Cationic amphiphilic calixarenes:
nanoscopic micelle formation
and gene delivery
Roman Rodik, Stanislav Miroshnichenko, Vitaly Kalchenko
Institute of Organic Chemistry National Academy of Sciences of Ukraine
Namrata Jain, Ludovic Richert, Yves Mely, Andrey Klymchenko
Laboratory of Biophotonics and Pharmacology, Faculty of Pharmacy,
University of Strasbourg
Application pathways of calixarenes in biology and medicine
Picture from: Calixarene-based multivalent ligands. L. Baldini, A. Casnati, F.
Sansone and R. Ungaro Chem. Soc. Rev., 2007, 36, 254–266
Calixresorc[4]arene glycoclusters.
DNA binding and transfection
Y. Aoyama, Trend. Glycosci. Glycotech. 2005, 17, 39-47.
Gen transfection by polycationic multicalixarenes with
ammonium residues
CA
O
CA
NH
HN
O
Results of transfection pDs2-mito (Clontech)
plasmid in CHO cells
O
O
O
O
O
NH HN
O
CA CA
H3+N
-
Cl
+
H3 N
OO
Cl - Cl-
NH3
NH
HN
O
O
HN
-
+
Cl
NH3+
NH
H3+N
Cl
Cl
H3 N
+
NH3+
Cl- Cl- NH3+
CA =
O
(H 2C) 3
O
O
O
O
(H 2C) 3
O
O
O
a FuGene®; b control; c cone multicalixarene; d
1,3-alt multicalixarene
R. Lalor, J. L. DiGesso, A. Mueller and S. E. Matthews, Chem. Commun. 2007, 4907.
Gen transfection by tetraguanidinium calixarenes
R
R
R
O
O
O
O
NH2
NH2
H2N + N
H
Cl-
Results of transfection pEGFP-C1 plasmid in RD-4
cells mediated by calixarene-DOPE formulation
R
ClH2N
Cl-
NH
HN
+
NH2
+
N + NH2
H
Cl-
NH2
H2N
a-c
a: R = t-Bu
b: R = H
c: R = n-Hex
V. Bagnacani, F. Sansone, G. Donofrio, L. Baldini, A. Casnati, R. Ungaro. Org.
Lett., 2008, 10, 3953-3956.
Water soluble tetracationic calix[4]arene
Cl
Cl
CH3
+
H3 C
CH3 H C
3
N
+
N
HO Cl
OH
OH
H3 C
+
N
H3 C
HO Cl
CH3
+
N
CH3
O
O
O
O
CX3
N. O. Mchedlov-Petrossyan, L. N. Vilkova, N. A.
Vodolazkaya, A. G. Yakubovskaya, R. V. Rodik, V. I.
Boyko and V. I. Kalchenko, Sensors 2006, 6, 962.
N. O. Mchedlov-Petrossyan, N. A. Vodolazkaya, L.
N. Vilkova, O. Y. Soboleva, L. V. Kutuzova, R. V.
Rodik, S. I. Miroshnichenko and A. B. Drapaylo, J.
Mol. Liq. 2009, 145, 197.
For tetrapropoxy calix[4]arene CX3
size of aggregates near 3 nm (DLS
data) n = 6
Synthesis of tetracationic tetraoctylcalix[4]arenes
N
Cl
N
+ Cl
N
+
N
N
+ Cl
N
N
+
N Cl
Cl OH
N
Cl
Cl
Cl
N
N
O O
O O
CX8im
N
Cl
O O
O O
1
Water solubility of CX8 and CX8im – high!!!
+
Cl OH
N
+
HO Cl
N
OH
O O
HO Cl
+
O O
CX8
N
+
8.0x10
water
2.9 M
23.4 M
69.8 M
92.6 M
0.14 mM
0.28 mM
0.98 mM
6
6.0x10
6
4.0x10
6
2.0x10
6
8
Fluorescence intensity (a. u.)
Fluoresscence intensity (a. u.)
Pyren fluorescence in CX8 water solution
2.4x10
8
1.6x10
7
8.0x10
0.01
350
400
1.0
450
500
Wavelenght, nm
I1
I3
0.6
0.4
380
0.1
1
C, mM
Slow then sharp growth of
fluorescence as well as changes
in I1/I3 relation indicates that
micelles arises in solution
0.8
370
550
390
CMC values obtained with pyren probe
Cl
Cl
OH
CH3
+
CH3 H C
3
N
+
N
H3 C
Cl
HO Cl
OH
H3 C
+
N
H3 C
HO Cl
CH3
O
O
O
CX3
+
N
+
H3 C
CH3 H C
3
N
+
N
HO Cl
OH
OH
CH3
CH3
O
Cl
N
H3 C
HO
H3 C
+
CH3
Cl
+
Cl
N
CH3
O
O
O
O
CX8
N
N
+
Cl
+
+
N
N
N
O
N
O
O
Cl
O
CX8im
Water
4·10-4 M
5·10-5 M
1·10-5 M
20 mM Hepes
buffer, pH 7.4
7·10-5 M
6·10-6 M
3·10-6 M
Previous and
150 mM NaCl
8·10-5 M
3·10-6 M
2·10-6 M
N
+
N
Cl
Size of micelles
(Data obtained from DLS measurements)
Size, nm
(polydispersity)
Zeta potential, mV
CX3
3-41
CX8
6.3 (0.4)
37
6.3 (0.25)
41
CX8im
Concentration of CX3 10-3 M, CX8, CX8im 10-4 M
1N.
O. Mchedlov-Petrossyan, N. A. Vodolazkaya, L. N. Vilkova, O. Y.
Soboleva, L. V. Kutuzova, R. V. Rodik, S. I. Miroshnichenko and A. B.
Drapaylo, J. Mol. Liq. 2009, 145, 197.
Fluorescence intensity (a.u.)
Fluorescence correlation spectroscopy experiments
buffer
10 M CX8
1.6
1.2
0.8
0.4
0.0
550
600
650
700
750
Wavelength (nm)
FCS data on CX8 micelles stained with Nile Red
[NR], M
N
Size, nm
0.2
17
5.4
CX8,
0.4
29
5.1
10M
0.8
50.5
6.6
1.6
51
6.3
Sample
5.10 nm
Aggregation number of CX8 in micelles
Geometrical estimation:
FCS Data: 50 particles per excitation
volume 0.34×10-15 L
CX8 approximately is truncated cone with D 1.53 nm,
d 0.51 nm and h 1.62 nm.
Obtained concentration ca 250 nM.
For micelle formation necessary regular cone
geometry with l 2.54 nm. So diameter of micelles 5.1
nm
Aggregation number 10 µM/250 nM is
Volume of micelle 69.5 nm3, volume of CX8 (total
cone) 1.5 nm3, void volume at hexagonal packing
near 10%. So the aggregation number
(69.5×0.9)/1.5 is 41.7.
Starting concentration 10 µM.
40
CT-DNA complexation
Ethidium Bromide displacement experiments
Dependence of integral intensity EB at different concentration of cationic calixarenes
Normalized graphs
1.0
1.0
CX3
CX8
CX8im
0.8
CX3
CX8
CX8im
0.8
0.6
0.6
0.4
0.4
0.2
1E-3
0.005
0.01
C mM
Tris-bufer 20mM, pH 7.4
[CT-DNA]=2•10-5 M
1E-3
0.005
0.01
C, mM
0.1
Tris-bufer 20mM, pH 7.4, 150mM NaCl
[CT-DNA]=2•10-5 M
Size of DNA-calixarene micelles
(Data obtained from DLS measurements)
CT-DNA
Sample
CX3
CX8
CX8im
Parameter
pDNA
N/P=2 N/P=5 N/P=2 N/P=5
Size (N), nm
962
1200
370
1300
Pld.
0.45
0.57
0.28
0.29
Size (N), nm
69
58
60
41
Pld.
0.17
0.32
0.14
0.16
Size (N), nm
72
61
54
41
Pld.
0.16
0.24
0.19
0.14
CX8/
DOPE
Size (N), nm
57
50
Pld.
0.16
0.18
CX8im/
DOPE
Size (N), nm
66
50
Pld.
0.11
0.17
Zeta potential, mV
N/P=2
N/P=5
-15
-6
42
42
42
43
-
-
-
-
AFM image of CT-DNA-calixarene complexes
V(CX8)
1.5(0.4)105 nm3
V(CX8im) 0.9(0.3)105 nm3
d(CX8) 65nm
d(CX8im) 55 nm
AFM topography (A and C) and phase (B and D) images of calixarene CX8 (A,B) and CX8im (C,D) complexes
with CT-DNA (N/P = 2) in 20 mM MES buffer (pH 7). Tapping mode in buffer was used. Circles highlight some
larger structures showing, which in phase images can be resolved as combination of smaller particles.
DNA-calixarene micelles analysis
(CT-DNA was used)
+ ++ +
Fluorescence intensity (a.u.)
++
+ + ++ + + +
+
-
+
+ + ++ + + +
+
+
+
+
+
buffer
+
+
+
1.6
+
+
+
10 M CX8
+
+ ++
+
CX8
/CT-DNA,
+
1.2
+
+
+
N/P
=
2
+
+
+
++
+
++
+
++
++ ++
0.8
+
++
+ + ++ + +
++
+
+
+
+
+
+
+
+
+
++
+ ++
+
++
0.4
++
+
+
+
+ + ++ + +
+
+
+
+
++
+
+
+
+
+
+
+
+
+
0.0 ++
+
+
+
+
+
550
600
700
750
+
+
+ 650
+
+
+
+
+
+
+
+
Wavelength
(nm) +
+
+
+
+
++
+
++
+
+
+
+
++
+
++
++
++
+
+
+
+
+
++
+ ++
++
+ + ++
++
++
+
+
+ + ++ + +
++
++
++
+
+
+
+
+
+
+
+
+
+
+
+
+
++
+
++
++
++
+
+
++
+
+
+
+
+
+
+
+
-
-
-
-
-
-
-
-
-
-
One DNA-CX8 micelle contains 700 CX8 micelles
Gene transfection
Transfection is the process of deliberately introducing nucleic acids into cells. The term is
used notably for non-viral methods in eukaryotic cells.
A plasmid (lat. plasmid) is a DNA molecule that is separate from, and can
replicate independently of, the chromosomal DNA. They are double stranded and,
in many cases, circular. Plasmids usually occur naturally in bacteria, but are
sometimes found in eukaryotic organisms
Protein biosynthesis
Transcription ► Processing and Transport ► Translation
Results of pCMV-Luc plasmid transfection mediated
by calixarenes and calixarene-DOPE formulations
/P
N
/P
/P
N
N
/P
2
5
+
N
A
3
el
ls
10
N
4
C
10
pD
5
O
PE
10
D
6
+
10
PE
7
O
10
CX3
CX8
CX8im
Controls
D
8
5
10
2
RLU/mg protein
Semi-quantitative analyses, amount of transfected cell in the view area
Transfection efficiency of calixarene/pDNA complexes in COS-7 cells. Gene expression
determined from the luciferase assay was expressed as RLU/mg of total protein. The
experiments were repeated three times.
Viability of cells
MTT cell viability assay
CX4
100
CX8
CX8im
Controls
% Cell Viability
80
60
40
20
ls
el
C
Je
tP
EI
M
0
10
M
.5
37
15
M
0
For transfection experiments were used solutions of 10 and 25 µM calixarene concentration
Conclusions and perspectives
•The present results propose a powerful two-step
hierarchical approach for generating small DNA
nanoparticles.
•The further development of gene delivery vectors
based on calixarenes capable to form stable cationic
micellar structures may discover more powerful or
selective DNA vectors for needs biology and
medicine.
Acknowledgements:
Rodik
Roman
is
grateful
to
ARCUS
program
(collaboration
of
Ukraine,
Russia
and
region
Alsace, France) for supporting his visit to the
French Laboratory.
Plasmid binding
Gel electrophoresis experiments
CX3
CX8
CX8im
Results of GFP plasmid transfection mediated by
calixarenes and calixarene-DOPE formulations
Semi-quantitative analyses, amount of transfected cell in the view area
CX3
CX8
CX8im
120
Quantity of cells
100
80
60
40
20
DO
PE
N/
P
5
+
DO
PE
N/
P
2
+
1
N/
P
+
DO
PE
5
N/
P
2
N/
P
N/
P
1
0
Transfection efficiency of calixarene/pDNA complexes in COS-7 cells. Gene expression was
determined from the count of green fluorescence cells in the quarter of microscope view area.