi-
calixarene systems offers the potential of combining high levels of
surface functionality, with a increased degree of pre-organisation,
amphiphilic characteristics and a range of fixed three-dimensional
architectures through the choice of calixarene conformation.
Whilst multicalixarene systems have been known for some time,11
apart from the excellent research from the groups of Bo¨hmer, on
the use of urea-functionalised multicalixarenes for self-assembly,12
and of Beer, on the preparation of cation binders,13 these systems
have not incorporated surface functionality and have been
principally of synthetic interest only.
In this communication, we present the synthesis of the first
amino surface-functionalised multicalixarenes (Fig. 1), evaluation
of their DNA-binding ability and toxicity and their exploitation as
gene transfection vectors.
School of Chemical Sciences and Pharmacy, University of East Anglia,
Norwich, UK. E-mail: susan.matthews@uea.ac.uk; Fax: 44 1603 592003;
Tel: 44 1603 593986
{ Electronic supplementary information (ESI) available: Full experimental
details for synthesis of multicalixarenes and biological studies. See DOI:
10.1039/b712100h
Scheme 1 Synthesis of functionalised outer surface. Reagents and
conditions: (a) n-3-(bromopropyl)phthalimide, NaH, DMF; (b), HNO3,
TFA, DCM, 0 uC; (c) SnCl2, EtOH, reflux; (d) Boc2O, DCM; (e)
BocGlyONSu, DCM; (f) hydrazine hydrate, EtOH.
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in DNA binding we further evaluated the binding of 1–3 over a
range of concentrations (0.2–1.0 mM) compared to an amine
equivalent concentration of the control single calixarene 5. On
reducing the concentration of vector, some discrimination is seen
between the different architectures of the MCs (Fig. 2(b)) with the
asymmetrically functionalised 3 showing stronger binding than the
symmetrical functionalised 1. Functionalisation with glycine MC 2
results in enhanced binding at lower concentrations compared to
the conformationally analogous MC 1 (see ESI,{ Fig. 5).
Interestingly, the glycyl amino derivative 5 possesses significant
DNA binding ability, comparable with that of MCs 1 and 3, but
less than that of 2, however, previous studies21 indicate that, unless
self assembly of the ligands occurs, vectors featuring less that 6–8
positive charges are unable to mediate transfection successfully.
These results indicate that there may be a degree of cooperativity
in the binding of DNA between the functionalised calixarenes in
multicalixarenes, which is not available with the single calixarenes,
and, additionally, that the binding is affected by the multi-
calixarene architecture and the nature of the amine and is more
efficient when the charged groups are provided on a mono-
directional surface.
Scheme 2 Synthesis of functionalised multicalixarenes. Reagents and
conditions: (a) NMe4OH, THF, reflux; (b) PFP, DCC, EtOAc; (c) Hu¨nig’s
base, DMAP, DCM; (d) HCl (g), DCM.
The toxicity of the multicalixarenes was determined in a range
of cell lines including Chinese Hamster Ovary (CHO), Human
Embryonic Kidney (HEK) and a Human monocytic cell line
(THP-1) using a cell viability MTS assay over 24–72 h.
Representative data for the MTS assay in HEK cells, after 48 h,
is shown in Fig. 3 (equivalent data was obtained in CHO and
THP-1 cells). All the multicalixarenes 1–3 showed a comparable
lack of cell toxicity to both the PBS control and b-cyclodextrin
over the full pharmaceutically relevant concentration range and
are thus suitable for use as in vitro vectors. It is interesting to note
that the single calixarene 4 showed significant toxicity, at high
concentrations, compared with controls and MCs 1–3 in both
HEK and CHO cells and this may indicate a different cell uptake
process for the single and multicalixarenes, a process we are
currently investigating.
carboxylic acid derivatives 1416 and 1917 providing the protected
multicalixarenes in yields of between 39 and 78% (Scheme 2) These
were then readily deprotected to reveal surface amines, in near
quantitative yields, by treatment with HCl. The two comparative
single calixarenes 4,18 5,19 as their hydrochloride salts, were
prepared according to literature procedures.
The DNA-binding ability of calixarenes 1–5 was initially
evaluated through gel electrophoresis studies20 using a 3000
Base-Pair plasmid (either pGEM-EYFP or pEGFP-N1).
Screening at a high ligand concentration of 2 mM indicated that
the MCs 1–3 were able to bind DNA through charge neutralisa-
tion and thus the plasmid DNA was unable to pass through the gel
in response to applied charge (Fig. 2(a)). In contrast, the single
amino calixarene 4 was unable to bind the plasmid DNA at this
concentration. In an effort to understand the role of cooperativity
Fig. 2 Gel electrophoresis studies were performed on a 1% agarose gel at
130 V. (a) High concentration (2 mM) pGEM-EYFP plasmid. (b)
Dilution study (concentrations shown in mM) pEGFP-N1. M is DNA
ladder, C is a DNA control with no calixarene present. Single calixarenes,
not featuring amino functionality, have been included as additional
controls: (a) p-tBu calix[4]arene tetrapropylether,23 (b) calix[4]arene
tetrapropylether.24
Fig. 3 (a) Representative cell viability assays (HEK cells tested for
proliferation after 48 h using an MTS assay) for MCs 1–3 and 4. (b) HEK
cells after 48 h incubation with PBS. (c) HEK cells after 48 h incubation
with MC 3. Images were acquired using a digital camera mounted on a
microscope.
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Fig. 4 Transfection of a plasmid expressing a fluorescent protein that
accumulates in mitochondria (pDs2-mito) in CHO cells after 48 h. (a)
Positive control (cells transfected with commercial FuGene1); (b) negative
control (PBS buffer); (c) transfection with 3; (d) transfection with 2.
Images acquired using a Zeiss Axiovision 2 microscope.
The ability of 1–5 to transfect plasmid DNA was investigated in
CHO cells using pDs2-mito (Clontech) a plasmid leading to
expression of a fluorescent protein that accumulates in mitochon-
dria. The conditions used22 were those for a standard transfection
with FuGene1 (Roche) a commercial positive control, and 1 mg
DNA, and are not optimised for MCs. Effective transfection
(10%), compared to FuGene (50%), was observed for 2, the
multicalixarene featuring aliphatic amines after 48 h (Fig. 4).
However, interestingly, no transfection was observed with 1 and 3,
where the amine is an arylamine, which show good binding in
electrophoresis studies, or with the single calixarene controls 4 and
5. This variation in transfection ability may be a consequence of
the degree of charge exhibited by the multicalixarenes in pH 7
buffer with only multicalixarene 2 being fully positively charged
and binding DNA effectively or a result of reduced DNA release
within the endosomal pathway in the case of multicalixarenes 1
and 3.
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The potential of functionalised multicalixarenes to effect gene
transfection has been clearly shown and the low toxicity of these
materials indicates that they are of great promise as delivery
agents. We are currently investigating the effect of charge loading
and scaffold architecture of the multicalixarenes on DNA
transfection.
This work was supported by the Association for International
Cancer Research (03-256) and the University of East Anglia. The
authors acknowledge the EPSRC Mass Spectrometry Service,
University of Swansea for providing characterisation.
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