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Self-Assembling Dendrimers for Gene Transfection
One important goal within the first phase of the NCCR has been the design and synthesis of innovative
DNA nano-carriers. The small amphiphilic cationic dendrimers 1 and 2 (Fig. 1) have been indeed
identified as promising transfection vectors, capable of delivering in vitro foreign DNA into the
nucleus of cells [1]. The project has now two objectives: the elucidation of biophysical mechanisms
and the optimization of biological activity. The ultimate goal is the use of this novel class of
dendritic compounds in gene therapy.
1
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2
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Fig. 1: Two promising dendrimers for gene transfection |
Physico-chemical analyses can afford a unique understanding of dendrimer-dendrimer and dendrimer-DNA
interactions. As amphiphilic dendrimers cannot easily be crystallized, Scanning Tunneling Microscopy
(STM) imaging of monolayers deposited on graphite surfaces (Fig. 2A) and study of Langmuir films at
the air/water interface were some good starting points. The -stacking of conjugated cores
and hydrogen bonds between amide functionalities are believed to govern the self-assembly process.
A prerequisite to efficient DNA protection and transport through cell barriers is its tight
compaction. Atomic Force Microscopy (AFM) allowed thus the direct observation on surface of strong
electrostatic interactions between 1 and DNA, resulting in the formation of dense spheroidal
polyplexes (Fig. 2B). Cryogenic Transmission Electron Microscopy (CryoTEM) was also carried out.
From dendrimer solutions, individual vesicles of dendrimer 1 with a bilayer could be observed. These
vesicles disappeared then in presence of DNA and aggregates became visible (Fig. 2C). It is possible
that the negatively charged DNA molecules are intercalated between the two positive lipid bilayers [2].
a
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b
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c
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c
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Fig. 2: A) STM images of 1 deposited by drop casting on HOPG
(Highly Oriented Pyrolitic Graphite) displaying a periodicity of about 4.5 Å
(25 nm x 25 nm, -0.7 V, 1 pA). B) AFM of DNA/1 polyplexes deposited on freshly cleaved
mica surface (diameter 50-80nm, height 5± 0.5 nm). C) CryoTEM image of DNA/1 polyplexes
and schematic view of the possible aggregates. |
In parallel, and in order to identify the biologically crucial structural parameters, a vast library of
new dendrimers has been designed and is currently being synthesized. Further in vitro transfection assays
will allow us to establish quantitative structure-activity relationships (QSAR) to optimize transfection
efficiency. The insertion of chemical moieties sensitive to pH variation that occurs while trafficking
within the cell or to external UV light is also targeted (Fig. 3). Finally, Confocal Laser Scanning
Microscopy (CSLM) on living cells might help to identify the limiting biological issues [3].
3
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4
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Fig. 3:Targeted stimuli sensitive dendrimers with acid-cleavable
(Schiff's base) (3) or photoswitchable azo-core (4). |
[1] |
Amphiphilic Dendrimers: Novel Self-Assembling Vectors for Efficient Gene Delivery.
Derk Joester, Myriam Losson, Raphaël Pugin, Harry Heinzelmann, Elke Walter, Hans P. Merkle, François Diederich
Angew. Chem. Int. Ed. 2003, 42, No. 13, 1486-1490
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[2] |
Lipid-DNA Complex Formation: Reorganization and Rupture of Lipid Vesicles in the Presence of DNA As Observed by Cryoelectron Microscopy
Stefan Huebner, Bronwyn Jean Battersby, Rudo Grimm, and Gregor Cevc
Biophys. J. 1999 76: 3158-3166
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[3] |
DNA–cationic amphiphile interactions
Maria G. Miguel, Alberto A. C. C. Pais, Rita S. Dias, Cecília Leal, Mónica Rosa and Björn Lindman
Colloids and Surfaces A: Physicochemical and Engineering Aspects, Volume 228, Issues 1-3 ,43-55
|
[4] |
Prospects for cationic polymers in gene and oligonucleotide therapy against cancer
Thomas Merdan, Jindrich Kopeek and Thomas Kissel
Adv. Drug Deliv. Rev. 2002, 54, 715
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Contact:
François Diederich |
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Laboratory of Organic Chemistry
ETH Zurich
Switzerland
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