Influence of Cationic Phosphatidylcholine Derivative on Monolayer

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Biological and Environmental Phenomena at the Interface

The influence of cationic phosphatidylcholine derivative on monolayer and bilayer artificial bacterial membranes Marzena Mach, Karolina W#der, Katarzyna H#c-Wydro, Micha# Flasi#ski, Joanna Lewandowska-Lancucka, Kinga Wójcik, and Pawel Wydro Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b04262 • Publication Date (Web): 13 Apr 2018 Downloaded from http://pubs.acs.org on April 14, 2018

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The influence of cationic phosphatidylcholine derivative on monolayer and bilayer artificial bacterial membranes. Marzena Mach1,Karolina Węder1, Katarzyna Hąc-Wydro2, Michał Flasiński2, Joanna Lewandowska-Łańcucka1, Kinga Wójcik3, Paweł Wydro1, *

1

Department of Physical Chemistry and Electrochemistry, Faculty of Chemistry, Jagiellonian University, Gronostajowa 2, 30-387, Kraków, Poland

2

Department of Environmental Chemistry, Faculty of Chemistry, Jagiellonian University, Gronostajowa 2, 30-387, Kraków, Poland

3

Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Kraków, Poland

Corresponding author (P. Wydro) e-mail: [email protected] Phone: +48 (12)686 25 19

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Abstract An increasing number of bacterial infections and the rise in antibiotic resistance of a number of bacteria speciesforces to search for new antibacterial compounds. The latter facts motivate the investigations presented herein and aimed at studying the influence of a cationic lipid:1palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (EPOPC) on model (mono,- and bilayer) membranes. The monolayer experiments involved the analysis of the interactions of EPOPC with bacterial membrane lipids in one component and mixed systems as well as Brewster Angle Microcopy studies. Theproperties of liposomes were analyzed based on the results of dynamic light scattering (DLS) and zeta potential measurements as well as on the experiments concerningthe release of calcein entrapped in liposomes after titration with surfactant solution and steady-state fluorescence anisotropy of DPH. The obtained results evidenced that EPOPC, even at low concentrations, strongly changes organization of model systems making them less condensed. Moreover, EPOPC decreases the hydrodynamic diameter of liposomes, increases their zeta potential and destabilizes model membranes increasing their fluidity and permeability. Also, the in vitro tests performed on Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive) strains prove that EPOPC has some bacteriostatic properties which seem to be stronger towards Gram-negative than Gram-positive bacteria. All these findings allow one to conclude that EPOPC mode of action may be directly connected with the interactions of EPOPC molecules with bacterial membranes.

Keywords: model bacterial membranes, Langmuir monolayers, liposomes, membrane fluidity, Brewster angle microscopy


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Introduction Pathogenic bacteria may cause serious infections in theliving organism, which, untreatedareharmful for human health or even life. To cure these bacterial infections various substances called antimicrobial agents are used. These compoundscan be classified according to their mode of action, chemical nature, andsource or spectrum of activity [1]. Considering the mechanism of action of antibacterial agents various cellular sites or cellular processes may be a target for these compounds. For example, they may affect cell wall, protein or nucleic acid synthesis or cause disorder at the level of cytoplasmic membrane [2]. The modifications make by antibacterial agents in the membranes architecture lead to the increase of the bilayer permeability and finally to its disruption. This kind of mode of action was confirmed for example for various antibacterial agents carrying a positive charge [2,3]. This is the consequence of the composition and organization of a pathogen membrane, since, in bacterial membranes anionic lipids are present in a significant amount in the outer layer of the membrane. Thus they are able to easily interact with cationic drugs [4]. One of the most serious problems in antibacterial therapy is the rise in antibiotic resistance of a number of bacteria species. This is the driving force in searching for the new compounds, which may serve as effective antimicrobial agents. It seems that due to lipid-like nature and cationic charge a cationic phosphatidylcholine derivatives may have potential to be effective antibacterial drug. It can be supposed that these compounds, like other cationic amphiphiles [5,6], are able to interact with pathogen membrane and in this manner alter the lipids organization and/or change the membrane net charge, which may finally lead to the membrane damage. The compound investigated herein, namely, 1-palmitoyl-2-oleoyl-snglycero-3-ethylphosphocholine

(EPOPC)

belongs

to

the

group

of

triesters

of

phosphatidylcholine (P-O-ethylphosphatidylcholine; EPCs) and it can be obtained from diacyl-phosphatidylcholines(PCs) by phosphate ethylation of the polar headgroup. It was evidenced that similarly to natural phosphatidylcholines also EPCs disperse readily in water forming vesicles, which are able to easily fuse with erythrocytes and anionic liposomes [7-9]. ACS Paragon Plus Environment

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EPCs have stronger surface activity and have higher surface potential than their parent compounds [10]. Moreover, as they are derived from natural phosphatidylcholines, they are well metabolized and exhibit reduced toxicity in cell cultures of mammals as compared to the other cationic surfactants, [8,12]. Since these compounds and their mixtures were found to be very effective in lipofection, the cationic triesters of phosphatidylcholine seem to be very attractive agents in various scientific and biomedical applications, for example to deliver genetic material (gene therapy) or drugs (drug delivery) to tissue or cells [8,10-12]. Antibacterial activity of these compounds has not been studied so far, however, it can be supposed that, by analogy to other cationic amphiphiles, they also can be considered as antimicrobial agents. Thus, antibacterial therapy seems to be novel promising area of potential application of these compounds. However, the effectivity of these substances as antibacterial agents as well as their mechanism of antimicrobial action require verification and systematic studies. A good and widely applied method to get first insight into the effect of various active molecules on a membrane is to perform the experiments in artificial, mono - or bilayer, membrane systems. Despite some limitations of model membranes and obviously their much simple composition as compared to natural systems they are useful in the studies of the properties of various membrane active compounds [13-16]. The model membranes, both bilayers and monolayers were applied also in the investigations of the agents having antibacterial activity [17,18]. We applied this approach in the studies presented herein. The aim of this work was to investigate the interactions of one of triesters of phosphatidylcholine derivative, that is 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (EPOPC) with the lipids representative for bacterial membranes, namely phospahtidylethanolamine - PE; and phosphatidylglycerol PG) as well as with the mixed PE/PG system being a widely used model of bacterial membrane. The influence of EPOPC on the lipids was studied in Langmuir monolayers and liposomes. Based on these investigations the modifications induced by EPOPC in artificial lipid membranes were analyzed. Moreover to verify the antibacterial


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activity of EPOPC the in vitro tests on Escherichia coli and Staphylococcus aureus strains were performed.

Experimental Materials 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-snglycero-3-phospho-(1'-rac-glycerol) (sodium salt) (POPG), 1-palmitoyl-2-oleoyl-sn-glycero3-ethylphosphocholine (chloride salt) (EPOPC) were synthetic products of high purity (≥99%) purchased from Avanti Polar Lipids, Inc. 1,6-Diphenyl-1,3,5-hexatriene (DPH), Bis[N,N−bis(carboxymethyl)aminomethyl]

fluorescein

(calcein)

4-(1,1,3,3-

Tetramethylbutyl)phenyl-polyethylene glycol (Triton X-100) as well as solvents (chloroform and methanol, both HPLC grade, ≥99.9%)were provided by Sigma-Aldrich. Methods Langmuir monolayer technique To prepare spreading solutions the investigated lipids were dissolved in chloroform/methanol (9:1 v/v)mixture. Mixed solutions of desirable compositions were prepared from the respective stock solutions and deposited onto water subphase with the Hamilton micro syringe, precise to 1.0 µL. After spreading, the monolayers were left to equilibrate for 10 min before the compression was initiated. Since practically no influence of the compression velocity (within the range of 5-30 cm2/min) was found for the investigated compounds, in all the experiments monolayers were compressed with the barrier speed of 20 cm2/min. Π-A isotherms were recorded with a NIMA (U.K.) Langmuir trough (total area = 300 cm2) equipped with one movable barrier, placed on an anti-vibration table. Surface pressure was measured with the accuracy of ± 0.1 mN/m using a Wilhelmy plate made of filter paper (ashless Whatman Chr1) connected to an electrobalance. The subphase temperature (20°C) was controlled thermostatically to within 0.1 °C by a circulating water system. All


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experiments were repeated at least twice to ensure consistent results. In all experiments MilliQ water (resistivity of 18.2 MΩ⋅cm) was used. Brewster Angle Microscopy (BAM) The visualization of Langmuir monolayersduring their compression was performed with ultraBAM instrument (Accurion GmbH, Goettingen, Germany) equipped with a 50 mW laser emitting p-polarized light at a wavelength of 658 nm, a 10x magnification objective, polarizer, analyzer and a CCD camera. The spatial resolution of the BAM was 2 µm. Liposomes preparation Liposome suspensions were prepared by hydrating dry lipid film. Lipid films of required composition were obtained by mixing appropriate volumes of stock lipid solutions and evaporation of the solvent under a gentle stream of nitrogen. Then lipid films were hydrated in phosphate buffer (pH 7.4). The final concentration of lipids in all suspensions was 1,4 µmol/mL. The obtained multilamellar vesicle dispersions were subjected to five freeze–thaw cycles from liquid nitrogentemperature to 60°C and then extruded six times (12 passes) throughthe polycarbonate membrane filters with 100-nm pores using a Liposofast extruder (Avestin, Ottawa, Canada) Hydrodynamic diameter and zeta potential of liposomes. The measurements of hydrodynamic diameter and zeta potential of vesicles were performed at 20 °C with Malvern Nano ZS light-scattering apparatus (Malvern Instrument Ltd., Worcestershire, UK). The z-averaged hydrodynamic mean diameters (dz), dispersity (DI) and distribution profiles of the samples were calculated using the software provided by Malvern. The zeta potential of liposomes was measured using the technique of laser Doppler velocimetry. Measurements were repeated three times for each sample. Transmission Electron Microscopy (TEM) TEM observations were performed on a JEOL, JEM 2100 transmission electron microscope (Japan) at an accelerating voltage of 200 kV. A negative staining technique was utilized to visualize the liposomes. A 200 mesh copper grid coated with Formvar/carbon film (Pacific ACS Paragon Plus Environment

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Grid-Tech) was dipped in the sample dispersion and left for about 15 min. The excess of the sample was blotted with filter paper and the samples were subsequently stained with a 1% solution of uranyl acetate in water and then allowed to dry. Steady-state fluorescence anisotropy Liposomes with the fluorescence probe embedded into membrane were obtained by the addition of DMF solution of 1,6-Diphenyl-1,3,5-hexatriene (DPH) to the liposome suspensions [19-21]. The final concentration of DPH was 0.14 µM. Then the suspensions was incubated about 60 min at room temperature with gentle stirring. The fluorescence intensity of samples was measured with spectrofluorimeter (Hitachi, F-7100) to which excitation and analyzing polarizerswere attached. The samples were excited with vertically polarized light (350 nm), and the emissionintensities at 428 nm both parallel (IVV) and perpendicular (IVH) tothe excited light were recorded. The fluorescence anisotropy (r) value of DPH wascalculated from the following equation: =

− + 2

wherein = ⁄ is the correction factor (instrumental function). Calcein-release measurement Liposomes with entrapped calcein were prepared similarly as described earlier in (section Liposomes preparation), however, hydration of thin lipid films were performed with 0.06 M solution of calcein (pH = 7.4). Calcein molecules which were unclosed in liposomes were removed from calcein-loaded liposomes by size-exclusion chromatography on a Sephadex G50 column using PBS buffer as an eluent. As expected, the fluorescence intensity of the suspensions of liposomes with entrapped calcein was very low due to the self-quenching effect. Then the suspensions of liposomes weresubjectedtothetitration with 1.2% solution of Triton-X100 and the changes in fluorescence intensity resulting from calcein release from the vesicles after addition of each portion ofsurfactant was measured with Hitachi F-7100 spectrofluorimeter. Excitation and emission wavelengths were set at 490 and 520 nm, respectively. ACS Paragon Plus Environment

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Determination of the minimum inhibitory concentration (MIC) of EPOPC liposome suspension Minimum inhibitory concentration (MIC) were determined by the microdilution method [22]. Overnight cultures of Escherichia coli ATCC 25922 (Gram-negative bacteria) and Staphylococcus aureus ATCC 6538 (Gram-positive bacteria) in Mueller Hinton II Broth (BD BBL™) (MH II broth) were diluted 10-fold and incubated at 37°C with shaking to log phase of growth. A series dilutions of EPOPC liposome suspension in distilled water (100 µl) were prepared in a 96-well microtiter plate and 100 µl of bacteria suspension in 2 x MHII broth (7.5 × 106 colony forming units (CFU)/mL, confirmed by viable counts) was added into each well, except the broth sterility and EPOPC sterility control column. Microdilution was prepared in duplicates for both bacterial species. The plate was incubated at 37°C with shaking for 18 h and the growth of the bacteria was monitoring turbidimetrically (optical density at 600 nm). Bacterial viability assay Diluted bacterial suspension in 2 x MH II broth (9 × 104 CFU/mL - E. coli and 2,7 × 105 CFU/mL – S. aureus) prepared as above was added to the same volume of EPOPC liposome suspension in distilled water (1.14 and 2.29 mg/mL - final concentration) in a 96-well plate. Wells with no EPOPC were used as positive growth controls. The all mixtures were performed in duplicates. The plate was incubated for 4 h at 37°C and viable counts were determined from each mixtures.


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Resultsand Discussion 1. Interactions of EPOPC with POPE and POPG in Langmuir monolayers In Fig. 1 a-b the surface pressure area curves recorded for one component lipid monolayers and binary POPE/EPOPC and POPG/EPOPC are shown.

π, mN/m

60 POPE xEPOPC=0.1 xEPOPC=0.3 xEPOPC=0.5 xEPOPC=0.7 xEPOPC=0.9 EPOPC

40

20

0 35

70

105

140

175

A, Å2/molecule

π, mN/m

60 POPG xEPOPC=0.1 xEPOPC=0.3 xEPOPC=0.5 xEPOPC=0.7 xEPOPC=0.9 EPOPC

40

20

0 30

60

90

120

150

180

0.6

0.8

1.0

0.6

0.8

1.0

A, Å2/molecule POPE/EPOPC

∆GExc, J/mol

300 0 -300 -600 -900 0.0

π=5 mN/m π=15 mN/m π=30 mN/m π=32.5 mN/m 0.2

0.4

xEPOPC

POPG/EPOPC

0

∆GExc, J/mol

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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-500

-1000

-1500 0.0

π=5 mN/m π=15 mN/m π=30 mN/m π=32.5 mN/m 0.2

0.4

xEPOPC

Fig. 1. π - A isotherms for POPE/EPOPC (a) and POPG/EPOPC (b); ∆GExc vs the monolayer composition plots for POPE/EPOPC (c) and POPG/EPOPC (d).


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As results from the analysis of the isotherm recorded for EPOPC monolayer, the lift off area for this film is ca. 150 Å2/molecule, the collapse surface pressure πcoll ≈ 45 mN/m and the maximalvalue of the compressional modulus calculated according to eq. 1 [23] is ca. 100 mN/m. C S−1 = − A ( dπ / dA)

(1)

wherein A is the mean area per molecule value at a given surface pressure π. The BAM pictures taken at any stage of film compression were found to be completely homogenous (not presented images). Thus, the analysis of the course of the curve obtained for one component EPOPC film and calculated parameters mentioned above indicates that these lipid molecules form the film in a liquid state. In comparison with a native molecule that is zwitterionic POPC (CS-1 ≈ 140 mN/m [24]), EPOPC molecules form more expanded monolayers. This is the consequence of the positive charge of hydrophilic group of EPOPC, which causes stronger electrostatic repulsions between EPOPC moleculesas compared to those between zwitterionic POPC molecules. The isotherms recorded for the films formed by the remaining compounds (POPE and POPG) are in agreement with those published previously [25]. Both POPE and POPG monolayers are in a liquid state in a wide range of the surface pressures, however, at higher surface pressures a phase transition appears and the monolayer becomes more condensed (please see Fig. S2 in the Supporting Information). This phenomenon can be easily observed in BAM pictures taken for both films (Fig. 2). As it can be observed in Fig. 1 the isotherms for the mixed films are localized between those for the one component films and with the increase of EPOPC level in the system, their shape and the collapse surface pressure values become similar to the curve for EPOPC film. Moreover, the addition of EPOPC into POPE or POPG films strongly affect the phase transitions of both monolayers which reflects in the vanishing of the plateau in the isotherms for the films of EPOPC molar fraction ≥ 0.3. All the above observations indicate that EPOPC may cause fluidization of phospholipid films and


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suggest miscibility of the components of the studied binary systems. This effect reflects well in BAM pictures taken at various stages of mixed film compression (Fig. 2).

Fig. 2. BAM images for POPE, POPG and selected POPE/EPOPC and POPG/EPOPC monolayers. As it can be observed both in the case of POPE and POPG the addition of EPOPC alters the morphology of condensed domains observed originally for one-component lipid films in the region of phase transition. Namely, the domains change their shape to be more oval and they are visibly much smaller as compared to those found within POPE and POPG monolayer. For both mixed systems the condensed phase at higher surface pressures was found to vanish at relatively low level of EPOPC (that is at the molar fractionof 0.3), which ACS Paragon Plus Environment

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indicates that fluidizing influence of the studied phosphatidylcholine derivative is pronounced. On the other hand, as can be seen in Fig. S2 (presented in the supplementary section), the addition of EPOPC into POPG film prevents the formation of a condensed phase (only one maximum in Cs-1 vs π curve) but up to xEPOPC< 0.9, in a wide range of surface pressures, practically does not influence the fluidity of the mixed film. To more deeply analyze miscibility between the components of these films and to quantitatively evaluate the interactions between the molecules in the monolayers the excess free energy of mixing ∆GExc values were calculated according to formula 2 [26]:

∆ =

(2)

= −

(3)

Aid = A1 x1 + A2 x2

(4)

In the foregoing equations A is the mean area per molecule for the mixed film determined from the isotherm at a given surface pressure, A1, A2 are mean molecular areas of the respective components in their pure films at a given surface pressure and x1, x2 are the mole fractions of components 1 and 2 in the mixed film, N is Avogadro’s number. The results of these calculations (Fig. 1 c, d) evidence that the values of this parameter are different from zero and for both systems in a wide range of monolayer composition they are negative. This fact proves miscibility of the monolayer components and suggests that the interactions between the lipids in the mixed films are more attractive than the interactions between molecules in the respective one-component monolayer. Moreover, the values obtained for POPG/EPOPC monolayer are more negative than those for POPE/EPOPC film. This proves stronger attractions of EPOPC with POPG than with POPE, which is not surprising taking into account that the interacting molecules are of the opposite charges (cationic EPOPC and anionic POPG) and electrostatic attractions determined the properties of the system. Moreover, the interactions are the strongest for equimolar composition of POPG/EPOPC film. In the case of POPE/EPOPC mixed films, at lower level of thecationic lipid ∆ valuesare slightly positive, which suggests that the interactions between EPOPC and POPE are less ACS Paragon Plus Environment

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attractive than those between lipid molecules in one component POPE film. As it is known, POPE molecules interact with neighboring molecules via hydrogen bonds [27] and the addition of cationic EPOPC into thisthermodynamically stable system is unfavorable. On the other hand, at higher level of EPOPC in POPE/EPOPC films the negative values of ∆ are observed. This effect seems to be clear when the properties of these monolayers are considered from the point of view of one component EPOPC monolayer. Namely, POPE molecules added into EPOPC film separate cationic lipid molecules and weakens electrostatic repulsions existing between EPOPCs which in turn is thermodynamically favorable and reflects in the negative values of ∆ .

2. The influence of EPOPC on POPE/POPG binary mixture a) Langmuir monolayer experiments In the next step of the investigations the influence of EPOPC on binary POPE/POPG = 3:1 monolayer was investigated. The surface pressure/area curves and the excess free energy of mixing vs composition plots for these monolayers are shown in Fig. 3a and b.


Langmuir

π, mN/m

60

40

POPE/POPG=3:1 xEPOPC=0.1 xEPOPC=0.3 xEPOPC=0.5 xEPOPC=0.7 xEPOPC=0.9 EPOPC

20

0 30

60

90

120

150

180

0.8

1.0

2

A, Å /molecule 250 POPE/POPG=3:1

0

∆GExc, J/mol

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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-250

-500

π=5 mN/m π=15 mN/m π=30 mN/m π=32.5 mN/m

-750 0.0

0.2

0.4

0.6

xEPOPC

Fig. 3. π - A isotherms (a) and ∆GExc vs the monolayer composition plots (b) for model POPE/POPG monolayer at different concentrations of EPOPC.

As far as the POPE/POPG = 3:1 mixture is concerned the monolayers of this composition are frequently used as a model of bacteria membrane and the properties of these systems are well described in literature [28-30]. Shortly, for these systems favorable mixing between phospholipids in the monolayer was found, which in our experiments reflects in slightly negative values of the excess free energy of mixing. The isotherm and BAM pictures (Fig. 4) taken for model system evidence the phase transition between liquid and condensed state at higher surface pressures. As it can be observed the domains of condensed phase start to ACS Paragon Plus Environment

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format ca. 32 mN/m and they systematically grow, in the number and size, with the compression. At the surface pressure of ca. 45 mN/m the condensed phase covers practically whole interface and then the brighter phase indicating monolayer collapse starts to form.

Fig. 4. BAM images taken for POPE/POPG model bacterial membrane and for POPE/POPG/EPOPC films at 10% of cationic lipid.

As it can be predicted based on the results obtained for binary systems the addition of cationic lipid into the model system causes that the film becomes more fluid. This reflects in the course and shape of the isotherms (which are more expanded and deprived of the kink characteristic of the phase transition) as well as in BAM pictures. The foregoing influence of EPOPC is strong because the condensed domains for ternary POPE/POPG/EPOPC films occurs only at 10% of cationic lipid in the mixed film and vanishes with further addition of EPOPC. Moreover, also at this composition (10% of EPOPC) pronounced differences in the film morphology can be noticed. These reflect in a lower number and smaller size of condensed domains formed at the interface incomparisonwith POPE/POPG monolayer (Fig. 4). Based on the calculated ∆GExc values it can be summarized that the addition of EPOPC ACS Paragon Plus Environment

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into the model membrane is thermodynamically favorable. As it can be found in Fig. 3b with the increase of the content of cationic lipid in themodel system the values of this parameter becomes more negative and they are lower than the values for POPE/POPG monolayer. This proves the mixing of monolayer components, confirmed also in BAM images, and evidences that the interactions between the molecules in ternary films are more favorable than the interactions between EPOPC molecules in one component film and between POPE and POPG in 3:1 monolayer. b. liposome characteristic The properties of POPE/POPG/EPOPC liposomes were characterized by their size measured by dynamic light scattering (DLS) and zeta potential. The results of these experiments are compiled in Table 1.

Table 1.The z-average diameter (dz), dispersity index (DI) values and zeta potentials (ζ) of liposomes differing in the level of EPOPC. PE/PG/EPOPC xEPOPC

dz ± 2[nm]

DI

ζ± 4[mV]

0

132

0.065

-64.5

0.1

126

0.072

-46.8

0.3

122

0.082

+40.5

0.5

120

0.087

+58.8

0.7

117

0.083

+65.5

0.9

109

0.080

+69.1

As can be found in Table 1, the average hydrodynamic diameter of liposomes decreases with the addition of EPOPC into the system. These differences are rather small and additionally concern the hydrodynamic diameter which may mean that these changes are connected with the variations of the thickness of the hydration layer whereas the diameter of liposomes (without hydration layer) practically does not change. The values of dispersity index was rather small (lower than 0.1), which indicates that the prepared liposomes were of small size distribution, they are stable and do notundergo aggregation. Moreover, with the increase of ACS Paragon Plus Environment

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EPOPC content in the system,zeta potential valuesincrease (which means that the surface chargeincreases) and at 30% or higher level of cationic lipid they were positive.The composition of liposomes for which the charge is neutralized is of xEPOPC≈ 0.2 (please see Fig. S2 in the Supporting Information). Considering the values of zeta potential, the fact that they are higher than+30 mV and lower than −30 mV allows one to conclude that liposomes are well stabilized by repulsion, which prevents from their aggregation [31]. In order to confirm vesicle formation and gain some information on morphology of the obtained liposomes Transmission Electron Microscopy (TEM) visualizations and AFM studies (please see Supporting Information) were performed. Figure 5 shows TEM micrographs of the pure POPE/POPG liposomes (A) and liposomes modified with EPOPC (xEPOPC = 0.5) (B).

Fig. 5. TEM micrographs of POPE/POPG liposomes (A) and POPE/POPG/EPOPC (xEPOPC= 0.5) (B) It seems that in both cases the morphology of the objects observed is quite similar. The POPE/POPG pristine liposomes, as well as, those POPE/POPG/EPOPC exhibit the good spherical shape with the sizes in the range of 80-160 nm. Thus one can conclude that the addition of cationic lipid into the POPE/POPG system does not affect noticeably the morphology of the resulted objects. The fluidity of liposome membranes was evaluated basedon the steady-state fluorescence anisotropy (r) experiments with the application of (DPH) as a fluorescent probe, which ACS Paragon Plus Environment

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responds on the order and packing of surrounding acyl chains [e.g. 32,33]. The changes of the steady-state fluorescence anisotropy with the increase of EPOPC molar fraction in liposomes membrane are shown in Fig. 5 a. As can be seen even a small addition of EPOPC (10 mol%) to POPE/POPG membrane strongly affects its fluidity which substantially increases with the addition of EPOPC up to its 50 mol% content in the membrane. Whereas further incorporation of EPOPC changes the fluidity of bilayer insignificantly. To investigate the influence of EPOPC on the permeability of model bacterial membranes we performed experiments of the release of calcein entrapped in the liposomes. Since the fluorescence anisotropy experiments showed that even small addition ofEPOPC strongly disturbs molecular organization of the model bacterial membrane, calcein release measurements were done only for low concentration of EPOPC (5, 10, 15 mol%). In Fig. 5b the changes in calcein fluorescence intensity after titration of the liposomes with the solution of Triton X-100 are presented. As it can be seen the addition of surfactant causes the increase of fluorescence intensity. This is due to a release of calcein from liposomes interior. Finally the intensity valueswere stabilized, which corresponds toatotal disintegration of liposome. One can see that the higher concentration of EPOPC in the system, the lower level of surfactant is required to disrupt the model membrane. This enables to conclude that the presence of EPOPC in POPE/POPG bilayer leads to its destabilization by the increase of permeability.


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DPH anisotropy (r)

a 0.16 0.14 0.12 0.10 0.08 0.06 0.04 0.0

0.2

0.4

0.6

0.8

1.0

XEPOPC Fluorescence intensity [a.u.]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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b 0.9

0.6 POPE/POPG=3:1 xEPOPC=0.05 xEPOPC=0.10 xEPOPC=0.15

0.3

0.0 0.0

0.2

0.4

0.6

0.8

1.0

[Triton X-100]/[lipids]

Fig. 6. The changes of the steady-state fluorescence anisotropy with the increase of EPOPC molar fraction in liposomes membrane (a); the changes in calcein fluorescence intensity after titration of the liposomes with the solution of Triton X-100 (b).

The studies performed on ternary POPE/POPG/EPOPC monolayers agree very well with those for mixed POPE/POPG/EPOPC liposomes. The results collected for both membrane models applied in ourexperiments clearly indicate that the incorporation of EPOPC strongly affects molecular organization of the investigated systems changing the packing and ordering of lipids, which in turn causes the increase of the fluidity and permeability of themodel membranes.


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3. Antibacterial properties of EPOPC liposome suspension To investigate the antibacterial properties of the liposomal suspension of EPOPC the dilution method against Gram-negative Escherichia coli ATCC 25922 and Gram-positive Staphylococcus aureus ATCC 6538 strains was applied. The obtained results indicated that the suspension of EPOPC vesicles, in the investigated range of concentrations, does not fully inhibit growth neither Escherichia coli ATCC 25922 nor Staphylococcus aureus ATCC 6538 strains. Thus, minimum inhibitory concentration (MIC) was found to be higher than 2.29 mg/mL for both microorganisms. Since we were not able to determine the exact MIC value, the additional in vitro test has been performed. For this purpose, the bacteria were incubated for 4h in medium containing various concentration of EPOPC (1.14 and 2.29 mg/mL) and in EPOPC-free medium (used as positive growth control). After incubation colony forming units (CFU) per 1 mL were determined for each mixture. The obtained results are collected in Table 2.

Table 2. The influence of the concentration of liposomal suspension of EPOPC on the number of colony forming units (CFU) per 1 mL for E. coli and S. aureus incubated for 4h. Incubation time Bacteria

Medium MH II broth

E. coli

4h

4.5×104 CFU/mL

4.5 × 106 CFU/mL

EPOPC 1.14 mg/mL

3.0 × 105 CFU/mL

EPOPC 2.29mg/mL

4.5 × 104 CFU/mL 1.35 × 105 CFU/mL

MH II broth S. aureus

0h

1.55 × 106 CFU/mL

EPOPC 1.14 mg/mL

6.0 × 105 CFU/mL

EPOPC 2.29 mg/mL

1.35 × 105 CFU/mL

As can be seen, a four-hour incubation of bacteria in the medium containing liposomal suspension of higher concentration of EPOPC causes that the number of colony forming units per 1 mL does not change, whereas it strongly increases in control sample. This means that suspension of EPOPC vesicles at this concentration prevents multiplication of both bacteria ACS Paragon Plus Environment

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strains. For lower concentration of EPOPC, one can see that bacterial growth is partially inhibited in comparison to control. However, the inhibition of multiplication is not complete and in addition it is stronger in the case of E. coli than S. aureus. This may indicate that EPOPC has stronger bacteriostatic activity against Gram-negative bacteria (E. coli) than Gram-positive ones (S. aureus), which may be due to differences in the structures of their membranes. However, since antibacterial properties of a given compound may strongly depend on the kind of the microorganism this general conclusion should be supported by investigations performed with the applications of a wider variety of bacterial strains.

Conclusions All the collected results evidence that EPOPC is of strong fluidizing and destabilizing influence on model bacterial membranes. The results of experiments performed on monolayers prove favorable interactions between this molecule and model bacterial membrane lipids as well as the ability of EPOPC to prevent the formation of condensed phase at the air/water interface. The addition of EPOPC into lipid films changes visibly characteristics of condensed domains formed within native lipid monolayers as well as reduces their number. The ability of EPOPC to alter properties of model bacterial membrane was further confirmed in the experiments on model bilayers. As it can be found, EPOPC decreases the average hydrodynamic diameter of liposomes and increases zeta potential values -the latter indicating the increase of the surface charge. Based on these results it can be concluded that EPOPC causes disorders at the level of model bacterial membranes, by changing its surface charge, lipid organization and increasing fluidity and permeability. Moreover, it is highly important to notice that these alterations in the properties of both mono,- and bilayer artificial membranes were pronounced even at very low concentration of EPOPC. Also, the in vitro tests performed on Escherichia coli ATCC 25922 (Gram-negative) and Staphylococcus aureus ATCC 6538 (Gram-positive) strains prove that EPOPC has some bacteriostatic properties which seems to be stronger towards Gram-negative than GramACS Paragon Plus Environment

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positive bacteria. This may result from the differences in the structure of their membranes. However, in order to draw general conclusions, it would be necessary to carry out studies on various strains of bacteria. Taking into account the results obtained for the investigated model membranes as well as stronger bacteriostatic activity of EPOPC towards E. coli (Gramnegative) as compared to S. aureus (Gram-positive) bacteria, it can be suggested that bacteriostatic influence of EPOPC molecules is connected with their activity at the level of bacterial membrane. The interactions of EPOPC with membrane lipids and resulting modifications in the organization of membrane leading to the increase of membrane fluidity and permeability may be considered as a mechanism of action of this compound.

Acknowledgment This project was financed by the National Science Centre, Poland (Grant No. 2016/21/B/ST5/00266).


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Influence of Cationic Phosphatidylcholine Derivative on Monolayer

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