CALIXARENE-BASED NANOSYSTEMS
FOR GENE DELIVERY
Francesco Sansone, Laura Baldini, Alessandro Casnati, Gaetano
Donofrio, Miroslav Dudič, Claudio Rivetti, Rocco Ungaro
Dipartimento di Chimica Organica e Industriale
Università di Parma
Unità INSTM
“Gene Therapy”
Vector
Vettore
Delivery of nucleic
acids to patients for
therapeutic purposes
Vector-DNA
Complesso
Vettore-DNA
complex
DNA
Therapeutic
terapeutico
DNA
Cellular membrane
Membrana cellulare
Gene Therapy can be
used either to treat
diseases
caused
by
defective
genes
(e.g.
Cystic Fibrosis) or to
express other genes for
therapeutic treatment (e.g.
to express genes inside
cancer cells to kill them)
Endosoma
Endosome
Sintesi
delle
Protein
proteine
Synthesis
Nucleo
Nucleus
DNA Delivery Systems for Gene Therapy
Genes, unlike most drugs, are degraded when introduced into the body.
To protect genes, we need to develop delivery systems (Vectors).
VIRAL VECTORS
SYNTHETIC NON-VIRAL VECTORS
Possible infection risk (remove as many genes as possible)
Immune reaction (memory from previous infections)
Protect therapeutic genes well
Show some specificity
No hazard from native gene expression
Less efficient gene transfer than viruses
Lower tissue specificity
Adenovirus / Adeno-associated Virus
Retroviral Vectors
Herpes Simplex Virus
Lentiviral Vectors
Liposomes
Cationic Polymers
Gemini Surfactants
Glycocluster Nanoparticles
“Gene Therapy”
with non-viral, synthetic vectors
Li and Huang Gene Therapy 2006
Felgner et al PNAS 1987
Behr et al PNAS 1989
Gao et al Biochim. Biophys. Res. Commun. 1991
Guanidinium-Calix[n]arenes
+ NH2
Cl
H2N
NH
Cl
+ Cl
H2N
+ Cl
H2N
NH
H2N
HN
Cl +
H2N
NH2
Cl +
NH2
NH2
HN
HN
Cl
H2N
+
H
N
H2N
OMe
OMe MeO
+
NH2
N
H
OMe
+ Cl
H2N
H2N
NH2
NH2
O O
R
R
O O
R R
R = C3H7: 4G4Pr-cone
R = C6H13: 4G4Hex-cone
R = C8H17: 4G4Oct-cone
CONE conformation
Tetrahedron 2004, 60, 11621-11626
JACS 2006, 128, 14528-14536
Cl
NH
HN
O
HN
NH2
+
NH2
NH2
O
O
O
n-3
Cl + NH
2
n = 4: 4G4Me-mobile
n = 6: 6G6Me-mobile
n = 8: 8G8Me-mobile
Conformationally MOBILE
NH
Cl
H2N
+
HN
NH2
H2N
4G4Pr-alt
Cl
NH2
+
1,3-ALTERNATE
Synthesis
1
t
R
Bu
t
t
Bu Bu
t
Bu
t
Bu
RI, base
O O
O
H
H
HNO3 or NaNO3
OR
OR RO
OR
t
Bu
1
t
R
Bu
H
1
t
R
1a: R = hexyl; cone
1b: R = propyl; 1,3-alt
1c: R = methyl; mobile
2a: R1= NO2;R = hexyl; cone
2b: R1= NO2;R = propyl; 1,3-alt
2c: R1= NO2;R = methyl; mobile
NH2NH2, Pd/C
_ + NH2
H2N
+
H2N
3a: R1= NH2;R = hexyl; cone
3b: R1= NH2;R = propyl; 1,3-alt
3c: R1= NH2;R = methyl; mobile
NBoc
BocHN
NH
H2N
H
N
1
R
O
H
Bu
Cl
OR
OR RO
OR
Cl
_
+
NH2
OR
OR RO
OR
NH
N
H
_ NH
Cl
2
BocHN
HCl
NBoc
OR
OR RO
OR
H
N
BocN
N
H
NHBoc
HN
NH2
+ NH2
Cl
HN
NHBoc
_
4G4-Hex-cone: R = hexyl; cone
4G4-Pr-alt: R = propyl; 1,3-alt
4G4-Me-mobile: R = methyl; mobile
NBoc
4a: R = hexyl; cone
4b: R = propyl; 1,3-alt
4c: R = methyl; mobile
HgCl2
or Mukayama's reagent
BocNHC(S)NHBoc
Water solubility
compound
e (dm 3 x mol -1 x cm -1)
cmax (mol x dm -3)
4G4Pr-cone
3300
1x10-2
4G4Oct-cone
-
 10-4
6G6Me-mobile
4640
8x10-3
8G8Me-mobile
5120
4x10-3
Cell Transfection
GenePORTER
transfection
4G4Oct-cone
reagent
4G4Oct-cone
4G4Oct-cone
4G4Oct-cone
5 mM
10 mM
20 mM
4G4Pr-cone
4G4Pr-cone
4G4Pr-cone
4G4Pr-cone
DOPE
15 mM
15 mM
+
DOPE 15 mM
15 mM
+
DOPE 30 mM
15 mM
+
DOPE 7.5 mM
30 mM
a
40 mM
b
without DOPE
Calix[4] octyl
Calix[4] hexyl
cone
Transfection
Calix[4] propyl cone
with DOPE
No Transfection
Calix[4] methyl mobile
Calix[4] propyl 1,3-alt
Calix[6] methyl mobile
Calix[8] methyl
Plasmid DNA and CONE Guanidinium-Calix[4]arenes
Plasmid
DNA
1 nM
Plasmid
DNA
+
4G4Hex-cone
(1mM)
JACS 2006, 128, 14528-14536
Plasmid
DNA
+
4G4Pr-cone
(1mM)
Plasmid
DNA
+
4G4Oct-cone
(1mM)
Plasmid DNA and “OTHERS” Guanidinium-Calixarenes
Plasmid
DNA
+
6G6Me-mobile
(1mM)
Plasmid
DNA
+
4G4Me-mobile
(1mM)
Plasmid
DNA
+
8G8Me-mobile
(1mM)
Plasmid
DNA
+
4G4Pr-alt
(1mM)
Effect of ETHANOL on CONE Guanidinium-Calix[4]arenes
Plasmid
DNA
1 nM
+
5% EtOH
Plasmid
DNA
+
4G4Hex-cone
+ 15% EtOH
Plasmid
DNA
+
4G4Pr-cone
+ 5% EtOH
Plasmid
DNA
+
4G4Oct-cone
+ 15% EtOH
H2N
H2N
H2N
+
+
NH
NH2
HN
H2N
NH2
+
HN
HN
NH2
O O
O O
R
+ +
+ NH2
=+
R R
R
R = C3H7: 4G4Pr-cone
R = C6H13: 4G4Hex-cone
R = C8H17: 4G4Oct-cone
Transfection !
+ +
+
+
+ +
+
+
+
+
+ +
+
+ +
+
+
+
+
+
+
+ +
+
+
+
+
+ +
+
+ +
+ +
+
+
+ +
+
+ +
+ +
+
+
+
+
+ +
+
+
+
Effect of ETHANOL on 1,3-ALTERNATE and MOBILE
Guanidinium-Calix[4]arenes
Plasmid
DNA
+
4G4Pr-alt
+ 5% EtOH
Plasmid
DNA
+
4G4Me-mobile
+ 5% EtOH
+
HN
NH2
O
=
O
+
O
NH
NH2
+
H2N
+
+
O
NH2
HN
+
+
NH2
H2N
Transfection !
4G4Pr-alt
+ DOPE
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
H2N
+
NH
+
H2N
Effect of ETHANOL on
MOBILE Guanidinium-Calix[6]- and Calix[8]arenes
Plasmid
DNA
+
6G6Me-mobile
+5% EtOH
Plasmid
DNA
+
8G8Me-mobile
+5% EtOH
NH2
+
HN
NH2
+
=
+
No Transfection!
n-1
NH
+
NH2
+
+
+
+
+
+
+
+
+
+
++
+
+
+
+
+
+
+
+
+
+
+
++
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
n = 2: 6G6Me-mobile
n = 4: 8G8Me-mobile
+
+
H2N
+
+
H2N
NH
+
NH2
+
H2N + N
H
+
OMe MeO
OMe
NH2
+
NH2
+
+
OMe
OMe MeO
+
+
+
H +
NH2
N
+
H2N
NH2
+
HN
Conclusions
A new series of calixarene-based ligands was synthesised
able to bind plasmid DNA
Some of them give cell transfection thanks to their DNA
condensation capability
Condensation and transfection properties are strongly
dependent on conformation, structure and size of the
ligands
relative fluorescence at 600 nm
0.21
1
+
HN
A at 260 nm
NH2
H +
NH2
N
OMe
OMe MeO
0.8
NH2
Linear Plasmid
OMe MeO
OMe
NH2
H2N + N
H
0.17
0.15
NH2
H2N
NH2
+
HN
0.19
DNA
0.23
8G8Me-mobile
1.2
4G4Pr-cone
6G6Me-mobile
Interaction with plasmid DNA
H2N
H2N
NH
+
NH2 3
NH
+
NH2
+ Cl
H2 N
H2 N
Cl
NH
HN
O
O
0.6
+ 4G4Pr-cone
linear plasmid
4G4Pr-alt
8G8Me-mobile
++ 4G4Pr-cone
+
NH2
NH2
4G4Pr-cone
O
O
NH
NH2
Cl
H2 N
+
0.13
0.11 0.4
+ 4G4Pr-alt
8G8Me-mobile
HN
Cl
H2 N
8G8Me-mobile
NH2
+
+
HN
4G4Pr-alt
NH2
NH2
H2N
NH2
+
HN
+ Cl
H
H2 N N +
OMe
OMe MeO
0.09
H2 N
NH
+
NH2Cl NH
2
NH2 HN
O
+ Cl
H2N
+ Cl
H2N
0.2
NH
0.07
H2N
HN
Cl +
H2N NH2 NH
2
NHN2
H
+N N
2
H
H
HN
OMe MeO
OMe
Cl +
NH2
NH2
H2N
O O
O O
NH
+
NH2
NH2
O
O
O
NH
NH
HN
+
Cl
ClH2N NHNH
2
3 H2 N
2
H2 N
NH2
+
+
+ Cl
H2N
+ Cl
H2N
NH
0.05
H2N
Cl +
NH2
HN
-1
30
4
40
9
50
14
60
temp (°C)
19
70
24
80
29
90
O O
O O
4G4Pr-cone
calixarene concentration (microM)
Melting
curves
Ethidium Bromide
Displacement
Assays
Electrophoresis Mobility Shift Assay
JACS
Tetrahedron
JACS2006,
2006,128,
2004,
128,14528-14536
14528-14536
60, 11621-11626
HN
Cl +
NH2
NH2
4G4Pr-cone
0
20
H2N
NH2
HN
1H
NMR spectra in D2O
(300 MHz, 300 K)
Cl +
HN
NH2
NH2
H2N
NH2
+
Cl
HN
Cl
H +
NH2
N
OMe
OMe MeO
NH2
OMe MeO
OMe
NH2
H2N + N
H
Cl
NH
+ Cl
NH2
H2N
H2N
NH
+ Cl
NH2
6G6Me-mobile
10.0
8.0
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0.0
-1.0
-2.0
9.0
8.0
7.0
6.0
5.0
4.0
3.0
2.0
ppm (f1)
-3.0
+ NH2
Cl
+ Cl
H2N
+ Cl
H2N
NH
H2N
HN
H2N
Cl +
H2N
NH2
Cl +
NH2
NH2
HN
HN
Cl
Cl
NH2
O O
NH
H2N
+
H
N
H2N
OMe
OMe MeO
OMe
NH2
O O
HN
NH2
Cl + NH
2
4G4Pr-cone
4G4Me-mobile
9.0
8.0
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0.0
N
H
+
NH2
Dilution experiments
by 1H NMR in D2O
(300 MHz, 300 K)
with 4G4Hex-cone
10.0 mM
5.0 mM
120
2.5 mM
Hax
OCH2
100
+ Cl
H2N
NH
H2N
HN
Cl +
H2N
NH2
Cl +
NH2
NH2
HN
HN
NH2
O O
O O
80
% of aggregate
+ Cl
H2N
1.6 mM
60
40
1.0
mM
cac
cmc
20
4G4Hex-cone
0.5
mM
0
1e-5
0.2 mM
0.2 mM
1e-4
1e-3
[4G4Hex-cone] (M)
1e-2
1e-1
NMR DOSY experiments with 4G4Hex-cone in D2O (300 MHz, 300 K)
LB510_3: dosy di DM148 1mM in D2O, T=298K
LB510_5: dosy di DM148 0.2mM in D2O T=298K
LogD
LogD
ppm
ppm
-10.2
-10.2
-10.2
-10.1
-10.1
-10.1
-10.0
-10.0
-10.0
-9.9
-9.9
-9.9
-9.8
-9.8
-9.8
-9.7
-9.7
-9.7
-9.6
-9.6
-9.6
-9.5
-9.5
-9.5
-9.4
-9.4
-9.4
-9.3
-9.3
-9.3
ppm
LogD
-9.2
-9.2
-9.2
8
7
6
5
4
3
2
1
ppm
8
7
6
5
4
3
2
1
ppm
8
7
6
5
4
3
2
1
ppm
Hydrodynamic radius rH Hydrodynamic radius rH
of monomer:
of aggregate:
8.9 Å
32.7 Å
Acknowledgments
Miroslav Dudič
Laura Baldini,
Alessandro Casnati
Rocco Ungaro
Dept. of Organic and Industrial Chemistry
Gaetano Donofrio
Dept. of Animal Health
Claudio Rivetti
Dept. of Biochemistry and Molecular Biology