Synthesis, physicochemical characterization, and interaction with DNA of long-alkyl-chain gemini pyridinium surfactants

Article abstract of DOI:10.1002/cplu.201500007

Abstract Pyridinium gemini surfactants with hexadecyl chains linked to nitrogen atoms and a tuned aliphatic spacer that bridges the two pyridinium polar heads in 2,2′-positions have been synthesized and characterized. A multitechnique approach allowed us to study the aggregation behavior, using conductivity, surface tension, and fluorescence. Graphs of the specific conductivity (κ) versus the surfactant molar concentration (C), and graphs of the molar conductivity (Λ) versus C^0.5 suggest pre-aggregation phenomena of these amphiphiles at very low concentration. The trends of A_min as a function of the spacer length confirm the hypothesis of a conformational change of the molecule with four methylene groups as spacer owing to stacking interactions between the two pyridinium rings mediated by the counterion. Moreover, the trends of A_min and counterion binding (β) suggest that the spacer must be longer than eight carbon atoms to fold efficiently toward the micellar core. The opportunity to tune the surfactant structure and aggregation properties make those surfactants - particularly the long-chain ones for which the DNA complexing ability was shown by means of atomic force microscopy (AFM) imaging - desirable candidates for gene-delivery experiments. Bringing it all together: This study on gemini pyridinium surfactants (see figure) contributes to the knowledge about the aggregation behavior of these compounds and their interactions with biological molecules. Structure-activity relationships were found in the context of the physico-chemical properties of these compounds and DNA compaction. These insights should improve the understanding of surfactant-based gene delivery.

Full text of DOI:10.1002/cplu.201500007

DOI: 10.1002/cplu.201500007
Full Papers
Synthesis, Physicochemical Characterization, and
Interaction with DNA of Long-Alkyl-Chain Gemini
Pyridinium Surfactants
[
a]
[a]
[a]
[a]
Nadia Barbero, Claudio Magistris, Pierluigi Quagliotto,* Luca Bonandini,
[
a]
[a]
[b]
[b]
[b]
Claudia Barolo, Roberto Buscaino, Carlotta Compari, Laura Contardi, Emilia Fisicaro,
[a]
and Guido Viscardi
Dedicated to Professor Ermanno Barni on the occasion of his 80th birthday
Pyridinium gemini surfactants with hexadecyl chains linked to
nitrogen atoms and a tuned aliphatic spacer that bridges the
two pyridinium polar heads in 2,2’-positions have been synthe-
sized and characterized. A multitechnique approach allowed us
to study the aggregation behavior, using conductivity, surface
tension, and fluorescence. Graphs of the specific conductivity
cule with four methylene groups as spacer owing to stacking
interactions between the two pyridinium rings mediated by
the counterion. Moreover, the trends of Amin and counterion
binding (b) suggest that the spacer must be longer than eight
carbon atoms to fold efficiently toward the micellar core. The
opportunity to tune the surfactant structure and aggregation
properties make those surfactants—particularly the long-chain
ones for which the DNA complexing ability was shown by
means of atomic force microscopy (AFM) imaging—desirable
candidates for gene-delivery experiments.
(
k) versus the surfactant molar concentration (C), and graphs
0.5
of the molar conductivity (L) versus C suggest pre-aggrega-
tion phenomena of these amphiphiles at very low concentra-
tion. The trends of Amin as a function of the spacer length con-
firm the hypothesis of a conformational change of the mole-
Introduction
[
21–23]
Gemini surfactants differ from the more conventional mono-
meric ones since they consist of two hydrophilic head groups,
each of which bears one hydrophobic chain, connected by
a spacer. Their properties are peculiar in comparison with mon-
omeric surfactants owing to their increased surface activity,
useful viscoelastic properties, lower C20 (molar surfactant con-
centration required to reduce the surface tension of the sol-
es.
The first step of GT is to compact and encapsulate the
therapeutic desoxyribonucleic acid (DNA) into soft nanoparti-
cles and to deliver it into the nucleus by means of a specific
[
19]
vector. Retroviral vectors (RVs) have been widely used to de-
[24–31]
liver therapeutic genes with clear therapeutic benefit,
but
severe adverse events linked to insertional mutagenesis owing
to aberrant vector-on-host interactions occurred, for example,
À1 [1]
[27,32–35]
vent by 20 mNm ), and lower critical micelle concentration
in clinical trials for primary immunodeficiencies.
[
2–10]
(
CMC) of aggregates of different shapes.
Owing to these
Nonviral gene vectors appear to be a promising alternative.
Cationic amphiphiles, also known as cationic lipids, have
shown DNA complexation properties useful for cell transfec-
properties, the interest in gemini surfactants, particularly cat-
ionic ones, has increased recently in the field of pharmaceuti-
cals, both as noncovalent components in carbon-nanotube-
[13,36–43]
tion.
A recent review evidenced the great potential of
[
11]
based formulations and as nonviral vectors in gene thera-
these nonviral vectors, in particular the gemini surfactants,
owing to tunability of their molecular moieties: hydrophobic
[12–20]
py.
[9]
Gene therapy (GT) is finding more and more clinical applica-
tions for monogenic disorders, cancer, and infectious diseas-
chains and hydrophilic head group, spacer, and counterion.
However, efficient nonviral delivery systems must still be realiz-
ed.
[
a] Dr. N. Barbero, C. Magistris, Dr. P. Quagliotto, L. Bonandini, Dr. C. Barolo,
Dr. R. Buscaino, Prof. G. Viscardi
Dipartimento di Chimica
Interdepartmental “Nanostructured Surfaces and Interfaces”
NIS Centre–Università di Torino
For some time, we have been collecting physicochemical
and biological data on gemini surfactants with the idea of cor-
relating their structure with their biological activity, particularly
their transfection ability, in a quantitative way to drive the syn-
[17,44–47]
Via Pietro Giuria 7, 10125 Torino (Italy)
E-mail: pierluigi.quagliotto@unito.it
thesis of more and more efficient vectors.
Encouraging
results in gene delivery have been obtained by some of us by
using ammonium gemini surfactants as derivatives of N,N-bis-
dimethyl-1,2-ethanediamine with the general formula
[
b] Dr. C. Compari, L. Contardi, Prof. E. Fisicaro
Dipartimento di Farmacia–Università di Parma
Parco Area delle Scienze 27A, 43124 Parma (Italy)
+
+
2n+1/2ClÀ
OOCCH (CH ) N CH CH N (CH ) CH COOC H
2 3 2 2 2 2 2 2 n
CnH
2n+1
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cplu.201500007.
(bis-C BEC), in which the subscript n stands for the number of
n
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carbon atoms of the alkyl chain bound to the carboxyl group,
when formulated with l-a-phosphatidylethanolamine dioleoyl
[
17]
(
(C18:1,[cis]-9); DOPE). We then synthesized cationic gemini
surfactants that contained two pyridinium groups with dode-
[10]
[48]
cyl and fluoroalkyl chains. The pyridinium group should
account for better interaction with the DNA phosphate group
owing to higher charge dispersion with respect to ammonium
head groups; chloride counterion is normally used since it is
[
9]
nontoxic.
In this study we describe the synthesis of pyridinium coun-
terparts that bear hexadecyl chains and the study of their ag-
gregative properties by conductivity, surface tension, and fluo-
rescence measurements. In general, the use of long alkyl
chains improves the complexation and transfection ability of
DNA. The purpose of this study is also to define and optimize
structure/biological activity relationships (i.e., the determina-
[
9]
tion of the chain length to be used).
Pyridinium gemini surfactants, which are structurally differ-
ent from the compounds presented here (they have a spacer
that bridges the heteroaromatic nitrogen atoms), have been
[
20]
recently proposed in the literature for transfection purposes.
Scheme 2. Synthesis and quaternization reactions of a,w-bis(2-pyridyl)-
alkanes.
Results and Discussion
The structure of novel surfactants 1–4 (called 16Py-n-Py16-X, in
which X=CF SO , Cl) is described in Scheme 1 along with the
yields were generally very high, sometimes nearly quantitative.
This kind of alkylating agent made it possible to quaternarize,
for the first time, similarly crowded structures with long alkyl
chains. The ionic exchange technique gave the desired chlor-
3
3
[10]
ides (1b–10b) (see the Supporting Information).
Amphiphilic characterization
For it to act as an efficient nonviral vector of DNA, a surfactant
should compact and encapsulate the DNA into soft nanoparti-
cles that are large and stable enough for preventing rapid leak-
age into blood capillaries but small enough to escape macro-
phages of the reticuloendothelial system.
Since the properties of DNA nanoparticles are heavily affect-
ed by the structure of the cationic surfactant and by its aggre-
gation ability, it is fundamental to characterize the potential
nonviral vectors from an amphiphilic point of view to find
Scheme 1. Surfactants prepared in the present study.
[
9]
structure–activity relationships. Firstly, the critical micelle con-
centration (CMC), defined as the concentration at which a sur-
factant starts to form aggregates as micelles, has to be deter-
mined.
previously reported surfactants 5–8 (called 12Py-n-Py12-X, in
[
10]
which X=CF SO , Cl) and the structurally related monomers
3
3
[
49,50]
9
b and 10b.
Their synthesis (Scheme 2) was performed by
the use of hexadecyltrifluoromethanesulfonate as the quater-
The CMC values of current gemini surfactants were deter-
mined by using specific conductivity (k, which measures the
bulk properties of the system), surface tension (g, which is re-
lated to the surface properties of the system), and fluorescence
measurements (determined by the probe–micelle interaction).
In fact, the evaluation of the CMC in the case of gemini surfac-
tants is not as straightforward as for monomeric surfactants,
and discussion on this point is still open in the litera-
nizing agent of suitable a,w-bis(2-pyridyl)alkane, prepared as
[
10]
previously described.
enough, as noted previously.
were sufficiently reactive to prepare surfactants 5 and 6,
whereas their use was completely unsuccessful in obtaining 1–
Alkyl halides were not reactive
[
10,48]
Alkyl methanesulfonates
[10]
4
and 7,8.
The quaternization was performed in chloroform at 708C by
[9,10,44,45,47,48,51–65]
slowly dropping the base into the hexadecyltrifluoromethane-
sulfonate solution. The product was obtained as a solid by
simple cooling of the reaction mixture. After filtration, the solid
was crystallized from an acetonitrile/ethyl acetate mixture. The
ture.
Trifluoromethanesulfonate surfactants were not studied
from the amphiphilic point of view owing to their high Krafft
point: 80–858C for dodecyl gemini surfactants 5b–8b, where-
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Figure 1. Specific conductivity (k) versus C plot of surfactant 16Py-3-Py16 2
Cl (1b).
Figure 2. Surface tension as a function of the logarithm of the surfactant
concentration for the gemini surfactants 1b (
at 258C.
&
), 2b (~), 3b (*), and 4b (”)
as for the hexadecyl ones, the Krafft point was im-
possible to measure because it was higher than
1008C.
Table 1. Amphiphilic properties of surfactants 1b–10b at 258C.
Figure 1 (see also Figures S1–S11 of the Supporting
[
a]
[b]
[c]
[d]
[e]
[f]
[g]
Surf. Chain
n
CMCc
b [%]
CMCs
CMCc/CMCs CMC
CMC
I
1
/I
3
Information) shows, as an example, the dependence
1
2
3
4b
5b
6
7
8
b
b
b
C
C
C
16
16
16
H
H
H
33
33
33
33
25
25
25
3
4
8
12
3
8.51
9.42
8.38
5.95
61
53
49
56
68
69
44
70
55
61
1.2
2.1
7.1
4.5
8.87 195
10.2 143
1.11
1.08
of the specific conductivity (k) on surfactant molar
[
66]
concentration (C) for surfactant 1b. An evident in-
flection of the plot allowed us to extrapolate the
CMC value.
0.15 55.9
0.078 76.3
8.34 40.4 1.05
5.91 54.6 1.07
132
124
103
26.2
101
1570
C H
1
6
C
C
C
C
12
12
12
12
H
H
H
H
145
111
28
12
2.3
–
–
1.3
3.8
8.8
9.1
–
166
137
1.19
1.11
b
b
b
4
8
107
106
21
102
1270
Two different methods were applied to extract the
CMC values from specific conductivity (k) versus C
data. The first more classical approach allows the
CMC to be determined by the intersection of the two
97.2 0.96
35.7 1.44
25 12
9b
C H
1
6
33
–
–
139
991
1.34
0.94
10b
C
12
H
25
–
[
66]
linear regimes at low and high concentrations.
A
5
À1
[
a] Number of methylene groups in the spacer. [b] CMC”10 [molL ], obtained by
few years ago, a nonlinear fit method was proposed
specific conductivity (k) versus C data following the nonlinear fit method. [c] Degree
of counterion binding. [d] CMC”10 [molL ], obtained by surface-tension measure-
5
À1
[
51]
by Ruiz et al. This became a standard method to
deal with smooth specific conductivity versus C data
plots. Recently, we also applied it successfully to
5
À1
5
À1
ments. [e] CMC”10 [molL ], obtained using coumarin 6. [f] CMC”10 [mol L ], ob-
tained by using pyrene. [g] I /I ratio of the intensity of the first (373 nm) and third
384 nm) vibronic bands of pyrene fluorescence at the CMC.
1
3
(
[
10,52,53]
some monomeric and gemini surfactants.
Both
methods gave consistent results, but the nonlinear fit
method was straightforward in obtaining reliable
CMC values, since sometimes the transition between the two
linear regimes was gradual and it was difficult to extrapolate
the two lines (see the Supporting Information for details about
part of the micelle palisade at some relative distance from the
polar heads of the pyridinium cation.
Sigmoidal plots were obtained by plotting the emission in-
[
51]
the extrapolation method used by Ruiz et al.).
tensity (I ) versus C and the CMC values, extrapolated by
em
[70]
The CMC values, determined by both methods for the
gemini surfactants and their related monomers, are reported in
Table 1. The CMC values obtained by surface tension measure-
ments, shown in Figure 2, are also reported in Table 1. They
are taken as the concentrations at the point of intersection of
the two linear portions of the g versus logC plots (dotted
straight lines in Figure 2).
using Boltzmann sigmoidal fitting developed by Ruiz et al.
These are reported in Table 1. The Iem versus C plot for surfac-
tant 1b (16Py-3-Py16-Cl), taken as an example, is reported in
Figure 3a. CMC values obtained by using coumarin 6 as probe
are in agreement with those obtained by conductivity meas-
urements, which supports the use of conductivity as a tech-
nique to study micelle formation phenomena.
Since the CMC values extrapolated from specific conductivity
When using pyrene, one needs to consider the fluorescence-
quenching properties of pyridinium cations owing to pyrene
localization in the micelle near the cationic head groups fol-
lowing the interaction of the pyrene aromatic electrons with
(
k) and surface tension appear significantly different, we also
evaluated this aggregative parameter by use of the fluorescent
[
52,53,67–70]
probes coumarin 6 and pyrene.
values are reported in Table 1.
Extrapolated CMC
[55,71,72]
the positively charged pyridinium.
Kalyanasundaram
[73]
Coumarin 6 is a fluorescent probe that is highly hydrophobic
and insoluble in water; it is normally situated in the interior
et al. demonstrated that the quenching, while being a diffu-
sional quenching mechanism that operates among pyridinium
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literature, it seems that this stabilizing effect would be ob-
tained for chains with a length between 16 and 18. A similar
[56]
effect was observed for ammonium gemini surfactants, but
the effect of the hexadecyl versus dodecyl chain is approxi-
mately halved for pyridinium gemini surfactants series relative
[56]
to ammonium gemini ones, probably because the pyridini-
um ring reduces the conformational freedom. Its planar shape
and large dimension can hinder the micellar surface, thereby
[10]
increasing the CMC.
This lower efficiency of pyridinium
gemini surfactants than ammonium ones in forming micelles is
supported also by the CMC/C20 parameter reported in the Sup-
porting Information, the values of which appear higher for pyr-
idinium gemini surfactants relative to values of ammonium
[56,59]
gemini ones.
The effect of spacer length on CMC is worth noting: the
spacer seems to act as a third hydrophobic chain that affects
[45,46]
the aggregation and the packing parameter.
The lowering
of the CMC in the presence of hexadecyl chains seems less evi-
dent than in the presence of dodecyl chains. This hydrophobic
effect of the spacer has been already observed for ammonium
gemini surfactants and is confirmed by the pC20 values (see
the Supporting Information), which generally becomes higher
when both the alkyl chains linked to nitrogen atoms and the
[10]
spacer length increase in a series.
This suggests that the
spacer elongation can also significantly affect the properties of
surfactant–DNA lipoplexes.
The CMC values extrapolated from the surface tension are
generally lower than those extrapolated from the specific con-
ductivity (k), particularly for surfactants with hexadecyl chains
and long spacers (8–12 methylene groups); that is, for more
hydrophobic surfactants. This phenomenon has been already
Figure 3. Determination of CMC for surfactant 1b: a) Iem versus C plot using
coumarin 6 as the probe; b) I /I versus C plot using pyrene as the probe.
1
3
[48,57,58,74,75]
reported in the literature.
Rosen et al. tentatively in-
cations and pyrene, does not influence the I /I vibronic band
troduced the CMCc/CMCs ratio to describe the extent of the
1
3
[74]
ratio. By applying the conditions of Kalyanasundaram et al., de-
scribed in the Experimental Section, the measurements were
performed without any particular problem.
difference of the CMC determined by different methods.
Analogously, we reported this parameter in Table 1.
[
48,54,57–61,74,75]
In agreement with the literature,
we ascribed
The pyrene intensities of emissions at 373 (I ) and 384 nm
the difference between CMC data obtained by conductivity
and surface tension to the formation of premicellar aggregates,
not surface active, such as dimers, trimers, and so on. When
the surfactant concentration was increased, the premicellar ag-
gregates progressively turned into conventional micelles. To
confirm our hypothesis, an examination of conductivity meas-
urements in diluted solutions proved meaningful. For a mono-
meric surfactant in dilute solution, an increasing and a decreas-
ing linear correlation was obtained in specific conductivity (k)
1
(
I3) (first and third vibronic peaks, respectively) are influenced
by the polarity of its solubilization environment. Below the
CMC, the pyrene I1/I3 ratio value corresponds to the polar
water environment; as the surfactant concentration increases,
the pyrene I /I ratio decreases rapidly, thus indicating that the
1
3
pyrene senses a more hydrophobic environment, ultimately
reaching a roughly constant value owing to probe incorpora-
tion into the micelles. A typical sigmoidal curve was obtained
by plotting the I /I ratio as a function of the total surfactant
0.5
versus C and molar conductivity (L) versus C plots in very di-
luted solutions (Figure 4). Otherwise, anomalously higher
values of specific conductivity than that expected (inset of
Figure 1 and Figures S1–S11 of the Supporting Information) or
maxima in molar conductivity (L) plots (Figure 5 and Figur-
es S12–S22 of the Supporting Information) are due to preag-
1
3
concentration; the CMC was extrapolated by means of the
[
70]
method reported by Ruiz et al. in the literature. In Figure 3b,
the curve obtained for surfactant 1b (16Py-3-Py16-Cl) is report-
ed as an example.
The examination of CMC values obtained by specific con-
ductivity (k), surface tension, and coumarin 6 fluorescence (re-
ported in Table 1) confirms the higher efficiency of pyridinium
gemini surfactants in forming micelles relative to the corre-
sponding monomeric ones; it suggests that, as often found in
[57,62]
gregation phenomena.
Counterion binding and surface minimum area
[
5,56]
the literature,
longer chains (16Py-s-Py16 series) stabilize
The DNA complexation and DNA release in the cell is also
mediated by the optimal interaction of the cationic vector
the micellar aggregates and reduce CMC. On the basis of the
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us to evaluate how close the polar head groups are to each
[10,48,52–54]
other, that is, the micelle surface compactness.
In addition, the presence of a delocalized charge in pyridini-
um surfactants positively influences the aggregation properties
and the DNA transfection activity. The DNA complexation and
its release in the cell are also mediated by the optimal interac-
tion of the cationic vector with the negative charges of the
[12]
DNA phosphate group.
A head group with a delocalized
positive charge is a softer ion than the ammonium one and
thus should interact better with a reasonably soft anion such
[9]
as the phosphate that is present in DNA.
The degree of counterion binding (b) is calculated by the ex-
pression b=1Àa, in which a is the degree of counterion disso-
ciation obtained by the ratio of the two slopes directly deter-
mined from the definition of the two linear regimes after and
before the CMC of specific conductivity (k) versus C plot
[
Eq. (1)].
a ¼ ðdk=dCÞC>CMC=ðdk=dCÞC<CMC
ð1Þ
In Figure 6, b values obtained from the slopes evaluated by fol-
lowing the nonlinear fit are reported; b values obtained from
the slopes evaluated by following the classical method are re-
[51]
ported in the Supporting Information.
Figure 4. (a) Specific conductivity (k) versus C and (b) molar conductivity
0
.5
versus C plots for N-dodecylpyridinium chloride.
Figure 6. Degree of counterion binding (b) as a function of the spacer
length (n) for C12 (red squares) and C16 (dark blue diamonds) pyridinium
gemini surfactants.
0
.5
The b values vary along with spacer length and show a mini-
mum for n=4,8 in the case of hexadecyl surfactants and n=8
for dodecyl ones. For short spacers, that is, three methylene
groups long, the two charged head groups are quite close and
thus the surface charge density in the micelles is high, and
counterions are recruited from the solution to neutralize the
positive charges and stabilize the aggregate showing a relative-
ly high b value. When the spacer has four methylene groups,
Figure 5. Molar conductivity (L) versus C plot for surfactant 1b.
[12]
with the negative charges of the DNA phosphate group. The
study of the degree of counterion binding (b), that is, how
many counterions are tightly bound to the micelle in the Stern
layer, can be significant in evaluating the potential of a surfac-
tant to form lipoplexes. A high b value implies the presence of
a high charge density on the micellar surface, which requires
counterions to be neutralized. This parameter can also allow
[44,45]
we already suggested
that the molecule can double up,
thereby surrounding the counterion and taking it firmly in be-
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tween the pyridinium rings. The difference in b values between
dodecyl and hexadecyl surfactants could be due to the larger
hydrophobic interaction of hexadecyl chains.
in the evaluation of the physicochemical parameters such as
[77,78]
the Amin, which can be derived from the surface tension.
Absolute methods to determine the concentration of surfac-
tant molecules at the surface, the so-called “surface excess”
(G), such as neutron reflectivity (NR), were already assessed at
The surfactants that have a spacer made of eight methylene
groups have sufficient conformational freedom and they
occupy a larger space to the micellar surface, and the charge
density is decreased and b is lower.
[76]
the end of nineties. Their use allows us to compare their re-
sults with those obtained from the surface tension data. In
very recent years, the reliability of the Gibbs model [Eq. (3)]
was questioned on the basis of some apparent discrepancies
When the spacer is long enough to fold towards the micellar
core, and thus the two pyridinium rings can stay a bit closer,
the surface charge density increases along with the b value
[79–83]
and the problem was debated.
Careful use of the NR tech-
(
surfactants 4b, 8b).
The degree of counterion binding is related to the compact-
nique gave sufficient evidence that, at least in case of nonionic
surfactants, the Gibbs model was valid, whereas in the case of
ionic surfactants the ionic nature of the molecules made the
[
1,52,53]
ness of the micellar surface.
The values of b suggest that
[83]
the micellar surface of the 16Py-s-Py16 series has a lower com-
pactness than the 12Py-s-Py12 one. The use of pyrene is infor-
mative in this sense. The CMC determined with this fluorescent
probe for hexadecyl surfactants (1b–4b) was not in agreement
with the values determined by conductivity (from 5 to
results more complicated to interpret. Notwithstanding, the
debate on always better techniques and careful revision of
physicochemical models is useful for the progress of research
and knowledge. Since our more hydrophobic surfactants 3b
and 4b show CMC values ranging at a concentration of about
À5
À6
22 times higher). In fact, their less compact micellar surface
10 –10 m, they could also be affected by phenomena such
as the depletion of the adsorbed water–air surface layer owing
to adsorption of surfactants to the solid–liquid surface, thereby
changing considerably the bulk surfactant concentration and
also kinetic effects could be possibly affecting the process of
allows pyrene to interact with cationic head groups still re-
maining hydrated. As a consequence the I /I ratio does not
1
3
change significatively until the micelles show an adequately
high compactness and this occurs at higher surfactant concen-
tration. Noteworthy is the comparision of the I /I ratio of pyri-
[83]
surface-tension determination. However, resorting to an ab-
solute method such as NR to study adsorption phenomena is
difficult since it requires instrumentation that is currently not
easily available, therefore simpler and cost-effective benchtop
instrumentation that could permit the correct study of adsorp-
tion even at low concentrations is necessary, but still not avail-
able. The results we present, however, show a trend in reason-
able agreement with the literature, apart from some peculiar
behavior for compound 2b.
1
3
dinium and ammonium gemini surfactants. Ammonium gemini
surfactants show higher values ranging between 1.21 and
[
53,54,67]
1.35.
This means that the presence of a flat pyridinium
ring as the head group instead of trimethylammonium can
better help to accommodate and dehydrate the pyrene probe.
The literature suggests that the counterion can be bound to
a single surfactant molecule before micelle formation; that is,
[
62]
ion-pair formation occurs. The analysis of conductivity data
is informative also to evidence the ion-pair formation that de-
termines a departure of the experimental points from linearity
of the specific conductivity (k) versus C plots in diluted solu-
tions before the CMC; for example, compare Figure 1 (surfac-
tant 1b) with Figure 4a (N-dodecylpyridinium chloride). Zana
defined this behavior as a curvature towards the C axis and as-
signed it to ion-pair association, which is easily achieved for
gemini surfactants since the two cationic head groups can
In Figure 7, the Amin values as a function of the spacer length
(n) for both series are reported. The 16Py-s-Py16 surfactants
show greater values of A
than the 12Py-s-Py12 series, in
min
65]
[
agreement with literature. The Amin values increase with in-
creasing spacer length. This increase seems to reach a plateau
on going from a spacer length of eight to twelve methylene
groups, which suggests that, similarly to gemini ammonium
[
62,76]
keep a counterion tightly between them.
Similar behavior
for all the gemini surfactants 1b–8b was observed (see the
Supporting Information).
The surface minimum area (Amin), that is, the area occupied
by a single molecule at the air–solution interface, was extrapo-
lated from surface tension measurements and the maximum
surface excess concentration (Gmax) through Equations (2) and
[
1,59]
(3) (Gibbs model).
1
6
Amin ¼ 10 =ðN Á GmaxÞ
ð2Þ
ð3Þ
Gmax ¼ Àð1=2:303nRTÞð@g=@logCÞT
Before discussing the relationships between the minimum area
and behavior of the surfactants, it is worth mentioning that
the uncertainty in the surface purity can provoke great errors
Figure 7. Amin as a function of the spacer length (n) for C12 (red squares)
and C16 ( dark blue diamonds) pyridinium gemini surfactants.
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surfactants, the spacer tends to fold toward the micellar core.
Similar behavior has been already observed for gemini ammo-
nium surfactants with spacer lengths of 10–12 methylene
spacer. The AFM images (Figure 8) show that only 2b is able
to condense the DNA into nearly spherical particles. Surfactant
3b was able to succeed to a minor extent, and also gave rise
to some particles that were not homogeneous in size nor in
shape. The 1b and 4b hexadecyl surfactants along with the 5–
8 dodecyl surfactants produced no DNA collapse or only par-
tial collapse without achieving full DNA condensation. For
a more detailed discussion about the gene-delivery ability of
the compounds presented here, see Ref. [64].
[
59]
groups. Noteworthy is the minimum value of Amin for surfac-
tants with a spacer of four methylene groups, in agreement
with an analogous effect observed for dodecyl pyridinium
gemini surfactants that have methanesulfonates as counter-
[
44,45]
ions.
The conformational freedom of a four-methylene-
group spacer allows the molecule to fold like a book, which
gives rise to stacking interactions between the two pyridinium
rings, mediated by the counterion, the charge of which screens
the positive rings from each other.
Transfection experiments in which pyridinium gemini surfac-
tants have been formulated with DOPE ([l-a-phosphatidyletha-
nolamine dioleoyl (C18:1,[cis]-9)]) seem to indicate better per-
formance for the hexadecyl surfactants series with a particular
[64]
dependence on the spacer length. Higher activity has been
observed when the transfecting agent has four or eight meth-
ylene groups, which suggests a possible correlation of biologi-
cal activity with counterion binding (b) and Amin parameter and
also with the visual inspection of AFM images for which only
Surfactant–DNA interaction
To check the ability of the new gemini surfactants to induce
structural changes in the DNA, we used the atomic force mi-
croscopy (AFM) technique. The experiments were performed
using plasmid DNA imaged in air in the tapping mode before
and after incubation with surfactants 1b–8b. In Figure 8, as an
example, some images of the circular DNA and of the struc-
tures obtained after addition of the surfactants are shown.
They suggest a very nice structure–activity relationship, in that
the compacting ability is strictly related to the length of the
[64]
those surfactants showed an ability to compact DNA.
The variation of the spacer length is known to modify the
[
46,59]
packing parameter
and to tune the aggregation proper-
[
84,85]
ties
(CMC, micellar aggregation number, micellar shape,
etc.) and these preliminar results indicate that it affects the
properties of the surfactant–DNA lipoplexes. This structural fea-
ture, which determines the pe-
culiar behavior of surfactants 2b
and 3b for Amin and the degree
of counterion binding b, also
seems to be highly relevant for
their activity in transfection ex-
periments as suggested in our
recent paper where the DNA
shift and the interaction with
membranes of present series of
[64]
surfactants are described.
In view of these results, it ap-
pears interesting to explore simi-
lar structures that bear a hydro-
lyzable bond such as amide or
[86]
ester,
to improve the biode-
gradability and possibly the via-
bility to the cells, since usually
quaternary compounds show
bactericidal activity and limited
biodegradability that, however,
do not hamper their use as gene
carriers.
Conclusion
A series of hexadecyl pyridinium
gemini surfactants was prepared
by extending the generality of
a quaternization method based
on alkyl trifluoromethanesulfo-
nates. Peculiar aggregation be-
haviors were studied by a multi-
Figure 8. AFM images showing the effect of the interaction of different hexacecyl surfactants on the DNA com-
paction: a) native plasmid DNA; b) plasmid DNA with compound 2b; b) plasmid DNA with compound 3b; b) plas-
mid DNA with compound 4b. The behavior of 1b and dodecyl surfactants 5b–8b (not shown) is similar to that
of 4b.
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technique approach: conductivity, surface tension, and fluores-
tion activity shows that some surfactants of the present series,
formulated with DOPE, have high DNA transfection activity.
The chain-length-dependence study for the interaction with
DNA was important, since dodecyl analogues did not show
DNA complexing ability and only two hexadecyl analogues
formed lipoplexes in an efficient way. The effect of the spacer
length, for compounds that had four and eight methylene
groups, on both Amin and b is peculiar not only in relation to
the surfactant properties but also the transfection activity.
Although several structural features contribute to the surfac-
tant behavior, this relationship cannot be disregarded and
should enforce research to establish structure–activity relation-
ships.
À5
cence. The CMC values, in the order of 10 m, suggest
a higher efficiency of pyridinum gemini surfactants in compari-
son with the corresponding monomeric ones. Long alkyl
chains (16Py-s-Py16 series) stabilize the micellar aggregates.
The comparison of CMC values extrapolated by coumarin 6
and pyrene as fluorescent probes and specific conductivity (k)
0.5
versus C and molar conductivity (L) versus C plots suggests
the occurrence of premicellar aggregates for pyridinium
gemini surfactants with hexadecyl chains (16Py-s-Py16 series).
The data suggest also that pyrene is a less useful fluorescent
probe to determine the CMC for the present series of surfac-
tants, but it helps to evidence the lower micellar compactness
of the hexadecyl series with respect to dodecyl ones. In gener-
al, the application of fluorescent probes can give very useful
information also for surfactant systems that show nonconven- Experimental Section
tional behavior, thereby improving the knowledge of the com-
General procedures and materials
plex aggregation of surfactants.
For some terms of the surfactants series, a more complex
behavior was found. Formation of an ion pair and of pre-ag-
gregates has been supposed.
The 2,2’-alkybipyridines were prepared as described in the litera-
[10]
ture and products 5–10 were prepared according to previously
[10,48]
established procedures.
Also see the Supporting Information.
The study of the degree of counterion binding (b) could be
an interesting parameter for a structure–activity relationship
study, also with respect to gene vector activity. In this context,
the pyridinium ion is a softer cation than ammonium and its
delocalized charge can better interact with a reasonably soft
anion such as phosphate, thereby influencing the aggregation
properties and the DNA transfection activity. For DNA complex-
ation and its release in the cell, an optimal interaction of the
cationic vector with the negative phosphate group of DNA is
needed.
[
10,87]
Synthesis of hexadecyl triflate
This product was obtained by following previously stated proce-
[10]
dures. Chloroform was found to perform better than dichloro-
methane in the solubilization of the hexadecyl alcohol, thus im-
proving the procedure. After flash chromatography on silica with
petroleum ether, the product was obtained by solvent evaporation
and was used immediately for quaternizations. Yield 6.27 g (85%);
1
H NMR (CDCl ): d=0.88 (t, 3H; CH ), 1.26 (brs, 26H; 13CH ), 1.83
3
3
2
(
t, 2H; CF SO CH CH ), 4.53 ppm (t, 2H; CF SO CH CH ).
3 3 2 2 3 3 2 2
The degree of counterion binding decreased with the
lengthening of the alkyl chains and was strongly affected by
the spacer length. A minimum for the degree of counterion
binding was found for molecules that had a spacer made of
eight methylene groups, irrespective of the differences in the
alkyl chains. This peculiarity was related to the compromise of
the conformation that these gemini surfactants can adopt at
the micellar surface and the electrostatic interactions between
the pyridinium rings, which gives a poorly packed micellar
structure as a result.
General procedure for the quaternization of a,w-bis(2-pyri-
dyl)alkanes
A solution of hexadecyl triflate in anhydrous chloroform was intro-
duced into a three-necked flask under argon, and warmed at 708C
under magnetic stirring. A solution of the appropriate a,w-bis(2-
pyridyl)alkane in anhydrous chloroform was slowly dropped into
the warm solution. The reaction was stopped after 0.5–1.5 h. The
resulting solution was treated with ethyl acetate to give a white
precipitate that was filtered to give the pure product. Chlori-
des 1b–4b were obtained from products 1a–4a by ion exchange
The trends of Amin as a function of the spacer length for the
didodecyl and dihexadecyl gemini surfactants are in perfect
agreement with what we reported for the same didodecyl
gemini surfactants that had methanesulfonates as counterions,
which confirms the hypothesis of a conformational change of
the molecule when the spacer is four methylene groups long,
owing to stacking interactions between the two pyridinium
rings, mediated by the counterion. Moreover, the trends of
both Amin and b suggest that the spacer must be longer than
eight carbon atoms to fold towards the micellar core.
[10]
on an anionic exchange resin as previously described.
1
,1’-Dihexadecyl-2,2’-trimethylenebispyridinium ditrifluoromethane-
sulfonate (1a): Yield 87%; m.p. 206–2098C; R =0.31 on silica
f
(
eluent BAW: butanol/acetic acid/water 4:1:5, organic phase);
1
H NMR ([D ]DMSO): d=0.85 (t, 6H; 2CH ), 1.23 (m, 52H; 26CH ),
6
3
2
+
1.86 (m, 4H; N CH CH ), 2.19 (m, 2H; PyCH CH ), 3.29 (t, 4H;
PyCH
8
2
2
2
2
+
), 4.58 (t, 4H; N CH
.54 (t, 2H; Hpara), 9.03 ppm (d, 2H; Hortho); C NMR ([D ]DMSO): d=
), 8.02 (t, 2H; Hmeta), 8.11 (d, 2H; H’meta),
2
2
13
6
1
4.20 (CH ), 22.35, 26.01, 28.83, 28.97, 29.19 (several C), 29.33 (sev-
3
The opportunity to tune the aggregation properties of the
surfactants—such as the CMC towards lower values—the
whole molecular hydrophobic character, the spacer length,
and the degree of counterion binding make these surfactants,
in particular the long-chain ones, desirable candidates for
gene-delivery experiments. A preliminary study of the transfec-
eral C), 30.65, 30.98, 31.55, 57.34, 126.09 (Ar), 128.83 (Ar), 145.52
(
1
Ar), 146.03(Ar), 157.26 ppm (Ar); FTIR (KBr): n˜ =3086, 2918, 2850,
630, 1512, 1468, 1226, 1166, 786 cm ; UV/Vis (ethanol): lmax
À1
=
2
69 nm, loge=3.99; MS (ESI+): m/z: calcd. 797.58; found: 797.66
+
[M ÀCF SO ]; elemental analysis calcd (%) for C H F N O S : C
3
3
47 80
6
2
6 2
59.59, H 8.51, N 2.96; found: C 59.62, H 8.53, N 2.90.
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1
,1’-Dihexadecyl-2,2’-trimethylenebispyridinium
dichloride (1b):
until no significant change occurred. Standard deviation of the sur-
face tension measurements was less than 0.15 mNm . The ab-
sence of a minimum in the surface tension versus log(concentra-
tion) plot in the post-CMC region (see Figure 2) showed that there
was very little or no surface-active impurity present in the final
products.
À1
Yield 99%; m.p. 200–2018C; R =0.35 on silica (eluent BAW);
f
1
H NMR ([D ]DMSO): d=0.84 (t, 6H; 2CH ), 1.23 (m, 52H; 26CH ),
6
3
2
+
1
.86 (m, 4H; N CH CH ), 2.19 (m, 2H; PyCH CH ), 3.33 (t, 4H;
2 2 2 2
+
PyCH ), 4.67 (t, 4H; N CH ), 8.02 (t, 2H; H ), 8.22 (d, 2H; H’meta),
8
2
2
meta
13
.55 (t, 2H; Hpara), 9.16 ppm (d, 2H; Hortho); C NMR ([D ]DMSO): d=
6
1
3.96 (CH ), 22.12, 25.75, 28.65, 28.74, 28.98, 29.05 (several C), 29.11
3
(
several C), 30.59, 30.77, 31.32, 57.09, 125.77 (Ar), 128.78 (Ar),
1
1
45.19 (Ar), 145.84 (Ar), 157.20 ppm (Ar); FTIR (KBr): n˜ =2918, 2851,
Sample preparation and AFM imaging
À1
630, 1514, 1466, 1173, 789 cm ; UV/Vis (ethanol): lmax =269 nm,
+
DNA samples were prepared by diluting the plasmid DNA to
a final concentration of 0.5 nm in deposition buffer (4 mm 4-(2-hy-
droxyethyl)-1-piperazineethanesulfonic acid (Hepes), 10 mm NaCl,
loge=4.18; MS (ESI+): m/z: calcd: 683.60; found: 683.75 [M ÀCl];
elemental analysis calcd (%) for C H Cl N : C 75.06, H 11.20, N
4
5
80
2
2
3
.89; found: C 75.06, H 11.16, N 3.87.
2
mm MgCl , pH 7.4) either in the presence or in the absence of
2
surfactants 1b–8b. The mixture was incubated for 5 min at room
temperature, then a 20 mL droplet was deposited onto freshly
cleaved ruby mica (Ted Pella, Redding, CA) for one minute. The
mica disk was rinsed with milliQ water and dried with a weak
stream of nitrogen.
Conductivity measurements
Conductivity measurements were performed on a AMEL 160 con-
ductivity meter equipped with a conductivity cell that had a cell
À1
constant of 0.943 cm , except for surfactants 1b–4b for which
À1
AFM imaging was performed on the dried sample with a Nano-
scope IIIA Microscope (Digital Instruments Inc. Santa Barbara, CA)
operating in tapping mode. Commercial diving-board-shaped sili-
con cantilevers (NSC-15 Micromash Corp., Estonia) were used.
Images of 512”512 pixels were collected with a scan size of 2 mm
at a scan rate of 3 to 4 lines per second and were flattened after
recording using Nanoscope software.
a conductivity cell with a cell constant of 9.895 cm was used, as
[10,60]
already reported.
The addition of concentrated surfactant solu-
tions by a CRISON automatic burette to pure water and the collec-
tion of the conductivity data were performed by using a comput-
er-controlled automated system, working with a homemade pro-
gram written in Quick Basic (available from the author).
Fluorescence measurements
Acknowledgements
The steady-state intensity fluorescence measurements were per-
formed on a PerkinElmer LS55 spectrofluorimeter. Sample solutions
were prepared by dilution of a surfactant stock solution, to which
We gratefully acknowledge the financial support of the Compag-
nia di San Paolo (Progetti di Ricerca di Ateneo 2011-Linea 1A,
project ORTO11RRT5), the University of Torino (Ricerca Locale ex-
1
0 mL of the probe stock solution was added. Pyrene and coumar-
in 6 were dissolved in ethanol. Pyrene spectra were collected in
the 360–600 nm range, checking carefully that no excimer was
formed: final concentration 5”10 m, excitation wavelength
60%, Bando 2013), the Italian Ministry of Education, University
À7
and Research (MIUR), and PRIN2011 “NANO Molecular Technolo-
gies for Drug delivery–NANOMED”. N.B. thanks MIUR for partial
financial support of her research grant.
3
20 nm; excitation slit 5 nm and emission slit 2.5 nm. Coumarin 6
spectra were collected in the 475–650 nm range: final concentra-
À7
tion 1.5”10 m, excitation wavelength 465 nm; excitation slit
2
.5 nm and emission slit 5.0 nm. To fulfill the conditions for a cor-
rect measurement of the CMC of pyridinium surfactants with
pyrene, it is better to try to adjust the pyrene concentration to
have enough of a fluorescence signal after the CMC, since pyridini-
um surfactants are known to quench pyrene fluorescence. In addi-
tion, too high a concentration of pyrene could produce excimers
when it transfers into the micelles.
Keywords: amphiphiles · DNA · fluorescence · micelles ·
surfactants
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