Role of aggregate size in the hemolytic and antimicrobial activity of colloidal solutions based
on single and gemini surfactants from arginine
L. Tavano ab, M.R. Infante c, M. Abo Riya d, A. Pinazo c, M.P. Vinardell e,f, M. Mitjans e,f, M.A.
Manresag, L. Perez c*.
a
Department of Pharmaceutical Sciences, University of Calabria, Edificio Polifunzionale, 87036
Arcavacata di Rende, Cosenza, Italy.
b
Department of Engineering Modelling, University of Calabria, Via P. Bucci - Cubo 39C, 87036
Arcavacata di Rende, Cosenza, Italy.
c
Department of Chemical and Surfactants Technology, IQAC-CSIC, Jordi Girona 18-26, 08034
Barcelona, Spain.
d
Chemistry Department, Faculty Of Science, Benha University, Egypt.
e
Department de Fisiologia, Universitat de Barcelona, Joan XXIII s/n, 08028 Barcelona, Spain.
f
Unidad Asociada al CSIC
g
Department de Microbiologia, Universitat de Barcelona, Joan XXIII s/n, 08028 Barcelona, Spain.
*Corresponding author: lpmste@cid.csic.es
1
GRAPHICAL ABSTRACT
The role of the aggregate size on the hemolytic and antimicrobial activity of colloidal dispersions formulated
with cationic surfactants from arginine is reported. This study shows that the biological activity of Gemini
surfactants from arginine can be modulated by changing the spacer chain length or adding membrane
additives.
2
ABSTRACT
Cationic colloidal systems composed by arginine based surfactants (single or gemini structures) and
membrane additive compounds such as DLPC or cholesterol have been characterized by means of
size distribution and zeta-potential measurements. The single or monocatenary surfactant (LAM) as
well as the gemini with the shortest spacer chain (C6(LA)2) formed micelles, while aqueous
solutions of pure gemini surfactants with longer spacer (C9(LA)2 and C12(LA)2) made up very big
aggregates. The addition of phospholipids or cholesterol changed drastically the aggregation
behaviour. In the case of LAM and C6(LA)2, the incorporation of additives gave rise to the
formation of cationic vesicles. For C9(LA)2 and C12(LA)2, this type of additives promoted the
formation of smaller aggregates. All the formulations had positive zeta-potential values and in
general exhibited high colloidal stability. We also evaluated the hemolysis and the antimicrobial
activity of these systems. The capability of disrupting the erythrocyte’s membrane depends on the
hydrophobicity of the molecules and the size of aggregates in the solution. Gemini surfactants with
short spacer chains are more hemolytic than the single chain homologue while gemini with long
spacers are much less hemolytic than the single counterpart. Moreover, for the same formulation,
the hemolysis depends on the initial concentration of the stock solution used to set up the
hemolysis/concentration curve. Results show that small aggregates interact easily with these
biological membranes. The alkyl spacer chain and the presence of additives also play an important
role on the antimicrobial activity, and, in general, the interaction with bacteria and erythrocytes is
affected by the same parameters. The physico-chemical and biological characterization of these
systems might be important for several biotechnological applications in which cationic vesicular
systems are involved.
Keywords: gemini surfactants, arginine, vesicles, hemolysis, antibacterial activity.
3
INTRODUCTION
In recent years the concept of drug-delivery is changing. It is no more a simple formulation that
allows the administration of the drug, but a much more complex system based on the use of
nanomaterials, which can present special properties. Cationic vesicles based on positive charged
surfactants are among other colloidal systems one of the most promising, showing great potential. 1
Cationic liposomes have been successfully employed to interact with negatively charged surfaces or
biomolecules such as prokaryotic 2 or eukaryotic cells, 3 antigenic proteins, 4 synthetic polymers, 5
latex6 and mineral surfaces7. At present, there is an unprecedented level of interest in the properties
and structures of complexes consisting of DNA mixed with oppositely charged cationic liposomes.8
This interest arises because the complexes mimic natural viruses as chemical carriers of DNA into
cells in worldwide human gene therapy clinical trials.9
Other very useful applications of cationic liposomes are the delivery of peptides, proteins or
antimicrobial agents.10,11 It has been demonstrated that these vesicles are able to overcome bacterial
resistance related to the permeability barrier and enzymatic hydrolysis by a fusion process between
the liposomes and bacterial membranes. 12 Cationic liposomes were also investigated for their
potential targeting ability to the bacterial biofilms produced by skin and oral bacteria.13 Furthermore,
it has been demonstrated that the same cationic lipids used in liposome preparations might act by
themselves as anti-infective agents.14
Among the classical cationic surfactants, quaternary ammonium compounds (QACs) and
bis(QACs) are usually employed to obtain cationic liposomes.15 Usually, they have the ability to
interact with bacterial species and cultured mammalian cells. Vesicles based on QACs also have
been found to solubilize amphotericin B and other hydrophobic drugs such as miconazol (MCZ)
and to transfer DNA into cells through fusion with the cell membrane. 2,16,17
Unfortunately, one of the major problems related with the use of cationic vesicles based on QACs is
their high degree of toxicity towards the host.18 In fact, QACs present acute toxicity, poor chemical
and biological degradation and hemolytic activity. Thus, they are not suitable for biomedical
applications.19
For these reasons, a large variety of synthetic cationic amphiphiles with low toxicity have
been synthesized as an interesting alternative to the conventional cationic surfactants.20 An obvious
strategy to increase the efficiency of surfactants and improve their environmental properties is to
build up structures from renewable materials. In this light, our group has reported the synthesis of
new cationic surfactants based on the amino acid arginine, with different structures (monocatenary,
gemini, and glycerolipid), characterized by relevant nontoxic and antimicrobial properties as well as
4
rapid biodegradability.21 In addition, gemini surfactants from arginine are extraordinarily active in
reducing surface tension and it has been reported that the single chain derivatives improve
transfection efficiency if conjugated to cationic liposome systems.22,23
The physico-chemical and biological properties of liposomal systems obtained from the argininebased surfactants have not yet been studied, so it would be very interesting to learn how colloidal
formulations can affect the hemolysis and the antibacterial activity of this class of surfactants.
In this work, we report on the physicho-chemical and biological properties of colloidal
systems obtained from this class of surfactants. Single-chain arginine based surfactants (LAM) and
arginine gemini derivatives (C6(LA)2, C9(LA)2, and C12(LA)2 (Figure 10) were synthesized and
used as pure components or formulated in cationic mixtures with membrane additives (cholesterol,
or dilauroylphosphatidylcholine (DLPC)). The influence of different parameters including
surfactant structure (gemini or monocatenary), spacer chain length and membrane additive type on
the physico-chemical properties of the cationic vesicles as well as on the hemolytic and
antimicrobial activity has been investigated.
RESULTS AND DISCUSSIONS
Vesicle diameter, size distribution as well as charge density are important parameters in liposome
applications and can affect the biological activity of liposome formulations. In order to study how
these parameters affect the hemolysis and antimicrobial activity of cationic surfactants from
arginine, different formulations have been designed: pure surfactant solutions of LAM, C6(LA)2,
C9(LA)2 and C12(LA)2 at two different concentrations (4 and 2.5 mM) and mixed surfactant/additive
suspensions at two different molar ratios have been prepared. Table 1 presents the composition of
every formulation.
Usually, there is an upper limit to cholesterol incorporation in lipid bilayers and above this
limit cholesterol starts to precipitate. It has been reported that for phosphatidylcholine vesicles the
maximum cholesterol solubility is about 66% while for phosphatidylethanolamine the limit is about
51%.24 Taking into account this behavior, two formulations containing cholesterol or DLPC with a
surfactant/additive ratio of 8/2 and 5/5 have been prepared.
5
Table 1. Details on sample preparations (n= number of moles).
Sample
Number
and Label
Concentration
Surfactant
Additive
(mM)
Amount (n)
Amount (n)
LAM 1
4
8*10-6
LAM 2
2.5
5*10-6
LAM/CHOL 1
5
8*10-6
LAM/CHOL 2
5
5*10-6
LAM/DLPC 1
5
8*10-6
LAM/DLPC 2
5
5*10-6
C6 1
4
8*10-6
C6 2
2.5
5*10-6
C6/CHOL 1
5
8*10-6
C6/CHOL 2
5
5*10-6
C6/DLPC 1
5
8*10-6
C6/DLPC 2
5
5*10-6
C9 1
4
8*10-6
C9 2
2.5
5*10-6
C9/CHOL 1
5
8*10-6
C9/CHOL 2
5
5*10-6
C9/DLPC 1
5
8*10-6
C9/DLPC 2
5
5*10-6
C12 1
4
8*10-6
C12 2
2.5
5*10-6
C12/CHOL 1
5
8*10-6
C12/CHOL 2
5
5*10-6
C12/DLPC 1
5
8*10-6
C12/DLPC 2
5
5*10-6
(C6: C6(LA)2; C9: C9(LA)2; C12: C12(LA)2)
2*10-6
5*10-6
2*10-6
5*10-6
2*10-6
5*10-6
2*10-6
5*10-6
2*10-6
5*10-6
2*10-6
5*10-6
2*10-6
5*10-6
2*10-6
5*10-6
Mole
Ratio
Surf/Add
1:0
1:0
8:2
5:5
8:2
5:5
1:0
1:0
8:2
5:5
8:2
5:5
1:0
1:0
8:2
5:5
8:2
5:5
1:0
1:0
8:2
5:5
8:2
5:5
Visual observation
It is known that a change in the turbidity of a surfactant solution can relate to the change in the
amount and/or size of the surfactant aggregates.
Figure 1. Pictures of LAM formulations at different concentrations: a) LAM 1 and LAM 2, b) LAM/CHOL 1 and
LAM/CHOL 2, c) LAM/DLPC 1 and LAM/DLPC 2.
6
The pure LAM and C6(LA)2 surfactants formed transparent solutions, this behaviour suggests that
these compounds do not form vesicular aggregates (Fig.1a and Fig.2a). In fact, these surfactants
yield 1H-NMR typical high-resolution spectra with Lorentzian band shape corresponding to the
presence of small micelles.25
Figure 2. Pictures of C6 formulations at different concentrations: a) C6 1and C6 2, b) C6/CHOL 1 and C6/CHOL 2, c)
C6/DLPC 1 and C6/DLPC 2.
Solutions of pure C9(LA)2 (Fig.3a) and C12(LA)2 (Figure not shown) appeared in translucent and
viscous dispersions without sedimentation after several months. Moreover, the 1H-NMR spectra of
the C9(LA)2 and C12(LA)2 indicated the presence of larger aggregates. The slow motion of alkyl
chains in the vesicles or big aggregates resulted in extremely broad NMR signals with low
intensity.26 Previous Cryo-TEM studies showed that aqueous solutions of LAM and C6(LA)2 mainly
contain classical spherical micelles at the concentrations used in this work. However, different
results were obtained for the C9(LA)2 and C12(LA)2. For these two surfactants no spheroidal
micelles were seen in any of the concentrations examined. Gemini Cn(LA)2 solutions contain
cylinder micelles, twisted ribbons, flat ribbons or threadlike micelles and the concentration at which
these aggregates appear decreases as the spacer chain increases. 27 It has been reported that the
formation of these types of aggregates is favored by the presence of chiral head groups and
intermolecular H-bonding. 28,29,30
Figure 3. Pictures of C9 formulations at different concentrations: a) C9 1and C9 2, b) C9/CHOL 1 and C9/CHOL 2, c)
C9/DLPC 1 and C9/DLPC 2.
7
In general, the use of DLPC or cholesterol often helps in forming self-closed bilayers.31,32 Some
surfactants can not assemble to form vesicles due to their critical packing parameter, i.e. relative
space requirements of the hydrophobic and the hydrophilic parts of the amphiphiles. The
incorporation of phospholipids or cholesterol into the surfactant aggregates leads to appropriate
molecular geometry and hydrophobicity for vesicle formation. 33 Cholesterol also changes the
fluidity of the hydrophobic chains in the bilayer, thus promoting the formation of surfactant vesicles.
In this work, we have prepared aggregates formed by cationic arginine surfactants and cholesterol
or DLPC to study how these additives affect the aggregation of these cationic surfactants.
Surfactant formulations with additives showed different appearances. In the case of the LAM, the
addition of cholesterol gave rise to milky and cloudy dispersions (Fig.1b). In the case of C6(LA)2,
formulations appeared slightly less milky than the ones based on LAM which suggests the presence
of smaller aggregates (Fig.2b). Some evidence of phase separation has been observed after 1 week
for the formulation LAM/CHOL 2.
The addition of 20% DLPC to LAM or C6(LA)2 solutions originates transparent formulations with
the same aspect that those of respective pure surfactants (Fig.1c and Fig 2c). The incorporation of
50% DLPC originates turbid and bluish dispersions with the typical aspect of vesicular formulations
for LAM, but in the case of C6(LA)2, transparent solutions were obtained, suggesting that these
compounds do not form vesicular aggregates even in presence of 50% DLPC (Fig.2c).
For C9(LA)2 and C12(LA)2 surfactants, changes are different for every formulation. Given that both
surfactant solutions are milky and cloudy, the introduction of additives did not change in a relevant
manner the final aspect of the formulations. The presence of cholesterol gave rise to an increase in
sample turbidity, while the formulations containing DLPC seem to contain smaller aggregates since
they appear slightly less turbid than those of pure gemini surfactants (Fig. 3b, 3c).
Size and charge density of the aggregates
All the formulations previously described were characterized by Dynamic Light Scattering
measurements to determine particle size and size distribution. Figure 4 shows the results obtained
for some LAM formulations. According to visual observations, that showed only transparent
solutions for both concentrations of pure LAM solutions, the DLS measurement did not show any
peak related to vesicles, but only peaks of low intensity which refer to spherical micelles (Fig. 4c).
The low scattering intensity of this solution could be attributed to the small amount of aggregates
into the sample. Notice that the concentration of the LAM 1 solution is near the CMC of this
surfactant (4-6 mM), and then, it is expected that few micelles are present in the solution.34
8
Inclusion of cholesterol or phospholipids strongly affected the aggregate’s size. In particular, the
presence of cholesterol at 20% and 50% gave rise to higher aggregates. Figure 4a shows the narrow
peak centered at 200 nm obtained for the LAM/CHOL 2 sample. In the presence of 20% DLPC no
peaks referred to vesicular systems were found, probably mixed micelles are present in the solution.
In fact, it has been reported that the solubilization of water-insoluble compounds, such as lipids, in
the hydrophobic core of micellar aggregates leads to the breakdown of bilayered structures and
formation of lipid-surfactant mixed micelles.35 In particular, different studies on the properties of
mixed systems composed of cationic gemini surfactants and phospholipids have been conducted,
demonstrating that the origin of synergism of phospholipids with amino acid surfactants is based on
the reduction of electrostatic repulsions between the ionic head groups of the surfactant due to
intercalation of zwitterionic lipids in the mixed micelles.36 The formation of mixed micelles is the
basis of the wide application of surfactants in isolation and purification of membrane proteins, 37 in
DNA extraction38 and as drug delivery vehicles.39 On the other hand, the incorporation of 50 %
DLPC gave monodisperse aggregates with a peak centered at 100 nm, which is consistent with the
visual observation (Fig. 4b).
c
Figure 4. Size distribution profiles of LAM formulations: a) LAM/CHOL 2 b) LAM/DLPC 2 and c) LAM 1
The transparent C6 1 and C6 2 samples show only peaks centered at 0.5 and 5 nm, which can be
attributed to micelles (Fig. 5a). The CMC of this surfactant is around 0.4-0.6 mM34, because that,
9
the number of micelles at 4 mM is high and the intensity of the DLS graph is greater that those
obtained for the LAM 1 solution. With the introduction of DLPC similar results were obtained. The
incorporation of cholesterol promotes the formation of small and big aggregates with mean
hydrodynamic diameter (dH) higher than 1000 nm (Fig.5b).
a
b
Figure 5. Size distribution profiles of C6(LA)2 based formulations: a) C6 1, b) C6/CHOL 1
The intensity distribution graphs of C9(LA)2 formulations are shown in Figure 6. Different features
can be observed in every graph. The pure C9 1 has two different populations, aggregates with
diameters in the range of 100 nm and large aggregates with diameters around 900 nm (Fig.6a).
These large aggregates could correspond to twisted ribbons, flat ribbons, threadlike ribbons or
helical aggregates. These aggregates are consistent with the viscosity of the solutions and with
previous cryotem and NMR studies.27 Although forming large aggregates, such dispersions are
stable for an indefinitely long time (up to 1 year) and a tendency to phase separation was not
observed.
10
Figure 6. Size distribution profiles of C9 formulations: a)C9 1; b)C9 2; c)C9/CHOL 1; d) C9/CHOL 2; e) C9/DLPC 1; f)
C9/DLPC 2.
In order to establish a control on the aggregate´s size, this formulation was subjected to extrusion 20
times with an extruser device equipped with a 200 nm pore size polycarbonate membrane.
Extrusion of this solution was very difficult, moreover, HPLC analysis showed that some surfactant
was retained on the membrane. The extrused formulation also contained large aggregates. It is
probable that the big elongated aggregates, long thread-like twisted ribbons or helical aggregates
can cross the membrane retaining the original shape and size. The less concentrated C 9(LA)2
solution presents only a single population with the average hydrodynamic value centered at 200 nm
(Fig.6b). Perhaps this lower concentration is not enough to promote the formation of the large
aggregates observed at higher concentrations. The Cryo-TEM studies carried out with the C9(LA)2
and C12(LA)2 indicated that no spheroidal micelles were seen in any of the concentrations examined
in this work. The C9(LA)2 showed ribbons at very low concentrations (0.1 wt%) and when the
concentration was increased to 0.7 % the ribbons were tightly twisted. Similar results were obtained
for the C12(LA)2, but in this case aggregates appeared at lower concentrations (0.05%).27 These
types of aggregates have been also observed for other amino acid surfactants. At high
concentrations, 8.0 mM, the z-average dH value of aggregates formed by histidine based surfactants
is around 1.0 m and the optical microscopic images of this solutions reveals the existence of
11
helical and fiber-like aggregates. At lower concentrations, these histidine based surfactants form
aggregates with a z-average dH value of 100 nm.40
In addition, we decided to study the influence of a different method of preparation on the
physicochemical properties of these formulations. The experiments were performed by using both
the sonication method and the thin layer evaporation method (film), starting from the same
surfactant/additive composition, but no important differences in the aggregate’s size and
distribution have been found.
The inclusion of neutral amphoteric vesicle inducing agents into the aggregates formed by
this surfactant was found to have a strong influence on their shape and size. The addition of 20%
cholesterol to the C9(LA)2 solution drastically changes the intensity average size distribution plot.
The large aggregates present on the original solution (C9 1 sample) disappear and two populations
can be observed with dH values in the range of 30-200 nm (Fig.6c). The size of these aggregates
suggests that the incorporation of cholesterol on the surfactant assemblies promotes the formation
of vesicles. The formulation in which 50% cholesterol has been incorporated to the original solution
(C9/Chol 2, Figure 6d) shows very big aggregates. It has been observed that as the concentration of
cholesterol increases the system undergoes a process of phase separation to form cholesterol-rich
domains.40 The big aggregates observed on the figure could correspond to the cholesterol rich
domains. In fact, at this high concentration of additive the dispersion becomes more turbid and
some evidence of phase separation has been observed after 4 weeks. Usually there is an upper limit
to cholesterol incorporation in lipid bilayers, above which the system undergoes a process of phase
separation to form cholesterol rich domains. It has been observed that for membranes composed of
phosphatidylcholine, the maximum cholesterol solubility is about 66%,24 for vesicles formed by
rhamnolipid surfactants the maximum limit is about 50%41.
The addition of DLPC to the more concentrated C9(LA)2 solution also originates an
heterogeneous dispersion with dH values on the range of 40-200 nm (Fig.6e). Notice that also in this
case the large aggregates of the original C9 1 sample have disappeared probably due to the
formation of mixed vesicles with the DLPC. The z-average dH is slightly lower than that of the
similar formulation made up of cholesterol. The increase of the DLPC concentration decreases the
dH values and in the C9/DLPC 2 formulation there is no evidence of phase separation perhaps
because these phospholipids form stable vesicles (Fig.6f). Table 2 shows the different aggregates
present on every LAM and C9(LA)2 formulation.
12
Table 2. Aggregation behavior of colloidal solutions from LAM and C 9(LA)2
LAM 1
LAM/CHOL 1
LAM/DLPC 1
Micelles
Medium aggregates
Mixed micelles
(dH = <30 nm)
(dH =100 nm)
(dH =<30 nm)
LAM 2
LAM/CHOL 2
LAM/DLPC 2
Micelles
Medium aggregates
Mixed aggregates
(dH = <30 nm)
(dH =200 nm)
(dH = 100 nm)
C9 1
Medium and Big aggregates
(dH = 100-900 nm)
C9/CHOL 1
Small and Medium aggregates
(dH =30-200 nm)
C9 2
Medium aggregates
(dH = 200 nm)
C9/CHOL 2
Medium and Big aggregates
(dH =300-1000 nm)
C9/DLPC 1
Small and Medium aggregates
(dH =40-200 nm)
C9/DLPC 2
Small and Medium aggregates
(dH =5-20-200 nm)
The intensity graphs obtained for the C12-based formulations are similar to those of C9(LA)2
(Data not shown). The pure surfactant aqueous solutions are viscous and the DLS graphs indicate
the presence of very big aggregates and in general cholesterol and DLPC also promote the
formation of colloidal solutions with smaller aggregates, except for the C12/CHOL 2 formulation.
Zeta-potential
Zeta-potential measurements are a convenient way to characterize the electrostatic properties
of vesicles, since the Zeta-potential is a measure of the electrical charge close to the surface of the
vesicles. For all the formulations studied in this work, large positive Zeta-potential values (between
50 and 70 mV) have been obtained. The addition of neutral or amphoteric additives to pure
surfactant solutions does not affect significantly the Zeta-potential of the aggregates. The insertion
of the additive in the cationic aggregates causes the separation of the cationic polar head on the
assemblies and consequently decreases the electrostatic repulsion between the cationic charges.
This fact could promote a decrease of the counterions bonded to the aggregates that explains the
similar zeta potential of the different formulations. Similar performance has been reported for
dioctadecyldimethylammonium
bromide
(DODAB)/dipalmitoylphosphatidylcholine
(DPPC)
vesicles. Upon increasing the percentage of DODAB in the vesicles from 0 to 50% the z-potential
remained practically constant.42 In general, it was observed that the formulations presented high
colloidal stability and no phase separation or precipitation was observed up to 2 months after
preparation. The stability of these suspensions is probably due to the stabilizing effect produced by
the repulsive interaction among aggregates with positive surface charges. The positive charge
density of the aggregates is large enough to ensure that no coagulation occurs. In fact, one method
used to stabilize non-charged vesicles or niosomes is to add a charged molecule to the bilayer to
13
prevent aggregation of niosomes. Phosphatidic acid and stearylamine or cetylpyridinium chloride
are commonly used.43
Hemolytic activity
Hemolysis by surfactants is a process of great fundamental and practical importance. The
mammalian erythrocyte lacks internal organelles and since it is the simplest cellular model
obtainable, it is the most popular cell membrane system to study the surfactant-membrane
interaction. For all these reasons, it is adopted as a convenient model system.44 Gemini cationic
surfactants from amino acids have attracted much interest due to their excellent surface properties
(very low CMC values and large activity reducing surface tension) and their antimicrobial and
environmental behaviour. However, the influence of the spacer chain on the hemolytic character of
these surfactants has been rarely studied. Here we have evaluated the hemolysis behaviour of
different gemini surfactant solutions in which we have varied the spacer chain of the gemini, the
concentration of the stock solution employed to calculate the HC50 value and the additive used to
enhance the formation of mixed vesicles.
The experiments performed to study the hemolytic activity of our formulations were carried
out preparing stock of pure surfactant solutions of LAM, C6(LA)2, C9(LA)2 and C12(LA)2 and mixed
surfactant/additive suspensions at two different concentrations (Table 2). Then, the HC50 of all
stock formulations was calculated preparing, from each of these original solutions, samples with
decreasing concentrations of surfactant or surfactant/additive mixture. The experiments were
carried out the same day of the sample´s preparation. The degree of hemolysis induced by
surfactants was represented by a surfactant concentration-response curve, in which hemolysis is
expressed as a function of the logarithm of surfactant concentration.
Concentration-dependent surfactant effects on erythrocyte membranes are well known and
widely described in the literature. Conventional single-chain amphiphiles have the ability to induce
shape alterations, vesiculation and hemolysis in human erythrocytes, as well as to protect
erythrocytes against hypotonic hemolysis,45 but it is important to compare these membrane effects
with those of the corresponding gemini surfactant or lipid/surfactant mixtures. Figure 7 shows the
HC50 values for all formulations based on LAM. Results showed that regardless the initial
concentration of the stock formulations, no relevant difference in terms of HC50 was achieved. Pure
LAM solutions showed very similar hemolytic activity. When cholesterol is present in the sample,
hemolysis was found to be a little lower than that of the corresponding pure LAM solution, HC50 of
LAM 1 is 91 M while HC50 of LAM/CHOL 1 is 104 M. This behaviour could be ascribed to the
cloudy dispersions obtained in presence of cholesterol, indicative of the presence of bigger
14
aggregates with respect to the ones present in LAM 1 and 2. In fact, as mentioned before, the LAM
solutions appear as transparent solutions containing small micelles. Small aggregates seem to be
more toxic than bigger ones, perhaps because they interact better and more easier with erythrocytes,
causing higher hemolysis.
Compared with pure LAM, LAM/DLPC 1 is more hemolytic while LAM/DLPC 2 is less
lytic to erythrocytes. Notice that LAM/DLPC 1 contains mainly mixed micelles whereas
LAM/DLPC 2 has large size mixed vesicles. This fact indicates that incorporation of additives can
reduce the hemolytic character of cationic surfactants.
Figure 7. HC50 values of LAM based formulations (mean±S.E of three experiments). For HC50 of LAM/additive formulation
the value corresponds to the LAM concentration in the sample.
These results are in concordance with some reported in the literature showing that hemolytic
activity of surface active agents can be reduced by co-formulation with egg phosphatidylcholine
(PC); 46 in particular the combination of dodecyl betaineted and PC gives very low hemolytic
activity. 47 Hemolysis due to diacylglycerol arginine based surfactants can be drastically reduced
when this surfactant is included in cationic vesicles formed by phosphatidylglicerol 48 and the
toxicity of dioctadecyl-dimethyl-ammonium bromide was drastically reduced when it was mixed
with cholesterol and phosphatidylcholine.49
In the case of C6(LA)2 and C6(LA)2/additive mixtures, the percentage of hemolysis at the
lowest concentration tested (20 M) was almost 100%. This means that this surfactant is much
more hemolytic than its single chain homologue LAM. It has been reported that the C 3(LA)2
homologue with three carbon atoms in the spacer chain, is also more hemolytic than LAM 50 and
gemini surfactants from lysine with spacer chains of six CH2 groups are more hemolytic than the
corresponding monocatenary counterparts 51 . Similar results have been reported for Bis(Quats)
surfactants in which the gemini surfactants are more toxic than the corresponding monomeric
15
ones.52,53 Gemini surfactants are more hydrophobic compounds and it has been described that when
the hydrophobicity of the surfactant increased, stronger membrane permeabilization was obtained
and the molecules became more hemolytic.54
Figures 8 and 9 show the HC50 data obtained for C9(LA)2 and C12(LA)2 based formulations.
Similar behaviour has been obtained for the two gemini surfactants. The most remarkable feature is
that the hemolytic activity of these two gemini is very low in comparison with those obtained for
C6(LA)2 and LAM indicating that the spacer chain length plays an important role in hemolysis. In
other words, the erythrocyte-disrupting ability of gemini with short spacers is considerably higher
than that of LAM while the gemini with longer spacer chains are rather less hemolytic than LAM.
This behavior seems to be related to the morphology and size of the aggregates formed by every
surfactant. As it has been described, pure LAM and C6(LA)2 form mainly spherical micelles while
C9(LA)2 and C12(LA)2 form viscous solutions that contain big aggregates like helicoidal or wormlike micelles. The reasons for this might be related to the conformation of the surfactant in aqueous
media. In fact the high hydrophobicity and flexibility of the spacer in C9(LA)2 and C12(LA)2 allows
the spacer chain to penetrate into the aggregated core, reducing the distance between the polar head
groups and forming a compact molecule with low surface area. This particular conformation, due to
the length of the spacer, can certainly influence the size of aggregates. In this way, C9 and C12 are
able to form closely packed structures at lower concentrations than the C6 homologue. 27 These
results indicate that big aggregates produce less hemolysis.
Another noteworthy result is that for C9(LA)2 and C12(LA)2 in which the HC50 depends on
the surfactant concentration in the initial stock solution used to determine the hemolytic activity.
For example, for the C9(LA)2, it can be observed that when the stock solution used is the C9 1 ( 4
mM), the HC50 is 200 M while for the C9-2 stock solution (2.5 mM), the HC50 is slightly lower
(170 M). This has also been observed in the case of gemini/additive formulations. Finally, it is
worth pointing out that addition of cholesterol does not affect the hemolytic activity of these two
surfactants while the adition of DLPC increases their hemolytic behavior.
All these results can be related with the aggregate’s size obtained by DLS and confirm that
hemolysis increases when the aggregate´s size decreases. In fact, it has been observed that in all
cases the solutions containing most gemini (C9 1, C9/CHOL 1 and C9/DLPC 1) have bigger
aggregates. For example, the C9 1 solutions contain aggregates with dH around 800 nm while the
C9 2 solution contains aggregates of about 200 nm.
A relevant difference in terms of HC50 values was found when phospholipids were added.
The presence of 20% DLPC gave an HC50 value of 87 M and the increase of additive up to 50%
gave polidispersed small aggregates with an HC50 value smaller than 20 M. Notice that the
16
solution containing phospholipids form vesicles with lower dH than the aggregates observed in pure
gemini or gemini/cholesterol formulations.
Figure 8..HC50 of C9 and C9 based formulations (mean±S.E. of three replicates)
Figure 9. HC50 of C12 and C12 based formulations (mean±S.E. of three replicates)
According to the bilayer couple hypothesis, amphiphiles induce shape alterations in erythrocytes by
being asymmetrically distributed between the bilayer leaflets, thereby expanding one leaflet relative
to the other. This transbilayer movement from the outer to the inner leaflet of membrane is easier
for single or gemini amphiphiles with a shorter alkyl chain, because of their small dimensions,
while amphiphiles with longer spacer are readily intercalated into the outer membrane leaflet, but
can not translocate to the inner membrane because of their limited mobility.55
Some studies have reported that the hemolytic activity is higher for the compounds with
more hydrophobic content48 but in some cases this trend is not followed. Fogt et al56 and Lv et al18,
for example, reported that traditionally for aliphatic chains, single-tailed cationic lipids are more
toxic and less efficient than their double-tailed counterparts. Pinnaduwage et al57 reported that cetyl
17
trimethylammonium bromide (CTAB) was more toxic and less efficient than DOTMA. Moreover,
the cytotoxicity of gemini cationic surfactants could be related to the length of the spacer, for
exemple Floch et al reported that an increase in the length of the linker segment led to decreased
toxicity in cell culture.58 The effect of hydrophobic domains on toxicity has not been adequately
addressed to date and many scientists have been trying to give a proper explanation; however, there
is still a long way to go.
Figures 7, 8 and 9 indicate that the hemolytic activity of these surfactants showed the
following trend: C12<C9<LAM<C6. These data confirm that it is difficult to predict the hemolytic
activity of surfactants and a lot of variables need to be considered. From the results obtained in this
work, it can be stated that aggregate size and morphology play an important role in the hemolytic
behaviour of these gemini surfactants, the bigger the aggregate’s size, the smaller the hemolytic
character of the solutions. It can also be concluded that it is essential to accurately characterize
colloidal formulations in order to obtain data that allow correlating results with toxic effects.
It is also worth mentioning that the hemolytic activity of LAM is lower than that of classical
monoQuat surfactants with comparable CMC values and the hemolytic character of C 9(LA)2 and
C12(LA)2 is much lower than that of bis(Quats) gemini surfactants53 or gemini surfactants based on
pyridinium salts19.
Antimicrobial activity
The common antimicrobial agents are extremely irritant and lethal and it is necessary to
formulate new types of safe and cost-effective biocidal materials. In this light, cationic surfactants
could be good alternatives to traditional antimicrobial drugs because of the possibility to combine
their biological properties with their surface features. It has been reported that the formulation of
mixtures of cationic lipids, like DODAB, with other neutral lipids could influence the bacterial
membrane lipids, modifying the final antibacterial activity.59 In fact, many studies have shown that
antimicrobial formulations in the form of vesicles could be successfully used as potential
bactericidal material or in combination with hydrophobic drugs, adequately conjugated.60
The arginine-based surfactants appear as good candidates for the preparation of
pharmaceutical formulations, thanks to their physico-chemical and biological properties.61 Within
the monocatenary surfactants, the most effective against gram positive and gram negative bacteria is
LAM, a homologue with 12 carbon atoms in the alkyl chain. The antimicrobial activity of pure
Cn(LA)2 surfactants depends on the spacer chain, compounds with short spacers appear to improve
the antimicrobial activity of their single chain homologue LAM while the antibacterial potency
decreases for longer spacer chains (C9(LA)2 or C10(LA)2).62 The hemolytic studies carried out in
18
this work suggest that the interaction with biological membranes depends not only on the
hydrophobic character of the molecules but also on the type and size of aggregates present at the
solution. To confirm this behaviour, the antimicrobial activity was evaluated. Taking into account
that the most anomalous results have been obtained for the longer spacer chains (C9(LA)2 and
C12(LA)2) the antimicrobial activity has been determined for the C9(LA)2 based formulations.
The antimicrobial activity of the formulations was determined in vitro on the basis of the
MIC values, 63 defined as the lowest concentration of antimicrobial agent that inhibits the
development of visible growth after 24 h of incubation at 37 °C. Stock of pure surfactant solutions
of C9(LA)2, and mixed surfactant/additive suspensions at two different concentrations, were
prepared as previously reported for hemolysis experiments, and then diluted in the concentration
range of 0.1-256 g/mL. The following bacteria were used: Gram positive bacteria: Bacillus
subtilis ATCC 663, Staphylococcus epidermidis ATCC 12228, Micrococcus luteus ATCC 9341;
Gram negative bacteria: Escherichia coli ATCC 8739; yeast: Candida albicans ATCC 10213.
The MIC values are shown in Table 3.
Table 3. Minimum inhibitory concentration (MIC) of formulations based on C 9(LA)2 surfactant. For the
surfactant/additive formulation the MIC value on the table indicates C 9(LA)2 amount.
Escherichia coli
ATCC 8739
Bacillus subtilis
ATCC 6633
Staphylococcus epidermidis
ATCC 12228
Micrococcus luteus
ATCC 9341
Candida albicans
ATCC 10231
C9 1
g/mL
C9 2
g/mL
C9/CHOL 1
g/mL
C9/CHOL 2
g/mL
C9/DLPC 1
g/mL
C9/DLPC 2
g/mL
>180
>112
>180
>112
>180
56
>180
>112
180
>112
>180
28
90
56
45
28
>180
28
90
56
45
28
>180
28
>180
112
90
>112
>180
56
The results of the antimicrobial tests for all the formulations are reported in Table 3. Usually
the Gram-negative bacteria were more resistant than the Gram-positive to cationic surfactants.64 As
expected, our systems showed low activity against Gram-negative bacteria, only the C9/DLPC 2
formulation was active against Escherichia coli. Gram-negative bacteria exhibit a complex structure
consisting of two bilayers, the outer membrane and the inner or cytoplasmic membrane. The outer
membrane has a highly asymmetric composition, formed by a polysaccharide layer located in the
outer leaflet and, to a large extent, by the zwitterionic phosphatidylethanolamine (PE) in the inner
one. The cytoplasmic membrane consists to a large extent of PE and to negatively charged
19
phosphatidylglycerol (PG). In contrast, Gram-positive bacteria only have a cytoplasmic membrane
that consists to a large extent of PG.
Large differences in the MIC values of these formulations have been found; this means that
the additive plays an important role in the antibacterial activity. The higher activity was obtained for
the C9/DLPC 2 sample, this solution is the only active against all microorganisms tested. This high
activity confirms the hemolysis results, in fact, as mentioned before, it has not been possible to
evaluate the EC50 of this formulation, due to its relevant toxicity against erythrocytes. It was
reported that mixtures of more than one surfactant (either similar or different types) provide many
advantages either in the physical or chemical properties over the use of a single surfactant.42
Mixtures of alkyltrimethylammonium bromide surfactants/sodium alkyl methyl ester sulfonate or
DODAB/DPPC at equimolar mixing ratio (5:5) have also the highest potency against bacteria.65
This could be explained by the close packing of the mixed surfactant molecules at this mixing ratio,
which was associated with high hydrophobicity, high adsorption tendency at the interface, smaller
aggregate size and hence with disruption of the bacterial membrane.66
In general, for these formulations, the antimicrobial activity as well as the interaction with
the erythrocyte’s membranes follows similar trends: a) the most active formulation is C9/DLPC 2, b)
C9/Chol samples are slightly more active that the corresponding C9 pure solutions and c) in all
cases the antimicrobial activity depends on the C9 concentration of the stock solution used to
determine the MIC, being the most active those with less quantity of surfactant (For example, the
MIC values of C9 1 are superior than those of C9 2).
In general, antimicrobial activity is attributed largely to the net hydrophobic character and
positive surfactant charge. Nevertheless, in all formulations, the active compound is the same
C9(LA)2, and the zeta-potential values are comparable. This suggests that as previously described,
the size of the aggregates in the solution strongly affects the interaction of this compound with
bacteria. These studies indicate that the antimicrobial activity increases as the aggregate’s size
decreases. The molecular details of the mechanism by which cationic liposomes interact with
biological membranes are still poorly understood. It has been reported that micelle-forming
quaternary ammonium surfactants react with phospholipids components in the membrane producing
membrane distortion and lytic effects.67 The mechanism of vesicles formed with bilayer forming
lipids as DODAB seems to be different. In this case the bacterial action possibly involves damage
to protein function at the bacterial external wall level, where vesicles do adhere without vesicle
rupture or cell lysis.68 Adsorption of DODAB cationic aggregates onto bacterial cells changes the
sign of the cell surface potential from negative to positive with a clear relationship between positive
charge on bacteria cells and cell death. It has been also reported that size, charge and transition
20
temperature (Tm) 69 of liposomes control the interaction with cellular membranes. Usually, for a
given concentration, smaller aggregates imply more surface area, thus, greater activity. Rejman et
al.70 also showed that particle size might be an important parameter in the pathway of entry in cells,
moreover the mechanism by which the particles were internalized was strongly dependent on
particle size. These authors found that cells took up particles up to size of 500 nm and no uptake
was seen of particles 1 μm in size. Moreover from 50-500 nm the internalization decreases as the
size increases. The experimental procedures carried out in this work do not allow us to establish the
mechanism of interaction. It can be speculated that in these systems several of the mechanism above
described are present. Notice that in our formulations, prepared with micelle-forming surfactants,
there are monomers, micelles and liposomes of very different size.
These data agree with those obtained by Carmona-Ribeiro et al14 who studied the
antimicrobial and hemolytic activity of DODAB bilayer fragments (dH=79 nm and =41 mV) and
DODAB large vesicles ( dH=500-800 nm and =48 mV) and found that small aggregates penetrate
more deeply on the cell surface and consequently have lower MIC and HC50 values. However,
different results have been obtained for small and large particles from cationic lipids and
polyelectrolytes. In this case, antimicrobial effect was dependent on the amount of positive charge
on the particles and independent of particle size.71
From these results it can be stated that the addition of additives modifies the size of
surfactant aggregates and this modification affects the interaction of these surfactants with
biological membranes. Moreover, results show that different variables have to be considered for
antimicrobial activity and hemolytic behaviour.
It is noteworthy that using lipid/cationic gemini surfactant mixtures, formulations with low
hemolytic character and moderate antimicrobial activity were obtained. Notice that the gemini
cationic surfactants used are easily biodegradable compounds that can be prepared from renewable
materials using sustainable procedures.21 Moreover, these formulations show antimicrobial activity
at lower concentrations than those in which they show erythrocyte toxicity, so that they can be
considered safe formulations for biological applications.
MATERIALS AND METHODS
Materials
Chemicals and reagents were supplied by Sigma Aldrich and used without further purifications. LDilauroylphosphatidylcholine (DLPC) and cholesterol (Chol) were obtained from Sigma Chemical
and used as received. Pure Millipore water from a Milli-Q four-bowl system was used in preparing
all sample solutions. Mueller-Hinton broth (MHB) was purchased from Difco Laboratories (USA).
21
The Nlauroyl arginine methyl ester (LAM) as well as the arginine-based gemini surfactants
(CnLA2) were synthesized according to the procedure described previously
72
and their
physicochemical properties are shown in Table 4. Gemini surfactants are made up of two
symmetrical long chain N-lauroylarginine residues of 12 carbon atoms linked by amide covalent
bonds to an alkylenediamine spacer chain of six, nine or twelve methylene groups (Figure 10). They
were obtained with a purity of 99 % by chemical condensation of the single N-lauroyl-L-arginine
previously protected, to the corresponding diamine in presence of an activating agent. A final
deprotection reaction was carried out to obtain the Gemini compounds. More details on the
synthetic procedures, purification, NMR and HPLC characterization are given elsewhere72. LAM
was obtained by a two step procedure: a) methylation of the acid group of the commercial Larginine and b) condensation of the lauryc acid to the amino group of the arginine methyl ester
using an activating agent. More details on the synthesis are reported in the literature.62
Figure 10. Molecular structure of a) Cn(LA)2 and b) LAM.
Elemental analysis of LAM and Cn(LA)2:
Lauroyl arginine methyl ester, LAM. Found: C, 55.58; H, 9.45, N, 13.77. Anal. Calcd for
C19H39N4O3Cl : C, 56.07; H, 9.66; N, 13.77
N, N-bis(N-dodecylarginine) ,-hexyl-diamide, C6(LA)2: Found: C, 58.03; H, 10.63; N, 16.12;
Cal. for C42H86N10O4Cl2: C, 58.33; H, 10.28; N, 16.20 .
22
N, N-bis(N-dodecylarginine) ,-nonyl-diamide, C9(LA)2. Found: C, 59.33; H, 10.33; N, 15.22.
Anal. Calcd for C45H92N10O4Cl2, C, 59.60; H, 10.15; N 15.50
N, N-bis(N-dodecylarginine) ,-dodecyl-diamide, C12(LA)2. Found: C, 60.32; N, 14.57; H,
10.42. Anal. Calcd for C48H98Cl2N10O4: C, 60.67; N, 14.74; H, 10.39.
Table 4. Physicochemical properties of LAM and (Cn(LA)2)
CMC*
Number of
Alkyl chain
alkyl chains
length
Cationic
1
C12
-
2.439
836.6
Cationic
2
C12
C6
0.460
C9(LA)2
906.7
Cationic
2
C12
C9
0.345
C12(LA)2
948.6
Cationic
2
C12
C12
0.163
Surfactant
MW
Charge
LAM
406.6
C6(LA)2
Spacer
M
* From reference nº 25
Sample preparation
Pure cationic surfactant solutions were prepared weighting a defined number of moles (8x10 -6 or
5x10-6) of every compound. Then 2 mL of Milliq water was added and the solution was gently
stirred for 5 minutes at room temperature.
Mixed cholesterol or phospholipid/cationic surfactant formulations were prepared using the
sonication method. Vesicles were prepared starting from different molar ratios between surfactant
and additive as reported in Table 1. Then, 2 mL of Milliq water was added and each sample was
strongly stirred for 5 minutes and sonicated for 15 minutes at 50 °C. After preparation, the
dispersion was left to equilibrate at room temperature overnight before use.
Zeta-potential and Size distribution analysis
The Z-potential of the formulations was measured with the laser Doppler electrophoretic mobility
measurements using the Zetasizer 2000 (Malvern Instruments Ltd., Malvern, U.K.), at 25°C. All
analysis was done in triplicate. Z-potential values and standard deviations (±S.D.) were elaborated
directly from the instrument.
The Dynamic Light Scattering technique (DLS) was used to measure the size distribution of
all formulations. The DLS unit determining vesicle size is a Malvern Zeta Nanosizer, working at
632.8 nm and 25ºC. The DLS measurements, started 5-10 min after the sample solution, were
placed in the DLS optical system to allow the sample to equilibrate at room temperature. The
23
scattering intensity was measured at a 173º angle to the incident beam. This is known as backscatter
detection and allows measurements in turbid dispersions, minimizing multiple scattering effects.
Depending on the system turbidity, the unit automatically determines the sample thinckness under
investigation and focuses the beam at a given position from the cell walls. At least 10 runs were
performed for each sample. The viscosity value (0.8872 mPa s) and the refractive index (1.33) of
water were used for all the measurements. The correlation function obtained for monomodal
distributions is different that those obtained for the multimodal distribution. After the correlation
function has been measured this information is used to calculate the size distribution. The software
supplied by the manufacturer (Zetasizer v6.20) automatically determines the optimum parameters
for the CONTIN73 algorithm for monomodal as well as multimodal distributions in order to produce
a size distribution profile.
The time decay of the autocorrelation function of the concentration fluctuations has a
characteristic decay rate , Γ, which is proportional to q2(Γ=Dq2) that characterizes a translational
diffusion with the mutual diffusion constant, D. The scattering vestor, q, is given by the equation:
Q=4πn/λ0sin(θ/2)
Where n is the refractive index of the solvent and θ is the scatter angle. The measured translational
diffusion constant was related to the average hydrodynamic radius, Rh, of the particles through the
equation:
D=kT/6πηRh
Were k is the Boltzmann constant, η the solvent viscosity, and T the absolute temperature.
Preparation of erythrocyte suspensions
Fresh blood was taken from rats and used the same day of the experiment. The procedure was
approved by the institutional ethics committee on animal experimentation. The erythrocytes were
washed three times in a phosphate buffer solution (PBS) containing 123.3 mM NaCl, 22.2 mM
Na2HPO4 and 5.6 mM KH2PO4 in distilled water (pH 7.4). The cells were then suspended at a cell
density of 8x109cell/mL.
Hemolysis assay
Hemolysis is the breakdown of red blood cells (erythrocytes) and the release of the hemoglobin,
occurring in the living organism or in a blood sample. To determine the hemolytic capacity of the
24
colloidal systems prepared in this work we followed an adaptation of the method described by Pape et
al. (1987).74
A series of different volumes of the samples (1-10 mg.mL-1), ranging from 10 to 80 μL, were
placed in polystyrene tubes containing 25 μL of erythrocyte suspension and phosphate buffered
saline were added to each tube, to a total volume of 1 mL. Samples were incubated at room
temperature while shaking for 10 minutes. Following incubation, the tubes were centrifuged (5 min
at 10000 rpm). The percentage of hemolysis was determined by comparing the absorbance (540 nm)
of the supernatant of the samples with that of the control totally hemolysed with distilled water.
Concentration-response curves were determined from hemolysis results (Figure 11), and
the
Haemolysis %
concentration inducing 50% hemolisys (HC50) were calculated from these curves.
100
90
80
70
60
50
40
30
20
10
0
1
10
100
1000
Surfactant concentration (g/ml)
Fig. 11 Haemolysis versus logarithm of concentration C, for LAM/CHOL 1 sample.
Antimicrobial activity
Antimicrobial activity was determined in vitro on the basis of MIC values, defined as the lowest
concentration of antimicrobial agent that inhibits the development of visible growth after 24 h of
incubation at 37°C. 75
The compounds tested were dissolved in MHB in a concentration range of 0.1-256 μg/mL,
and no precipitate was observed at the highest concentration of the surfactant. The MHB was
prepared according to the manufacturer’s instructions. Then 10 μL of a nutrient broth starter culture
of each bacterial strain was added to achieve final inoculums of ca. 5 10-4-510-5 CFU mL-1. The
cultures were incubated overnight at 37°C. Nutrient broth medium without the compound served as
control. Microorganism growth was determined visually after incubation for 24 h at 37°C compared
with an inoculate culture without antimicrobial compound. The development of turbidity in an
inoculated medium is a function of growth. A rise in turbidity reflects increase in both mass and cell
25
number. The lowest concentration of antimicrobial agent at which no visible turbidity was observed
was taken as the MIC.
CONCLUSIONS
Stable colloidal formulations based on arginine-single chain or gemini surfactants were prepared.
The size of the aggregates depends on the molecular architecture of the surfactants, the
concentration and the additives added to the formulation: aggregates of different dimensions and
stability can be obtained starting from pure surfactant solutions or surfactant/additive mixtures.
Single chain surfactants and Gemini with short spacer chains give rise to solutions with micellar
aggregates while Gemini with long spacers form big aggregates as twisted ribbons.
Antimicrobial and hemolytic activity is strongly affected by the size of the aggregates; small
aggregates penetrate more deeply into the cell surface and consequently have lower MIC and HC50
values. The length of the spacer chain modulates the aggregation type and consequently the
haemolytic and antimicrobial activities. Gemini with long spacer chains give rise to big aggregates
and have more difficulties to interact with biological membranes.
The introduction of additives such as cholesterol or DLPC also affects the biological
properties of these surfactants. Moreover, the concentration of the stock solution used to carry out
the biological tests is another important factor to take into account.
Several factors can be used to modulate the biological activity of these cationic surfactants.
In fact, cationic colloidal solutions (mixtures of C9/CHOL 1) have been obtained in this work that
are effective antimicrobial solutions with very low haemolytic character.
The results of this work suggest new possible pharmaceutical devices based on arginine
surfactants as a viable alternative to the classical formulations, showing good stability, low
hemolytic effects and also a natural antimicrobial activity, which is not provided by conventional
vesicles.
ACKNOWLEDGEMENTS
The authors would like to thank the financial support from Spanish Plan National I+D+I CTQ200914151-C02-01, CTQ2009-14151-C02-02, AGAUR 2009 SRG 246, CTQ2010-14897, CTQ201021183C02-01 and Unidad Asociada “Interacción de tensioactivos con membranes celulares”.
Moreover, the project has been co-funded with support from the Commission European Social Fund
and Region of Calabria (Italy) and the contract Estancia de Jóvenes Doctores Extranjeros en España,
MEC, SB2010-0129.
26
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