Cationic amphiphilic calixarenes to compact DNA into small nanoparticles for gene delivery

Article abstract of DOI:10.1039/c4nj01395f

Macrocycles have been attracting increasing attention as scaffolds for preparation of non-viral vectors for gene delivery. Here, following our earlier report, a series of amphiphilic calixarenes bearing cationic choline or N-(2-aminoethyl)-N,N-dimethylammonium groups at the upper rim and alkyl chains at the lower rim were synthesized. The effect of the length of aliphatic chains and the structure of the head group on their self-assembly, interaction with DNA, properties of their DNA complexes, transfection efficiency and cytotoxicity was studied. It was found that longer alkyl chains favor formation of small virus-sized DNA nanoparticles with low polydispersity. Moreover, longer alkyl chains, such as dodecyl groups, significantly improved the transfection efficiency, so transfection was observed in the presence of fetal bovine serum as well as with or without a helper lipid. Finally, we observed that the cytotoxicity of these calixarenes clearly decreased with increase of the chain length. On the other hand, the presence of four additional amino groups, which could be protonated at pH < 7, affected only the stoichiometry of the complexes with DNA without influencing their transfection efficiency or cytotoxicity. The results obtained provide new insights for designing non-viral vectors based on macrocyclic molecules.

Full text of DOI:10.1039/c4nj01395f

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Page 1 of 22
New Journal of Chemistry
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DOI: 10.1039/C4NJ01395F
Cationic amphiphilic calixarenes with longer alkyl chains are less cytotoxic and their virus-sized DNA
nanoparticles exhibit higher transfection efficiency.

New Journal of Chemistry
Page 2 of 22
Cationic amphiphilic calixarenes to compact DNA into small nanoparticles for
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DOI: 10.1039/C4NJ01395F
gene delivery
Roman V. Rodik,[a],[b] Ann-Sophie Anthony,[a] Vitaly I. Kalchenko,[b] Yves Mély[a] Andrey S.
Klymchenko[a],*
a
Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Université de Strasbourg,
Faculté de Pharmacie, 74, Route du Rhin, 67401 ILLKIRCH Cedex, France.
b Institute of Organic Chemistry, National Academy of Science of Ukraine, 02660, Murmanska str.,
5, Kiev, Ukraine.
* Corresponding author, Tel: +33 368 85 42 55; E-mail: andrey.klymchenko@unistra.fr.
Abstract
Macrocycles have been attracting increasing attention as scaffolds for preparation of non-viral
vectors for gene delivery. Here, following our earlier report, a series of amphiphilic calixarenes
bearing cationic choline or N-(2-aminoethyl)-N,N-dimethylammonium groups at the upper rim and
alkyl chains at the lower rim were synthesized. The effect of the length of aliphatic chains and the
structure of the head group on their self-assembly, interaction with DNA, properties of their DNA
complexes, transfection efficiency and cytotoxicity was studied. It was found that longer alkyl
chains favor formation of small virus-sized DNA nanoparticles with low polydispersity. Moreover,
longer alkyl chains, such as dodecyl groups, significantly improved the transfection efficiency, so
that transfection was observed in the presence of fetal bovine serum as well as with or without
helper lipid. Finally, we observed that cytotoxicity of these calixarenes clearly decreases with
increase of the chain length. On the other hand, the presence of four additional amino groups, which
could be protonated at pH<7 affected only the stoichiometry of the complexes with DNA without
influencing their transfection efficiency or cytotoxicity. The results obtained provide new insights
for designing non-viral vectors based on macrocyclic molecules.

Page 3 of 22
New Journal of Chemistry
Introduction
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Development of nanoscale vehicles to deliver therapeutic genes remains an importanDOtI:c1h0.a10ll3e9n/Cg4eNJ0in1395F
the field of gene therapy.1, 2 According to in vitro and in vivo studies, naked DNA and RNA exhibit
a very poor capacity to transfect cells, because of a number of barriers and their decomposition by
nucleases in biological media.3-5 Therefore, efficient gene delivery requires specially designed
nucleic acid carriers – gene delivery vectors.
Despite the success in the development of truncated viruses, so-called viral vectors,6 the problem of
their safety has stimulated the research on alternative concepts of gene delivery. For many decades
significant efforts have been made by chemists to develop artificial DNA carriers, so-called non-
viral vectors.1, 7 Particularly successful were developments of cationic lipids8-10 and polymers.11-13
Their complexes with DNA, so-called lipoplexes and polyplexes, respectively show high
transfection efficiency in vitro, weak immunogenicity and capacity to carry large quantities of
genetic information,1 though their application for gene therapy in vivo remains a challenge. One of
the key problems of cationic lipids and polymers is the poor structural control of their complexes
with DNA,14 which are generally large infinite and polydisperse structures.15-17
Therefore, the important unsolved problem is to control assembly of non-viral vectors with DNA
into small nano-objects of defined structure and architecture that remains stable in biological
media.1, 18-21 A particularly fruitful approach is to employ pre-organized building blocks such as
dendrimers,22-24 gold nanoparticles,25, 26 silica nanoparticles,27 carbon nanotubes,28, 29 and core-shell
organic nanoparticles.21 Among them, fullerenes30-32 and macrocyclic molecules, such as
cyclodextrins,33-35 calix[4]resorcinarenes,36-39 and calix[4]arenes40, 41 are particularly promising
because these synthetically available molecules have a well-defined 3D topology due to their
persistent shape and capacity to bear multiple groups for interacting with nucleic acids.42, 43
Recently, we described cationic amphiphiles based on calix[4]arenes and showed that their capacity
to form micelles allows preparation of virus-sized DNA nanoparticles with satisfactory transfection
efficiency.44 Moreover recently, Ungaro et al, presented an arginine analogues calix[4]arenes with
significantly improved transfection properties,45 and confirmed that cationic amphiphilic
calix[4]arenes are highly fruitful direction for development of nanoscopic non-viral vectors.
Here, we synthesized a series of amphiphilic calix[4]arenes with varied length of the alkyl chains
and the nature of the cationic group. Our studies showed that calix[4]arenes derivatives with clear
amphiphilic properties are capable to condense DNA into very small nanoparticles. Importantly, the
derivatives bearing relatively long chains exhibited higher transfection efficiency together with
lower cytotoxicity. Thus, the structure-property relationships were described suggesting new
insights for the rational design of non-viral vectors based on macrocycles.

New Journal of Chemistry
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Results and discussion
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DOI: 10.1039/C4NJ01395F
Design of calix[4]arenes
The design is based on our previous work,44 where the upper and lower rims are functionalized by
four cationic groups and aliphatic chains, respectively. A series of new derivatives with aliphatic
chains of varied length and with different cationic groups was synthesized. Calixarenes with hexyl,
dodecyl and hexadecyl alkyl chains were synthesized as the previously described CX3 and CX8
compounds, according to scheme 1.
OH
HO
OH
OH
OH
1
OH
HO
OH
N+
Cl-
C
Cl-
l
-
a
Cl-
N+
Cl
N+
Cl
N+
Cl
Cl
c
b
O
O
O
O
O
O
O
O
O
O
O
O
R
R
R
R
R
R
R
R
R
R
R
R
R = C3H7 - 2,
3
5
7
9
CX3,
CX6,
CX8,
CX12,
CX16
4
R = C6H13 - ,
R = C8H17 - 6,
8
R = C12H25
R = C16H33 - 10
- ,
11
a: Alk-Br, DMSO-NaOH; b: CH3-O-CH2Cl, SnCl4; c: (CH3)2N-CH2-CH2-OH
Scheme 1. Synthetic route to cationic calixarenes CX3-CX16.
The key step of synthesis is the quaternization reaction, which was done after alkylation of 1 and
further chloromethylation. This quaternization was performed in dry THF in all cases. Obtained
calixarenes precipitated directly from the reaction mixture despite the presence of highly lipophilic
groups. Water solubility of obtained calixarenes was also decreased with increase of alkyl chain
length. Thus, CX16 was practically insoluble in water and even in neat DMSO, and was only
soluble in methanol and DMSO-chloroform mixtures. To circumvent the solubility problem, we
shortened two of the four alkyl chains from hexadecyl to propyl. Calixarene CX3-16 was
synthesized according to scheme 2 and required an intermediate step, where two propyl groups
were initially introduced into distal position of the lower rim of 1.

Page 5 of 22
New Journal of Chemistry
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DOI: 10.1039/C4NJ01395F
Scheme 2. Synthetic route to calixarene CX3-16.
In our earlier work, we showed that substitution of tetrahedral tetraalkylammonium with planar N-
alkyl imidazolium did not influence the transfection efficiency.44 Therefore, we changed the
strategy by replacing the hydroxyethyl group by an aminoethyl group as shown in scheme 3 for
calixarene CX8N. These amino groups being in proximity of quaternized ammonium groups
should be much less basic than aliphatic amines, so that they could be protonated at the pH of
lysosomes, similarly to polyethyleneimine.46 Introduction of these groups to the macrocycle upper
rim was performed by reacting tetrachloromethyl calixarene 7 with trifluoroacetamide of N,N-
dimethyl ethylenediamine. The use of unprotected amine in this reaction was unsuccessful, leading
to a complex mixture of compounds. Interestingly, addition of a relatively polar amide group to the
tetracationic calixarene significantly increased its solubility in mid polar organic solvents. Besides
alcohols, DMF and DMSO, calixarene 15 was soluble in THF, chloroform, acetonitrile, but
insoluble in diethyl ether. This feature was used for its isolation. Deacylation of 15 was completed
in mild conditions by water ammonia. The final product CX8N could not be easily isolated from
trifluoroacetate ammonia.

New Journal of Chemistry
Page 6 of 22
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DOI: 10.1039/C4NJ01395F
Scheme 3. Synthetic route to calixarene CX8N.
Self-assembly
All synthesized calixarenes (except CX16) showed good water solubility. As these
amphiphiles are expected to self-assembly into micelles,44 we studied their critical micellar
concentration (CMC). Pyrene was used as an environmentally-sensitive fluorescent probe
molecule.47 The CMC was determined by plotting the ratio of I3/I1 peaks of pyrene (at 373 and 384
nm, respectively) as a function of the calixarene concentration. The typically plot presents
sigmoidal shape of the curve, as shown in Figure 1.
0.76
0.72
0.68
0.64
0.60
10-6
10-5
10-4
10-3
[CX6], M
Fig. 1. Intensity ratio I3/I1 of pyrene (1.5·10-7 M) vs CX6 concentration in 20 mM Tris buffer, pH
7.4.

Page 7 of 22
New Journal of Chemistry
The CMC values deduced from the point of growth of the I3/I1 ratio were found to decrease with the
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DOI: 10.1039/C4NJ01395F
increase in the chain length (Table 1), as expected. However, for calixarenes with long alkyl chains
(CX12, CX16 and CX3-16), the obtained CMC values were not significantly lower than that of
CX8 (1-4 µM), and were dependent on the pyrene concentration. We concluded that for these
calixarenes, we could not obtain reliable data, because the used pyrene concentration was much
higher than their CMC value. As a result, the point of I3/I1 ratio growth cannot be easily observed
because of the large excess of free pyrene molecules as compared to those bound to micelles.
Remarkably, the I3/I1 ratio also indicates the lipophilicity level of the pyrene environment in the
micelles. According to these data, the least lipophilic binding sites were observed for CX8N
micelles (I3/I1  0.72), whereas the most lipophilic ones were observed for CX3-16 micelles (I3/I1 
0.83).
Table 1. CMC and I3/I1 ratio values of investigated calixarenes in different media.a
Water
pH 7.4 buffer
pH 7.4 buffer,
150 mM NaCl
pH 5.0 buffer
CMC
µM
390
26
I3/I1
CMC, I3/I1
CMC,
I3/I1
ratio
0.82
0.81
0.78
0.70
0.83
CMC,
I3/I1
Ratio
0.79
0.76
0.73
0.72
0.84
µM
68
ratio
0.82
0.76
0.75
0.69
0.83
µM
64
µM
–
ratio
CX3
CX6
CX8
CX8N
9.8
4.4
3.6
1.0
4.4
2.7
3.0
–
–
19
–
17
14
–
0.69
0.75
CX3-16
a
pH 7.4 referes to 20 mM phosphate buffer (pH 7.4), pH 5.0 referes to 20 mM Acetate buffer (pH
5.0).
Next, the hydrodynamic diameter and polydispersity of the obtained micelles were evaluated by
dynamic light scattering (DLS). Obtained values of the hydrodynamic diameter in the range 5-8 nm
(Table 2) confirm the micellar nature of the assemblies, as in our earlier work on CX8.44
Remarkably, the size of the micelles increases with the chain length. Noticeably, CX12 gives
smaller particles than CX3-16, though the latter has 38 carbon atoms in their alkyl chains while
CX12 has 48 atoms. The micelles of CX8N are larger than those of CX8, probably due to the larger
hydration shell of amino groups as compared to hydroxyls. At pH 5, the size of the CX8N micelles
was further increased probably due to protonation of the amino groups. The relatively high
polydispersity observed in several samples is likely explained by the presence of a small quantity of
large particles (1000-5000 nm), which could be observed in the DLS data analysis by scattering

New Journal of Chemistry
Page 8 of 22
intensity. As DLS is much more sensitive to larger particles (scattering intensity is proportional to
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DOI: 10.1039/C4NJ01395F
the sixth power of the particle size), the detection of very small objects is usually perturbed even by
traces of large particles. According to statistics by volume, only the micellar fraction was
systematically observed for our calixarenes. In contrast, CX16 showed two populations of particles:
a fraction of 30-35 nm size which slowly transforms into larger particles of 300-400 nm size. Thus,
CX16 bearing much longer lipophilic chains is unable to form micelles, probably because the
volume taken by these chains is comparable or larger to that taken by the polar head groups. This
molecular topology is not suitable for micellar assembly and probably favors formation of larger
structures.
Table 2. Size and polydispersity of calixarene micelles and calixarene-DNA complexes obtained
from DLS data.a
Calixarene Alone
With pCMV-eGFPb
N/P 2
With CT-DNAb
N/P 2
N/P 5
N/P 5
d, nm PdI
d, nm
PdI
-
d, nm
PdI
-
d, nm
PdI
d, nm
PdI
2.93
5.47
5.63
6.07
6.56
0.42
0.68
0.25
0.34
0.51
-
-
1090
0.44
1280
90
0.57
CX3
CX6
CX8
CX8N
1380
72
0.46
0.12
0.14
-
66
51
62
59
0.12
0.11
0.10
0.25
1250
115
0.64
0.12
0.50
0.17
0.13
0.10
0.09
0.15
76
154
-
1070
103
112
76
CX8N
pH 5.0
7.37
8.28
0.30
0.17
117
107
0.13
0.12
55
57
0.10
0.10
106
102
0.14
0.11
76
75
0.04
0.10
CX12
CX3-16
a
d is hydrodynamic diameter, defined by volume statistics; PdI is polydispersity index. All
measurements were done in 20 mM Tris buffer at pH 7.4, unless indicated (20 mM acetate buffer
for pH 5). b 30 µM by phosphate groups.
Interaction with DNA
Gel-electrophoresis was used to study the complexation of calixarenes with DNA. Mixtures of
pDNA with calixarenes at N/P ratios 0, 0.2, 0.5, 1, 2 and 5 were prepared in Tris-buffer and tested

Page 9 of 22
New Journal of Chemistry
after 30 minutes of incubation. For CX8N acetate buffer with pH 5 was also investigated. Signal of
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DOI: 10.1039/C4NJ01395F
free pDNA disappeared at N/P 1 for all studied calixarenes (Fig. 2) indicating the efficient complex
formation independently of the calixarene structure. Remarkably, for CX8N at pH 5.0, complex
formation is observed already at N/P 0.5 (figure 3B), because full protonation of amino groups of
CX8N is expected to increase twice its charge.
Fig. 2. Gel electrophoresis of pDNA-calixarene complexes at different N/P ratios. A – CX6, B –
CX8, C – CX12, D – CX3-16. L is the ladder standard.
Fig. 3. Gel electrophoresis of pDNA-CX8N complexes at different N/P ratio. (A) pH = 7.4 (20 mM
Tris buffer), (B) pH = 5.0 (20 mM acetate buffer). L is the ladder standard.
Interaction of DNA with transfection agents and its further condensation can be monitored using
ethidium bromide displacement assay. For all studied calixarenes, a rapid decrease of ethidium
bromide fluorescence was observed at N/P ratios around 1 (Fig. 4). Above N/P 2, the fluorescence
was nearly constant showing that DNA condensation was completed around N/P 2. The only
exception was CX8N, especially at pH 5, where the plateau was reached at N/P 1, confirming the
results of gel electrophoresis. The final values of intensity for CX8N were significantly lower than
for other calixarenes indicating that the additional positive charges favor stronger DNA
condensation.

New Journal of Chemistry
Page 10 of 22
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DOI: 10.1039/C4NJ01395F
1.0
0.8
0.6
0.4
0.2
0.0
CX3
CX6
CX8
CX8N
CX12
CX8N pH5
CX3-16
0
2
4
6
8
10
12
N/P ratio
Fig. 4. Exclusion of ethidium bromide (0.4 M) from CT-DNA (20µM) in the presence of
calixarenes. Dependence of ethidium bromide intensity from calixarene concentration expressed in
N/P ratio. Concentrations of ethidium bromide and CT-DNA were 0.4 M and 20 µM, respectively,
in 20 mM Tris buffer (pH 7.4) or 20 mM acetate buffer (pH 5.0).
Size of calixarene-DNA complexes
For all calixarenes, except CX3 we observed small, stable, and unimodal particles at N/P ratio 5
(Table 2). At N/P 2, the particle size is larger, especially for CX6 and CX8N, which shows
microscopic particles. According to ethidium bromide data, the DNA condensation is completed at
N/P > 2. Therefore, the cationic agent should be in excess at higher N/P ratios, which increases the
positive charge of the complexes and thus favors their small size due to electrostatic repulsion
between particles. Moreover, the size of the complexes is smaller for pDNA compared to CT-DNA,
which correlates with the smaller size of the former. The effect of calixarene structure on the size of
complexes was observed mainly at N/P ratio 2, where CX6 and CX8N form larger complexes than
all other new calixarenes. In this respect, CX6 behaves similarly to CX3, likely as a result of its
insufficiently long aliphatic chains. Short chains decrease the capacity of calixarenes to form
micelles, which are building blocks for small DNA nanoparticles.44 Moreover, other reports
suggested that analogues of CX3 can form much larger structures, such as vesicles,48 and thus
unable to assemble with DNA into small nanoparticles. In case of CX8N, the additional amino
groups may induce additional interactions with nucleic acids through H-bonding, which could be a
factor producing aggregation of DNA into large particles.
Transfection study and cytotoxicity

Page 11 of 22
New Journal of Chemistry
According to our previous data with CX8, the transfection efficiency is significant only in the
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DOI: 10.1039/C4NJ01395F
presence of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). Therefore, we tested the
transfection efficiency of all new calixarenes together with CX8 in the presence of DOPE (1 mol
eq.) at N/P ratio 2. A plasmid encoding eGFP (pCMV-eGFP) was used for transfection, so that
transfected cells were detected by monitoring the expressed GFP using fluorescence microscopy
(Fig. 5). Remarkably, the most promising results were observed for CX12, which shows much
higher rate of transfected cells (~80%) than the previously reported CX8 (~36%), being comparable
to that of jetPEI (~70%), one of the most efficient transfection agents. The rate of transfected cells
grows in the following order: CX8N<CX6<CX3-16~CX8<CX12 (Fig. 6). In the presence of serum,
only CX12 showed detectable transfection among the studied calixarenes (Fig. 5). Moreover, CX12
was the only calixarene that showed detectable transfection without DOPE. Thus, the long alkyl
chains and the large hydrophobic domain of CX12 significantly improve the transfection efficiency.
Fig. 5. Fluorescence (A1, B1, C1, D1, E1, F1, G1, H1) and transmission (A2, B2, C2, E2, F2, G2,
H2) images of HeLa cells transfected with pCMV-eGFP plasmid complexed with calixarene (N/P 2
with 1eq. of DOPE) or commercial JetPEI. (A) JetPEI; (B) CX6; (C) CX8; (D) CX8N; (E) CX12;

New Journal of Chemistry
Page 12 of 22
(F) CX3-16; (G) CX12 without DOPE; (H) CX12 in the presence of fetal bovine serum. The images
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DOI: 10.1039/C4NJ01395F
were recorded 48 h after transfection.
100
80
60
40
20
0
12
CX
8N
CX
CX6 CX8
3-16
CX
JetPEI pDNA
naked
CX
12-FBS
12-w/o DOPE
CX
Fig. 6. Rate of transfected cells for calixarenes complexed with pDNA (pCMV-eGFP) in the
presence of 1eq. DOPE compared to commercial agent jetPEI and naked pDNA. The complexes or
naked DNA were first incubated for 3h with HeLa cells in Opti-MEM without FBS and then for
additional 45h with 10% FBS. For CX12, the data with 10% FBS (for all 48h) and the data without
DOPE (w/o DOPE) are shown.
Finally, the toxicity of new calixarenes was tested on HeLa cells using MTT assay. The cytotoxicity
decreases rapidly with the increase in the length of the aliphatic chains for all concentrations
studied, i.e. the following tendency was observed: CX6>CX8~CX8N>CX12>CX3-16. The high
cytotoxicity of CX6 is in line with the systematic observation of high cell mortality in the
transfection experiments with this calixarene (see Fig. 5B2). However, in these measurements the
first 3h of incubation were done in Opti-MEM solution without serum and the remaining 45h with
10% of serum. When the incubation was done in the presence of serum for all 48h, the cytotoxicity
of all calixarenes was significantly decreased (Fig. 5B). Nevertheless, the calixarenes with the
longest aliphatic chains (CX12 and CX3-16) showed the lowest cytotoxicity. This is the first
observation of a clear effect of the aliphatic chain length on the cytotoxicity of calixarenes. Longer
aliphatic chains decrease the CMC of CX12 and CX3-16, so that the concentration of free
molecules is lower than for calixarenes with shorter chains. Moreover, due to high lipophilicity,
CX12 and CX3-16 may stronger interact with the lipid components of the cells, which may prevent
them from interacting with other biomolecules, such as proteins and nucleic acids. Thus, CX12,
presenting the most hydrophobic domain, exhibited higher transfection efficiency combined with
lower cytotoxicity, compared to previously reported CX8.

Page 13 of 22
New Journal of Chemistry
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DOI: 10.1039/C4NJ01395F
CX6
CX8
CX8N
CX12
CX3-16
100
80
60
40
20
0
100
80
60
40
20
0
10
2
40
5
Calixarene concentration (M)
Fig. 7. Cytotoxicity of calixarenes without (A) and with 10% of serum (B) after 3h of incubation.
Cell viability was determined by MTT assay after 48 h of incubation.
Conclusions
A series of amphiphilic calixarenes bearing cationic groups at the upper rim and alkyl chains at the
lower rim were synthesized. The effect of the length of aliphatic chains and the head group structure
on their self-assembly, interaction with DNA, properties of their DNA complexes, transfection
efficiency and cytotoxicity was studied. It was found that longer alkyl chains favor formation of
smaller particles with DNA, characterized by higher transfection efficiency and lower cytotoxicity.
On the other hand, the presence of four additional amino groups, which could be protonated at
pH<7 affected only the stoichiometry of the complexes with DNA without affecting their
transfection efficiency or cytotoxicity. The results obtained provide new insights for designing new
artificial viruses based on macrocyclic molecules.
Experimental
Synthesis
All reactions were carried out in anhydrous solvent under dry atmosphere. NMR spectra were
recorded on a spectrometer “Varian VXR-300” using hexamethyldisiloxane and tetramethylsilane
as internal standards. Chemical shifts are given in  scales (1H and 19F). Mass spectra were recorded
using Mass Spectrometer Mariner System 5155.

New Journal of Chemistry
Page 14 of 22
Tetraalkoxy-calixarenes 2, 4, 6 were obtained accordingly literature data.49 Dipropoxycalixarene 12
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50
DOI: 10.1039/C4NJ01395F
was synthesized and described earlier. Synthesis of tetrachloromethylcalixarenes 3 and 7 as well
as calixarenes CX3 and CX8 was described in literature previously.51, 52
General procedure for synthesis of tetraalkoxy-calixarenes 8, 10 and 13. Sodium hydroxide
(50% water solution, 3.53 ml, 50.05 mmol NaOH) and then calixarene 1 or 12 (5.40 mmol) were
added to DMSO (25 mL). The reaction mixture was stirred for 15 minutes. Alkyl bromide (45.0
mmol) was added in three portions every 30 minutes and the mixture was stirred for 8-12 h at 70–80
°C. After cooling to 20 °C the diluted hydrochloric acid was poured into the reaction mixture.
Compounds 8, 10, 13 were extracted by chloroform, dried with sodium sulfate. Chloroform was
evaporated and the residue was washed with methanol.
25,26,27,28-Tetradodecyloxycalix[4]arene (8). Crude product was dried in vacuo (0.5 mm Hg) at
120 °C for 3 hours and crystallized from acetonitrile. Obtained as colorless solid. Yield 96%. M.p.
52 °С. 1H NMR (CDCl3, 299.94 MHz), δ ppm: 6.60 (d, J = 5.3 Hz, 8H, m-ArH), 6.59 (t, J = 5.3 Hz,
4H, p-ArH), 4.42 (d, J = 13.2 Hz, 4H, ArCHaxAr), 3.87 (t, J = 7.5 Hz, 8H, O-CH2-CH2-(CH2)9-
CH3), 3.17 (d, J = 13.2 Hz, 4H, ArCHeqAr), 1.86 (brm, 4H, O-CH2-CH2-(CH2)9-CH3), 1.44-1.24
13
(brs, 72H, O-CH2-CH2-(CH2)9-CH3), 0.89 (t, J = 6.5 Hz, 12H, O-CH2-CH2-(CH2)9-CH3) C NMR
(CDCl3, 125.73 MHz), δ ppm: 156.32, 134.90, 127.81, 121.58, 74.89, 31.72, 30.75, 30.10, 29.77,
29.74, 29.62, 29.58, 29.52, 29.20, 26.14, 22.46, 13.86.
25,26,27,28-Tetrahexadecyloxycalix[4]arene (10). Crude product was recrystallized twice from
1
acetonitrile. Obtained as colorless solid. Yield 83%. M.p. 75-75.5 °С. H NMR (CDCl3, 299.94
MHz), δ ppm: 6.59 (d, J = 5.5 Hz, 8H, m-ArH), 6.57 (t, J = 5.5 Hz, 4H, p-ArH), 4.42 (d, J = 13.2
Hz, 4H, ArCHaxAr), 3.87 (t, J = 7.3 Hz, 8H, O-CH2-CH2-(CH2)13-CH3), 3.16 (d, J = 13.2 Hz, 4H,
ArCHeqAr), 1.85 (brm, 4H, O-CH2-CH2-(CH2)13-CH3), 1.44-1.21 (brs, 104H, O-CH2-CH2-(CH2)13-
CH3) 0.88 (t, J = 6.4 Hz, 12H, O-CH2-CH2-(CH2)13-CH3) 13C NMR (CDCl3, 125.73 MHz), δ ppm:
156.60, 135.16, 128.07, 121.83, 75.14, 31.96, 31.03, 30.00, 29.89, 29.85, 29.83, 29.81, 29.79,
29.72, 29.41, 26.40, 22.71, 14.12.
25,27-Dihexadecyloxy-26,28-dipropoxycalix[4]arene (13). Macrocycle 13 was precipitated from
iso-propanol-ethanol (1:5, 50 ml), then crystallized from this mixture and obtained as pale-yellow
1
solid, which contains near 20% (weight) hexadecylbromide. Yield 39%. M.p. 68 °С. H NMR
(CDCl3, 299.94 MHz), δ ppm: 8.57 (m, 12H, m-ArH and p-ArH), 4.42 (d, J = 13.3 Hz, 4H,
ArCHaxAr), 3.87 and 3.84 (two t, J = 7.8 Hz, 8H, O-CH2-CH2-CH3, O-CH2-CH2-(CH2)13-CH3), 3.16

Page 15 of 22
New Journal of Chemistry
(d, J = 13.3 Hz, 4H, ArCHeqAr), 1.91 (brm, 4H, O-CH2-CH2-CH3, O-CH2-CH2-(CH2)13-CH3), 1.26
View Article Online
DOI: 10.1039/C4NJ01395F
(brs, 52H, O-CH2-CH2-(CH2)13-CH3) 0.98 (t, J = 7.4 Hz, 6H, O-CH2-CH2-CH3) 0.88 (t, J = 6.9 Hz,
6H, O-CH2-CH2-(CH2)13-CH3) 13C NMR (CDCl3, 125.73 MHz), δ ppm: 156.62, 156.56, 135.18,
135.09, 128.10, 128.05, 121.83, 75.09, 70.97, 31.93, 31.00, 30.25, 29.85, 29.79, 29.73, 29.71,
29.68, 29.63, 29.52, 29.37, 26.26, 26.21, 23.29, 22.70, 14.11, 10.39.
General procedure for synthesis of tetrachlorometnyl-calixarenes 5, 9, 11 and 14. To a solution
of calixarene 4, 8, 10 (1 mmol) in dry chloroform (40 ml) or 13 in mixture of dry chloroform (40
ml) and dry n-hexane (20 ml) was added methyl-chloromethyl ether (50 mmol) and the resulting
solution was cooled to -60 °C under argon atmosphere. Tin tetrachloride (8 mmol) was added
dropwise to the mixture under vigorous stirring. Agitation was continued for 30 min at temperatures
-60 °C, and then the mixture was allowed to reach a room temperature. Then, distilled water (25 ml)
was added and the mixture was stirred to color fading. Organic layer was separated, washed with
3% hydrochloric acid (20 ml), water (20 ml) and brine (20 ml) and then dried overnight by sodium
sulfate. Crystallization (from acetonitrile) of yellow solid obtained after solvent removal gave
calixerenes 5, 9, 11 or 14.
5,11,17,23-Tetrachloromethyl-25,26,27,28-tetrahexyloxycalix[4]arene (5). Obtained as pale
yellow solid. Yield 73%. M.p. 178-179 °С. 1H NMR (CDCl3, 299.94 MHz), δ ppm: 6.65 (s, 8H, m-
ArH), 4.38 (d, J = 13.2 Hz, 4H, ArCHaxAr), 4.30 (s, 8H, Ar-CH2Cl), 3.87 (t, J = 7.5 Hz, 8H, O-CH2-
CH2-(CH2)3-CH3), 3.16 (d, J = 13.2 Hz, 4H, ArCHeqAr), 1.89 (brm, 4H, O-CH2-CH2-(CH2)3-CH3),
1.44-1.25 (brs, 24H, O-CH2-CH2-(CH2)3-CH3) 0.92 (t, J = 6.6 Hz, 12H, O-CH2-CH2-(CH2)3-CH3),
13C NMR (CDCl3, 125.73 MHz), δ ppm: 156.77, 135.13, 130.86, 128.53, 75.39, 46.62, 32.02,
30.89, 30.19, 25.88, 22.82, 14.06.
5,11,17,23-Tetrachloromethyl-25,26,27,28-tetradodecyloxycalix[4]arene (9). Obtained as pale-
yellow solid. Yield 42%. M.p. 69 °С. 1H NMR (CDCl3, 299.94 MHz), δ ppm: 6.64 (s, 8H, m-ArH),
4.37 (d, J = 13.2 Hz, 4H, ArCHaxAr), 4.30 (s, 8H, Ar-CH2Cl), 3.86 (t, J = 7.5 Hz, 8H, O-CH2-CH2-
(CH2)9-CH3), 3.15 (d, J = 13.2 Hz, 4H, ArCHeqAr), 1.86 (brm, 4H, O-CH2-CH2-(CH2)9-CH3), 1.44-
1.12 (brs, 72H, O-CH2-CH2-(CH2)9-CH3) 0.88 (t, J = 6.3 Hz, 12H, O-CH2-CH2-(CH2)9-CH3) 13C
NMR (CDCl3, 125.73 MHz), δ ppm: 156.43, 134.77, 130.50, 128.17, 75.04, 46.25, 31.60, 30.53,
29.92, 29.62, 29.58, 29.49, 29.45, 29.39, 29.07, 25.95, 22.34, 13.74.
5,11,17,23-Tetrachloromethyl-25,26,27,28-tetrahexadecyloxycalix[4]arene (11). Obtained as
1
colorless solid. Yield 50%. M.p. 86 °С. H NMR (CDCl3, 299.94 MHz), δ ppm: 6.65 (s, 8H, m-
ArH), 4.38 (d, J = 13.4 Hz, 4H, ArCHaxAr), 4.30 (s, 8H, Ar-CH2Cl), 3.86 (t, J = 7.3 Hz, 8H, O-CH2-

New Journal of Chemistry
Page 16 of 22
CH2-(CH2)13-CH3), 3.15 (d, J = 13.4 Hz, 4H, ArCHeqAr), 1.86 (brm, 4H, O-CH2-CH2-(CH2)13-
View Article Online
DOI: 10.1039/C4NJ01395F
CH3), 1.41-1.19 (brs, 104H, O-CH2-CH2-(CH2)13-CH3) 0.88 (t, J = 6.7 Hz, 12H, O-CH2-CH2-
(CH2)13-CH3), 13C NMR (CDCl3, 125.73 MHz), δ ppm: 156.42, 134.76, 130.51, 128.17, 75.03,
46.24, 31.58, 30.54, 29.92, 29.62, 29.58, 29.49, 29.46, 29.45, 29.42, 29.41, 29.34, 29.03, 25.95,
22.33, 13.74.
5,11,17,23-Tetrachloromethyl-25,27-dihexadecyloxy-26,28-dipropoxycalix[4]arene
(14).
Obtained as a colorless solid. Yield 93%. M.p. 89 °С. 1H NMR (CDCl3, 299.94 MHz), δ ppm: 6.65
and 6.64 (two s, 8H), 4.38 (d, J = 13.4 Hz, 4H, ArCHaxAr), 4.29 (brs, 8H, Ar-CH2Cl), 3.86 and 3.83
(two t, J = 7.5 Hz, 8H, O-CH2-CH2-CH3, O-CH2-CH2-(CH2)13-CH3), 3.16 (d, J = 13.4 Hz, 4H,
ArCHeqAr), 1.90 (brm, 4H, O-CH2-CH2-CH3, O-CH2-CH2-(CH2)13-CH3), 1.26 (brs, 52H, O-CH2-
CH2-(CH2)13-CH3) 0.98 (t, J = 7.5 Hz, 6H, O-CH2-CH2-CH3), 0.88 (t, J = 6.7 Hz, 6H, O-CH2-CH2-
(CH2)13-CH3), 13C NMR (CDCl3, 125.73 MHz), δ ppm: 156.77, 156.76, 135.12, 135.10, 130.89,
130.86, 128.56, 75.35, 70.96, 46.60, 31.92, 30.89, 30.16, 29.82, 29.78, 29.76, 29.71, 29.66, 29.61,
29.51, 29.36, 26.20, 26.18, 23.23, 22.69, 14.10, 10.30
General procedure for synthesis of calixarenes CX6, CX12, CX16 and CX3-16. To a solution of
tetrachloromethylcalix[4]arene 5, 9, 11 or 14 (1 mmol) in tetrahydrofuran (20 ml) a solution of
N,N-dimethylethanolamine (5 mmol) in THF (5 ml) was added under stirring. The reaction mixture
was refluxed for 18 hrs. After cooling, the precipitate of calixarene CX6, CX12, CX16 or CX3-16
formed was filtered off. The product was dried in vacuum (0.05 mm Hg, 20 °С, 4 hrs) to give a
microcrystalline compound.
5,11,17,23-Tetra(N,N-dimethyl-N-hydroxyethylammonium)-methylene-25,26,27,28-
tetrahexyloxycalix[4]arene tetrachloride (CX6). Obtained as a pale-yellow microcrystalline
1
hygroscopic compound. Yield 74%. M.p. 270-273 °С (decomp.) H NMR ((CD3)2S=O, 299.94
MHz), δ ppm: 6.99 (s, 8H, m-ArH), 5.76 (brs, 4H, OH), 4.54 (s, 8H, Ar-CH2-N), 4.34 (d, J = 12.8
Hz, 4H, ArCHaxAr), 3.88 (brt, 16H, O-CH2-CH2-(CH2)3-CH3, CH2-OH), 3.29 (brs, 12H, ArCHeqAr,
N-CH2), 2.95 (s, 24H, N-CH3), 1.90 (m, 4H, O-CH2-CH2-(CH2)3-CH3), 1.45-1.21 (brs, 24H, O-
13
CH2-CH2-(CH2)3-CH3) 0.89 (brt, 12H, O-CH2-CH2-(CH2)3-CH3), C NMR ((CD3)2S=O, 125.73
MHz), δ ppm: 157.28, 134.69, 133.23, 121.93, 75.03, 66.78, 64.22, 54.80, 49.76, 31.53, 29.80,
2+
29.75, 25.39, 22.28, 13.85. MS: (m/z) found [M-2Cl]2+ = 1238.8496 (calcd for C72H120Cl2N4O8 ;
[M-2Cl]2+ = 1238.8483).

Page 17 of 22
New Journal of Chemistry
5,11,17,23-Tetra(N,N-dimethyl-N-hydroxyethylammonium)-methylene-25,26,27,28-
View Article Online
DOI: 10.1039/C4NJ01395F
tetradodecyloxycalix[4]arene tetrachloride (CX12). Obtained as colorless microcrystalline
1
hygroscopic compound. Yield 39%. M.p. 285-290 °С (decomp.) H NMR ((CD3)2S=O, 299.94
MHz), δ ppm: 6.93 (s, 8H, m-ArH), 5.63 (brs, 4H, OH), 4.53 (s, 8H, Ar-CH2-N), 4.35 (d, J = 12.8
Hz, 4H, ArCHaxAr), 3.88 (brt, 16H, O-CH2-CH2-(CH2)9-CH3, CH2-OH), 3.32 (brs, 12H, ArCHeqAr,
N-CH2), 2.97 (s, 24H, N-CH3), 1.89 (m, 4H, O-CH2-CH2-(CH2)9-CH3), 1.47-1.14 (brs, 72H, O-
13
CH2-CH2-(CH2)9-CH3) 0.84 (brt, 12H, O-CH2-CH2-(CH2)9-CH3), C NMR ((CD3)2S=O and 10%
CDCl3, 125.73 MHz), δ ppm: 157.24, 134.63, 133.16, 121.78, 75.02, 66.78, 64.14, 54.86, 49.69,
29.85, 29.82, 29.79, 29.55, 29.52, 29.36, 29.27, 29.17, 28.81, 25.89, 22.07, 13.73. MS: (m/z) found
2+
[M-2Cl]2+ = 1575.2250 (calcd for C96H168Cl2N4O8 ; [M-2Cl]2+ = 1575.2239).
5,11,17,23-Tetra(N,N-dimethyl-N-hydroxyethylammonium)-methylene-25,26,27,28-
tetrahexadecyloxycalix[4]arene tetrachloride (CX16). Obtained as colorless microcrystalline
1
hygroscopic compound. Yield 67%. M.p. 222-225, decomposition 235 °С. H NMR ((CD3OD,
500.07 MHz), δ ppm: 7.03 (s, 8H, m-ArH), 5.57 (brs, 4H, OH), 4.51 (d, J = 12.9 Hz, 4H,
ArCHaxAr), 4.47 (s, 8H, Ar-CH2-N), 4.03 (brs, 16H, O-CH2-CH2-(CH2)13-CH3, CH2-OH), 3.39 (brs,
12H, ArCHeqAr, N-CH2), 2.979 (s, 24H, N-CH3), 2.01 (m, 4H, O-CH2-CH2-(CH2)13-CH3), 1.56-
1.23 (brs, 104H, O-CH2-CH2-(CH2)13-CH3) 0.91 (brt, 12H, O-CH2-CH2-(CH2)13-CH3). MS: (m/z)
4+
found [M-4Cl]4+/4 = 432.64 (calcd for C112H200N4O8 ; [M-4Cl]4+/4 = 432.70), 13C NMR (CDCl3
+ 20%CD3OD, 125.73 MHz), δ ppm: 158.14, 135.64, 134.05, 121.83, 76.26, 68.09, 65.59, 56.00,
50.36, 31.25, 30.69, 30.29, 30.14, 30.09, 30.08, 30.07, 30.03, 30.01, 29.98, 29.92, 29.91, 29.60,
3+
26.50, 22.89, 14.23. MS: (m/z) found [M-3Cl]3+ = 1764.5095 (calcd for C112H200ClN4O8 ; [M-
3Cl]3+ = 1764.5055).
5,11,17,23-Tetra(N,N-dimethyl-N-hydroxyethylammonium)-methylene-25,27-dihexadecyloxy-
26,28-dipropoxycalix[4]arene tetrachloride (CX3-16). Obtained as colorless microcrystalline
1
hygroscopic compound. Yield 75%. M.p. 260-265 °С (decomp.) H NMR ((CD3)2S=O, 299.94
MHz), δ ppm: 7.09 and 6.85 (two s, 8H, pm-ArH), 5.70 (brs, 4H, OH), 4.57 and 4.49 (two s, 8H,
Ar-CH2-N), 4.35 (d, J = 13.3 Hz, 4H, ArCHaxAr), 3.91-3.83 (brm, 16H, O-CH2-CH2-CH3, O-CH2-
CH2-(CH2)13-CH3, CH2-OH), 3.35 (brs, 8H, ArCHeqAr, N-CH2), 3.35 (brs, 4H, N-CH2), 3.00 and
2.90 (two s, 24H, N-CH3), 1.89 (m, 4H, O-CH2-CH2-CH3, O-CH2-CH2-(CH2)13-CH3), 1.24 (brs,
52H, O-CH2-CH2-(CH2)13-CH3) 1.01 (t, J = 7.4 Hz, 6H, O-CH2-CH2-CH3) 0.85 (t, J = 6.9 Hz, 6H,
O-CH2-CH2-(CH2)13-CH3) 13C NMR (CD3OD, 125.73 MHz), δ ppm: 160.02, 159.46, 137.40,
136.62, 135.31, 134.84, 123.23, 123.07, 78.70, 77.01, 70.27, 66.74, 64.87, 57.00, 51.26, 51.04,
33.24, 31.66, 31.51, 31.17, 31.00, 30.97, 30.94, 30.64, 27.51, 25.43, 24.78, 23.90, 14.66, 11.26.

New Journal of Chemistry
Page 18 of 22
5,11,17,23-Tetra(N-trifluoroacetamido-N’,N’-dimethyl-ethylenediamino)-methylene-
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DOI: 10.1039/C4NJ01395F
25,26,27,28-tetraoctyloxycalix[4]arene
tetrachloride
(15).
To
a
solution
of
tetrachloromethylcalix[4]arene 7 (1 mmol) in tetrahydrofuran (15 ml) a solution of
trifluoroacetamide of N,N-dimethylethylendiamine (4.5 mmol) in THF (5 ml) was added under
stirring. The reaction mixture was refluxed for 12 hrs. After cooling, 30 ml of diethyl ether was
added. The pale-yellow precipitate (calixarene 15) formed was filtered off. The product was washed
by THF then redissolved in methanol (10 ml). Obtained solution was evaporated under reduced
pressure, resulted solid then re-suspended and sonificated in diethyl ether (15 ml). The ether was
evaporated under reduced pressure at room temperature to give the calixarene 15. Obtained as a
1
pale-yellow microcrystalline hygroscopic compound. Yield 72%. M.p. 280-285 °С (decomp.) H
NMR ((CD3)2S=O, 299.94 MHz), δ ppm:10.17 (s, 4H, NH-C(O)CF3), 6.93 (s, 8H, m-ArH), 4.62 (s,
8H, Ar-CH2-N), 4.33 (d, J = 12.5 Hz, 4H, ArCHaxAr), 3.88 (brt, 8H, CH2-NH-C(O)), 3.74 (brt, 8H,
O-CH2-CH2-(CH2)5-CH3), 3.42 (brs, 12H, ArCHeqAr, N-CH2), 3.01 (s, 24H, N-CH3), 1.86 (m, 4H,
O-CH2-CH2-(CH2)5-CH3), 1.50-1.19 (brs, 40H, O-CH2-CH2-(CH2)5-CH3) 0.85 (brt, 12H, O-CH2-
19
CH2-(CH2)5-CH3). F NMR ((CD3)2S=O, 110.4 MHz), δ ppm: -74.42 (s, CF3C(O)), 13C NMR
((CD3)2S=O, 125.73 MHz), δ ppm: 157.62, 134.95, 133.26, 121.65, 116.94, 114.65, 75.06, 66.30,
60.07, 49.56, 33.83, 31.56, 29.95, 29.56, 29.21, 26.01, 22.25, 13.90.
5,11,17,23-Tetra(N,N-dimethyl-ethylenediamino)-methylene-25,26,27,28-
tetraoctyloxycalix[4]arene tetrachloride (CX8N). To tetra(trifluoro)acetamido-calixarene 15 (0.8
mmol) was added water (20 ml), methanol (20 ml) and ammonia solution (35%, 20 ml). The
reaction mixture was refluxed under vigorous stirring for 24 h. After cooling the reaction mixture
was evaporated from the 1l flask. Precipitate was washed by diethyl ether (100 ml), solvent was
decanted and solid was dried by in vacuum at room temperature. The calixarene CX8N is pale
yellow microcrystalline compound was obtained as mixture with ammonium trifluoroacetate. Yield
1
90%. M.p. 290-295 °С (decomp.) H NMR ((CD3)2S=O, 299.94 MHz), δ ppm: 6.83 (s, 8H, m-
ArH), 4.61 (s, 8H, Ar-CH2-N), 4.32 (d, J = 12.5 Hz, 4H, ArCHaxAr), 3.87 (brt, 8H, CH2-NH2), 3.56
(brt, 8H, O-CH2-CH2-(CH2)5-CH3), 3.36 (brs, 12H, ArCHeqAr, N-CH2), 3.07 (s, 24H, N-CH3), 1.83
(m, 4H, O-CH2-CH2-(CH2)5-CH3), 1.59-1.14 (brs, 40H, O-CH2-CH2-(CH2)5-CH3) 0.85 (brt, 12H,
O-CH2-CH2-(CH2)5-CH3). 19F NMR ((CD3)2S=O, 110.4 MHz), δ ppm: -73.61 (s, CF3COO-), 13C
NMR ((CD3)2S=O, 125.73 MHz), δ ppm: 157.62, 134.95, 133.26, 121.65, 116.94, 114.65, 75.06,
66.30, 60.07, 49.56, 33.83, 31.56, 29.95, 29.56, 29.21, 26.01, 22.25, 13.90.
General methods
Dynamic light scattering measurements. The complexes of calixarenes with CT-DNA (Sigma, D
1501, highly polymerized DNA from calf thymus, with molecular weight between 10106 and

Page 19 of 22
New Journal of Chemistry
15106 daltons, verified by gel electrophoresis on agarose) or pDNA (pCMV-eGFP plasmid) were
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DOI: 10.1039/C4NJ01395F
prepared by mixing equal volumes of DNA and calixarene in 20 mM Tris (pH 7.4) buffer. The final
DNA (phosphate) concentration was 30 M, while the calixarene concentration was varied to
provide the final N/P. The N/P ratio between calixarene and DNA was expressed as the molar ratio
between all the charged Nitrogen atoms of the calixarene and the phosphate groups of the DNA.
DLS measurements were done after 30 min of incubation at room temperature. The average size of
the complexes was determined with a Zetamaster 3000 (Malvern Instruments, Paris, France) with
the following specifications: sampling time, 30 s; medium viscosity, 1.054 cP; refractive index (RI)
medium, 1.34; RI particle, 1.45; scattering angle, 90; temperature, 25 C.
Gel electrophoresis. The calixarene/pDNA complexes were prepared as for DLS measurements,
but at 30 M pDNA concentration. Gel electrophoresis was performed on a 1% agarose gel in
0.5×TAE (Tris-acetate-ethylenediaminetetraacetic acid) buffer at a constant voltage of 100 mV.
After electrophoresis, the gel was incubated in a 1 μg/mL solution of EtBr for 30 min and then
visualized by an UV transilluminator.
Fluorescence measurements. Absorption spectra were recorded on a Cary 4000 spectrophotometer
(Varian) and fluorescence spectra on a FluoroMax 3.0 or Fluorolog (Jobin Yvon, Horiba)
spectrofluorometer. All the spectra were corrected for the fluorescence of the blank (corresponding
solution without the fluorescence dye). For CMC measurements with pyrene, a 20 mM Tris buffer
(pH 7.4) solution, containing 150 (for CX3-CX8) or 30 nM (for CX8N and CX3-16) of pyrene was
titrated with increasing quantities of calixarenes, added from the stock solution in water. The
spectra were recorded 1 min after each addition. In the ethidium bromide exclusion assay, an
aliquot of CT-DNA (20 M final concentration of DNA phosphate) was added to the solution of
ethidium bromide (0.4 M). After 2 min, increasing quantities of calixarenes were added from the
stock solution. Fluorescence intensity at 600 nm, excited at 550 nm, was recorded 2 min after each
calixarene addition and then plotted as a function of the corresponding N/P ratio.
Transfection and cytotoxicity. HeLa cells (ATCC CCL-2) were grown in Dulbecco’s modified
Eagle medium (DMEM, Gibco-Invitrogen), supplemented with 10% fetal bovine serum (FBS,
Lonza) and 1% antibiotic solution (penicillin-streptomycin, Gibco-Invitrogen) at 37 C in
humidified atmosphere containing 5% CO2. Cells were seeded onto 24-well plate at a density of
2104 cells/well 24h before the transfection. The transfection was done by using calixarene/pDNA
(eGFP-Luc plasmid, 1 μg/well) complexes at different N/P ratios in serum-free Opti-MEM (Gibco-
Intvitogen), followed by 3 h incubation at 37 °C. Calixarene/pDNA complexes were obtained as for
DLS measurements, but in MES (pH 7.0) buffer. In some formulations, DOPE (1:1 molar ratio with
respect to calixarene) was used. For this purpose, a mixture of DOPE and calixarene in ethanol was

New Journal of Chemistry
Page 20 of 22
evaporated in a round bottom flask to obtain a film. Then, Tris buffer (pH 7.4) was added and the
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DOI: 10.1039/C4NJ01395F
samples were hydrated overnight at rt. Then, the samples were vortexed vigorously for 1 min and
further sonicated in an ultrasonic bath for 15 min. The obtained suspensions were mixed with an
equal volume of pDNA in Tris buffer to obtain a desired N/P ratio. All formulations were added to
cells 30 min after incubation at rt. After 3 h, 10% of FBS was added and cells were harvested for
another 45 h. In case of incubation with serum, the complexes were added to cells in DMEM
medium containing 20% FBS and incubated for 48h. Then, the fluorescence images were recorded
using BioRad epifluorescence microscope.
Cytotoxicity of
calixarenes
was
evaluated
using
3-(4,5-dimethyl-2-thiazolyl)-2,5-
diphenyltetrazolium bromide (MTT, Sigma-Aldrich) assay. HeLa cells were seeded and after 24 h
supplemented with calixarenes at different concentrations in serum-free Opti-MEM and further
incubated for 3 h at 37 °C. Then, 10% of FBS was added and cells were harvested for another 45 h.
Alternatively, calixarenes were added to the cells in DMEM medium with 10% FBS and incubated
for 48h. Then, cells were washed with Phosphate Buffer Saline (PBS) and incubated with serum
free medium containing 0.5 mg/mL MTT for 3 h at 37°C. Media was discarded and formazan
crystals were re-suspended in 0.5 mL MTT solvent (Sigma-Aldrich). Absorbance of formazan
solution was measured at 570 nm with respect to the background at 690 nm, using a Cary 4000
spectrophotometer. Viability was expressed as relative absorbance (%) of the sample vs. control
non-treated cells.
Acknowledgement
This work was supported by the joint project of NAS, Ukraine and CNRS, France (PICS #6053)
and University of Strasbourg.
1
13
Electronic supplementary information (ESI) available: original FT-IR, H NMR and C NMR
spectra are presented for all novel compounds.
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