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, Universitatde Barcelona, Joan XXIII s/n, 08028 Barcelona, Spain.

fUnidad Asociada al CSIC

gDepartment de Microbiologia, Universitatde Barcelona, Joan XXIII s/n, 08028 Barcelona, Spain.

*Corresponding author:

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.

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.

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] latex[6] and mineral surfaces[7]. 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 aminoacid arginine, with different structures (monocatenary, gemini, and glycerolipid), characterized by relevant nontoxic and antimicrobial properties as well as 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 arginine-based 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 ofliposome 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.

Table 1. Details on sample preparations (n= number of moles).

Sample Number
and Label / Concentration
(mM) / Surfactant
Amount (n) / Additive
Amount (n) / Mole Ratio
Surf/Add
LAM 1 / 4 / 8*10-6 / - / 1:0
LAM 2 / 2.5 / 5*10-6 / - / 1:0
LAM/CHOL 1 / 5 / 8*10-6 / 2*10-6 / 8:2
LAM/CHOL 2 / 5 / 5*10-6 / 5*10-6 / 5:5
LAM/DLPC 1 / 5 / 8*10-6 / 2*10-6 / 8:2
LAM/DLPC 2 / 5 / 5*10-6 / 5*10-6 / 5:5
C6 1 / 4 / 8*10-6 / - / 1:0
C6 2 / 2.5 / 5*10-6 / - / 1:0
C6/CHOL 1 / 5 / 8*10-6 / 2*10-6 / 8:2
C6/CHOL 2 / 5 / 5*10-6 / 5*10-6 / 5:5
C6/DLPC 1 / 5 / 8*10-6 / 2*10-6 / 8:2
C6/DLPC 2 / 5 / 5*10-6 / 5*10-6 / 5:5
C9 1 / 4 / 8*10-6 / - / 1:0
C9 2 / 2.5 / 5*10-6 / - / 1:0
C9/CHOL 1 / 5 / 8*10-6 / 2*10-6 / 8:2
C9/CHOL 2 / 5 / 5*10-6 / 5*10-6 / 5:5
C9/DLPC 1 / 5 / 8*10-6 / 2*10-6 / 8:2
C9/DLPC 2 / 5 / 5*10-6 / 5*10-6 / 5:5
C12 1 / 4 / 8*10-6 / - / 1:0
C12 2 / 2.5 / 5*10-6 / - / 1:0
C12/CHOL 1 / 5 / 8*10-6 / 2*10-6 / 8:2
C12/CHOL 2 / 5 / 5*10-6 / 5*10-6 / 5:5
C12/DLPC 1 / 5 / 8*10-6 / 2*10-6 / 8:2
C12/DLPC 2 / 5 / 5*10-6 / 5*10-6 / 5:5

(C6: C6(LA)2; C9: C9(LA)2; C12: C12(LA)2)

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 1and LAM 2, b) LAM/CHOL 1 and LAM/CHOL 2, c) LAM/DLPC 1 and LAM/DLPC 2.

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.

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]

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 extraction[38] 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).

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, 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).

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.27Although 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.

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 C9(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 aminoacid 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 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/additivecomposition, but no important differences in the aggregate’s size and distributionhave 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].