Interactions of a Hydrophobically Modified Polycation with Zwitterionic Lipid Membranes

Published: November 15, 2011

r 2011 American Chemical Society 676 dx.doi.org/10.1021/la203748q | Langmuir 2012, 28, 676–688

ARTICLE

pubs.acs.org/Langmuir

Interactions of a Hydrophobically Modified Polycation withZwitterionic Lipid MembranesMariusz Kepczynski,*,† Dorota Jamr�oz,*,† Magdalena Wytrwal,† Jan Bednar,‡,§ Ewa Rzad,† andMaria Nowakowska†

†Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Krak�ow, Poland‡Charles University in Prague, First Faculty of Medicine, Institute of Cellular Biology and Pathology, Albertov 4, 128 01 Prague 2,Czech Republic§University of Grenoble 1/CNRS, LIPhy UMR 5588, 140 Av. de la Physique, Grenoble, F-38041, France

bS Supporting Information

’ INTRODUCTION

The interactions between polycations and lipid or cell mem-branes play an important role in many biophysical applications.The most important of them include (i) delivery of geneticmaterials to cells in a targeted and safe manner,1�3 (ii) usage asbiocidal agents,4�6 and (iii) obtaining stabilized vesicles bycovering the liposomes surface with multilayer films.7,8 Becauseof these issues, the impact of both natural and synthetic polycationson phospholipid bilayer has been extensively studied using variousexperimental techniques.9

The success of gene therapy largely depends on the availabil-ity of suitable delivery vehicles. Polycations, especially poly-(ethylenimine) (PEI), are frequently studied as nonviral syn-thetic transfectants, which are characterized by excellent genecomplexing ability (formation of polyplexes) and eminent trans-fectant properties.3 Unfortunately, they show in vitro cytotoxi-city, which constitutes the major challenge for their clinicalapplications.3,10 Transport of molecules to and from the cellnucleus, mediated by polycations, is likely to be the result ofnonspecific interactions between the polyplexes and the nuclearmembrane.2 To date, research has focused rather on the

efficiency of transfecting the cell membranes. The molecularmechanisms of both the transfection and the polycation-inducedcytotoxicity are yet unknown. Much empirical evidence suggeststhough that the important role in these processes is the ability ofpolycations to disrupt the nuclear membrane and to increase themembrane permeability. It is known that the transfection processoccurs when small pores open in the nuclear membrane to admitgenetic materials.2

It is well-known that most antimicrobial peptides disrupt thebacterial membrane via transmembrane pore formation and/ormembrane destabilization.11,12 The majority of these peptidesare cationic or amphipathic. Recently, polymers containingquaternary ammonium or alkyl pyridinium moieties have beencommonly used as biocides.5 The quaternary amino groups inantimicrobial polymer are believed to be responsible for causingcell death by disrupting the cell membrane, thus allowing releaseof the intracellular contents.

Received: September 24, 2011Revised: November 8, 2011

ABSTRACT: The interactions between synthetic polycations and phospho-lipid bilayers play an important role in some biophysical applications such asgene delivery or antibacterial usage. Despite extensive investigation into thenature of these interactions, their physical and molecular bases remain poorlyunderstood. In this Article, we present the results of our studies on the impact ofa hydrophobically modified strong polycation on the properties of a zwitter-ionic bilayer used as a model of the mammalian cellular membrane. The studywas carried out using a set of complementary experimental methods andmolecular dynamic (MD) simulations. A new polycation, poly(allyl-N,N-dimethyl-N-hexylammonium chloride) (polymer 3), was synthesized, and itsinteractions with liposomes composed of 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC) were examined using dynamic light scattering (DLS),zeta potential measurements, and cryo-transmission electron microscopy(cryo-TEM). Our results have shown that polymer 3 can efficiently associate with and insert into the POPC membrane. However,it does not change its lamellar structure, as was demonstrated by cryo-TEM. The influence of polymer 3 on the membranefunctionality was studied by leakage experiments applying a fluorescence dye (calcein) encapsulated in the phospholipid vesicles.The MD simulations of model systems reveal that polymer 3 promotes formation of hydrophilic pores in the membrane, thusincreasing considerably its permeability.

677 dx.doi.org/10.1021/la203748q |Langmuir 2012, 28, 676–688

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It was shown that polycations associate strongly with orpenetrate into negatively charged liposomes bilayer, mostlydue to the electrostatic interactions.6,9 The interactions betweenpositively charged macromolecule and a zwitterionic membrane,commonly used as a mammalian membrane model, are morepuzzling. Quemeneur et al.13 studied adsorption of chitosan (acationic polysaccharide) and chitosan alkylated with hydrocar-bon chains of different lengths on phosphatidylcholine (PC)liposomes. It was demonstrated that the alkyl chains do notinteract with the lipid membrane and the chitosan adsorption isgoverned by electrostatic interaction mechanism. Eren et al.studied amphiphlic polyoxanorbornene functionalized with qua-ternary pyridinium side moieties substituted with various alkylchains.4 The authors showed that the polymers modified withshort alkyl chains (ethyl, buthyl) introduced only little disruptionof PC membranes, while those with longer side chains (hexyl todecyl) were much more active toward the PC vesicles. Ding et al.studied the interactions of antimicrobial cationic conjugatedpolyelectrolytes (CPEs) with membranes composed of lipidswith varying headgroup charge.6 Their results showed that thepolymers can efficiently associate with and insert into anionicphosphatidylglycerol (PG) membranes. However, these poly-mers did not interact with the zwitterionic lipid membranescomposed of PCs. Wang et al. studied a series of CPEs ofsignificant structural diversity.14 They concluded that the func-tional groups on the side chains dominate the polymer ability formembrane perturbation and that the macromolecules with thehigh charge density and hydrophobic alkyl side chains have theaffinity to the zwitterionic membranes. Sikor et al. studied theinteractions between PEI and the zwitterionic lipid vesicles.2 Itwas demonstrated that at low salt concentration the introductionof the polymer leads to aggregation of vesicles with formation ofstable clusters, while at high salt content there is no aggregationand PEI can penetrate into the bilayer. On the basis of thepresented above literature, one can conclude that further studiesgiving an insight into the molecular aspects of the interactionsbetween cationic polymers and zwitterionic membranes arenecessary to better understand the nature of these interactions.Molecular dynamics (MD) simulations can provide the neces-sary insight into several aspects of the interaction betweenbilayers and various compounds and have been extensively usedin the past few years to describe lipidmembranes in terms of theirstructure and dynamics.15 MD simulation of interaction betweenpolycations and lipid membranes has been addressed in a fewstudies. Lee and Larson performed coarse-grained MD simula-tions of polyamidoamine (PAMAM) dendrimers of different sizeand with different terminal groups put in contact with dipalmi-toylphosphatidylcholine (DPPC) and dimyristoylphosphatidyl-choline (DMPC) bilayers.16�18 Their simulations show thatcharge on the terminal groups plays a crucial role in thisinteraction. The dendrimers with charged terminal groups de-form and insert into the bilayer, whereas those with unchargedterminals remain on the membrane surface. The bigger size andhigh concentration of the dendrimer facilitate its penetration intothe membrane. On the contrary, a linear polycation, poly-L-lysine(PLL), was not observed to insert into the bilayer despite itshigh charge density.17 The importance of electrostatics in thePAMAM�lipid interaction has been supported by all-atomMD study of PAMAM approaching a DMPC surface.19,20 Freeenergy calculations performed for dendrimers with three dif-ferent terminal groups show that the gain in free energy on bindingto the membrane surface is much larger for the charged molecules.

This study also points out the role of lipid chain mobility in thebinding of dendrimers to the bilayer surface. Membrane in thefluid phase was found to bind the dendrimer molecules strongerthan while in the gel phase.20

In this Article, we examined the effect of a hydrophobicallymodified strong polycation on the oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC) bilayer. For that purpose, a newpolymer, poly(allyl-N,N-dimethyl-N-hexylammonium chloride)(3), was synthesized in a two-stepmodification of a commerciallyavailable poly(allylamine hydrochloride) (PAH). PAH was cho-sen because that polymer has found a wide variety of applicationsin both nanotechnology and biomedical fields.7,8 The POPCvesicles were treated with aqueous solutions of polymer 3 atvarious concentrations, and several experimental techniqueswere employed to study the polymer�membrane interactions.We used dynamic light scattering (DLS) to measure the size ofthe lipid vesicles and verify the possibility of vesicle aggregation.Additionally, we measured zeta potentials of the liposomes toconfirm adsorption of polymer 3 on the liposome surface. Cryo-transmission electron microscopy (Cryo-TEM) was applied toobserve the morphology of the polymer-covered liposomes.Change of the permeability of the POPC membrane inducedby the presence of polymer 3was monitored using a fluorescencemethod. Finally, we performed MD computer simulations to getinsight into the polycation�lipid interactions at the molecularlevel. TheMD simulations allowed one to determine the positionand orientation of the studied molecule and to follow the molec-ular motion at a level typically not accessible to the experimentalobservation.

’EXPERIMENTAL SECTION

Materials. 2-Oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC,g99.0%) and calcein were obtained from Sigma. Poly(allylamine hydro-chloride) (1) with average molecular weight of ∼15 000, hexanal (98%),NaBH4,N-methyl-2-pyrrolidone (NMP, spectrophotometric grade,g99%),iodomethane (99%), DMSO-d6 (99.9 atom % D), and D2O (99.9atom % D) were purchased from Aldrich and used as received. NaI(g99.0%) was received from POCh (Gliwice, Poland). Benzoylateddialysis tubing (2 000 g/mol molecular weight cutoff) and SephadexG-50 (20�50 μm) were purchased from Sigma-Aldrich. Millipore-qualitywater was used for all solution preparations.Apparatus. 1H and 13C NMR spectra were recorded on a Bruker

AMX 500 Hz instrument. The NMR spectra were taken at 80 �C inD2O/DMSO-d6 mixture (1:3 v/v) using DMSO-d6 residual peaks asinternal standards. IR spectra were recorded on a Nicolet IR200FT-IRspectrophotometer with an ATR attachment. Elemental analysis wasperformed on a EuroEA 3000 Elemental Analyzer. Fluorescence spectrawere recorded on a Perkin-Elmer LSD50B spectrofluorimeter equippedwith a thermostatted cuvette holder.Synthesis of 3. Synthesis of poly(allyl-N,N-dimethyl-N-hexylam-

monium chloride) (3) is schematically presented in Figure 1. 0.5 g ofcompound 1 (5.34 mmol of the amino groups) was dissolved in 5 mLof water. One milliliter of 5% NaOH solution was added, and thenthe pH of the solution was adjusted to 3�4 using 1% acetic acid. After30 min of stirring, 1.6 mL of hexanal (1.5-fold excess to the aminogroups of 1) was added, and the reaction mixture was stirred for 24 hat room temperature. Next, 0.52 g of NaBH4 dissolved in water wasdropwise added (1.5-fold excess to the added aldehyde), and themixture was stirred for 12 h. The crude intermediate product waspurified by dialysis against water. Finally, water was evaporated undera vacuum to give poly(allyl-N-hexyl-amine) (2) as white crystals(0.8 g, 53.1%).


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IR ν̅max

(cm�1): 3395 (N�H); 2926 (C�H); 1600 (N�H); 1511,1460 (C�C). 1H NMR (DMSO-d6:D2O (3:1 v/v)): δ (ppm) 0.76�1.03(m, 1.0H, CH2�CH3), 1.12�1.44 (m, 2.6H, (CH2)4�CH3), 1.47�2.30(m, 3.1H, N�CH2�CH�CH2; N�CH2�CH�CH2), 2.66�3.18 (m,10H, N�CH2�CH�CH2; N�CH2�CH2).

13C NMR (DMSO-d6:D2O(3:1 v/v)): δ (ppm) 13.7 (CH3); 21.8, 25.1, 25.9, 31.0 (4 � CH2); 30.8(CH�CH2�CH); 35.3 (N�CH2�CH�CH2); 42.5 (N�CH2�CH�CH2); 48.5 (N�CH2�(CH2)4�CH3). Anal. Calcd for polymer 2 with33% content of hexyl groups: C, 47.11; H, 9.99; N, 11.03. Found: C, 47.11;H, 9.87; N, 11.03.

Compound 2 was dissolved in 25 mL of NMP, and the solution wasstirred for 0.5 h at room temperature. Next, 1.4 M NaOH, 6.7 mL ofCH3I (10-fold excess to the amino groups), and 1.5 g of NaI were added.Reaction was carried out with stirring for 12 h at 50 �C. Next, toexchange the iodide counterions of the methylated derivative of polymer2 for chloride ones, the reactionmixture was placed in dialyzing tube anddialyzed in the following sequence (4 days in each environment): againstdeionized water, 0.1 M KCl, and again deionized water. Finally, waterwas evaporated under a vacuum to give the desired product 3 as fine paleyellow crystals (0.77 g, 66.2% yield).

IR ν̅max

(cm�1): 3374 (N�H); 2934 (C�H); 1633 (N�H); 1481(C�C). 1HNMR (DMSO-d6:D2O (3:1 v/v)): δ (ppm) 0.75�0.92 (m,0.8H, CH2�CH3), 1.17�1.37 (m, 1.7H, (CH2)3�CH3), 1.41�2.59 (m,3.9H, N�CH2�CH; N�CH2�CH�CH2; N�CH2�CH2), 2.82�3.90(m, 10H, N�CH2�CH�CH2; N�CH2�CH2; N�(CH3)2; N�(CH3)3).

13C NMR (DMSO-d6:D2O (3:1 v/v)): δ (ppm) 14.2 (CH3);22.3, 25.4, 25.9, 31.3 (4 � CH2); 26.1 (CH�CH2�CH); 31.3(CH2�CH�CH2); 51.2 ((CH3)2�N); 54.7 (3 � (CH3)3�N); 66.43(CH2�N�(CH3)2); 70.12 (CH2�N�(CH3)3).Preparation of Liposomes. Small unilamellar phospholipid

vesicles (SUV) were prepared by the extrusion technique.21

POPC (2.5 mg) was dissolved in chloroform. The lipid solution wasplaced in a volumetric flask, and the solvent was evaporated under agentle stream of nitrogen to complete dryness. One millimolar solutionof NaCl was added until a desired lipid concentration was attained(usually 2.5 mg/mL), and the sample was vortexed for 5 min. Theresulting multilamellar vesicle dispersion was subjected to five freeze�thaw cycles from the liquid nitrogen temperature to 60 �C and thenextruded six times through the membrane filters with 100 nm poresusing a gas-pressurized extruder. The pH value of the POPC dispersionswas determined to be 6.5.Coating of Liposomes with Polymer 3. A 0.5 mL dispersion of

liposomes (2.5 mg/mL) in 1 mMNaCl was placed in a sonication bath,and the appropriate volume of 0.5 mg/mL solution of the polymer wasquickly added. 1 mMNaCl was next added to the final volume of sampleof 1 mL.Cryo-Transmission Electron Microscopy (cryo-TEM). Cryo-

TEM was used to visualize the morphology of liposomes as described

previously.22 This technique allows the least perturbing and directimaging of the hydrated sample. The liposome dispersion was preparedat cPOPC = 2.5 mg/mL, and the concentrated solution of polymer 3 wasadded to the final concentration of 0.06 mg/mL (2.35 wt % content of3). Three microliters of the sample solution was applied to an electronmicroscopy grid covered with perforated supporting film. Most of thesample was removed by blotting for approximately 1 s, and the grid wasimmediately plunged into liquid ethane held at �183 �C. The samplewas then transferred without rewarming into a Tecnai Sphera G20

electron microscope using a Gatan 626 cryo-specimen holder. The imageswere recorded at 120 kV accelerating voltage andmicroscopemagnificationranging from 5000� to 14 500� using a Gatan UltraScan 1000 slow scanCCD camera and low dose mode with the electron dose not exceeding 15electrons per square Å. The microscopic observations were performedimmediately and 1, 2, and 24 h after the polymer introduction.Light Scattering and Zeta Potential Measurements. A

Malvern Nano ZS light scattering apparatus (Malvern Instrument Ltd.,Worcestershire, UK) was used for dynamic light scattering (DLS) and zetapotential measurements. The time-dependent autocorrelation functionof the photocurrent was acquired every 10 s with 15 acquisitions for eachrun. The samples were illuminated by a 633 nm laser, and the intensityof light scattered at an angle of 173� was measured by an avalanchephotodiode. The z-averaged hydrodynamic mean diameters (dz), poly-dispersity (PDI), and distribution profiles of the samples were calculatedusing the software provided by the manufacturer. The zeta potential ofthe liposomes was measured using the technique of laser Dopplervelocimetry (LDV).Calcein-Release Studies. Calcein-loaded (CL) liposomes were

prepared as described above for liposomes in the pure buffer solution,with the only difference being that the lipid film was hydrated with0.06 M solution of calcein (pH 8.5). The nonencapsulated calcein wasseparated from the CL liposomes by size-exclusion chromatographyon a Sephadex G-50 column using PBS buffer containing 1 mM NaCl(pH 7.4) as an eluent. The fluorescence intensity of the CL liposomeswas measured and found to be low due to the self-quenching effect. Theappropriate amount of the polymer solutionwas introduced, and the changein fluorescence intensity due to calcein release from the vesicles wasmonitored. Excitation and emission wavelengths were set at 490 and520 nm, respectively. The experiments were conducted at 25 �C. POPCliposomes display a single phase transition centered at �3.4 �C.23 There-fore, at 25 �C, the chains of lipids are fluid and the POPC membrane isin the liquid-crystalline state. A complete release of the dye was achievedby adding 30 μL of 15% solution of Triton-X100. The correspondingfluorescence intensity was used as 100% leakage. The amount of calceinreleased after time t, RF, was calculated using the following equation:24

RFðtÞ ¼ 100It � I0Imax � I0

in % ð1Þ

Figure 1. Synthesis of poly(allyl-N,N-dimethyl-N-hexylammonium chloride) (polymer 3).


Langmuir ARTICLE

where I0, It, and Imax are the fluorescence intensities measured withoutpolymer, at time t after the polymer introduction, and after the additionof Triton X-100, respectively.Molecular Dynamics Simulations. Initial Structure. A short

chain consisting of 10 units (decamer 3, Figure 2) was used in thesimulations as a model for polycation 3. A conformer of decamer 3 withan elongated main chain was generated, and its geometry was optimizedat the HF/6-31* level of theory. Two such molecules were introduced ata vertical orientation into an empty box with their centers of mass placedalong the box diagonal. The size of the box was the same as that of thesimulation box of a previously equilibrated POPC bilayer, consisting of128 POPC molecules. The molecules of decamer 3 were subsequentlyimmersed into the equilibrated POPC membrane, after removing someof the POPC molecules to create suitable compartments to accommo-date the decamer molecules. On completing the number of POPCmolecules to 128 (64 in each leaflet), a layer of water was added on top ofeach side of themembrane. The number of water molecules correspondsto a hydration ratio of ca. 30 H2O molecules per one POPC molecule,that is, a fully hydrated lipid membrane. Two initial systems, differing inthe depth of the decamer immersion into the hydrophobic region of themembrane and the number of water molecules (3910 and 3861,respectively), were prepared in this way. Prior to the MD simulations,both of the starting systems (hereafter referred to as membrane I and II)were energy minimized with the steep descent algorithm. To relax anypossible unfavorable contacts, they were submitted to a preliminaryconstant volume simulation run of 200 ps with a time step of 1 fs. ThePOPC molecule was described with the commonly used Berger lipidforce field.25 For the sake of compatibility, the parameters for thedecamer molecule were based on the same force field. A minor changein the charge distribution in the �C�N+(CH3)2�C� fragment wasintroduced due to a different number of methyl substituent in the POPCand decamer ammonium moieties. The Lennard-Jones parameters forthe Cl� anion were taken from Joung and Cheatham paper.26 Water wasdescribed using the simple point charge (SPC) model.27

Simulation Conditions. The MD simulations were performed withthe GROMACS 4.0.7 software package.28 The periodic boundaryconditions were applied in all three directions. The simulated systemwas maintained at the temperature of 298 K and the pressure of 1 baraccording to theNPT ensemble regime. The temperature was controlledby the velocity rescaling thermostat,29 and the pressure was keptconstant using the isotropic Berendsen barostat.30 The long-rangeelectrostatic potential was calculated using the particle-mesh Ewald(PME) method with the Coulomb cutoff radius of 1.0 nm.31 TheLennard-Jones potential was calculated with a twin range cutoff with theradii set at 1.0 and 1.4 nm and the pair list updated every 10 steps. TheLINCS constraints algorithm was employed for all bonds32 allowing fora 2 fs time step. The simulations were carried out for 110 ns in total, thefirst 10 ns considered as system equilibration and the remaining 100 nstreated as the productive run. The time step of 2 fs was used.Visualizations of the trajectories were made with the VMD package.33

To obtain a reference system for the decamer doped membrane, ashort simulation of 10 ns was performed on the equilibrated membraneof 128 POPC and 3820water molecules. To compare the hydration stateof the decamer molecules in the membrane and in bulk water, a smallsystem of four decamer 3 molecules embedded in a box of water wassimulated for 40 ns. Both supplementary simulations were carried outunder the same conditions as the studied membranes.

’RESULTS

Polymer 3 Synthesis. A quaternized derivative of poly-(allylamine hydrochloride) (1) with covalently attached alkylchains was synthesized using the modified procedure reported inthe literature for chitosan.34,35 Poly(allyl-N,N-dimethyl-N-hex-ylammonium chloride) (3) was obtained from commerciallyavailable polymer 1 in two steps as shown in Figure 1. Aminogroups of 1 reacted with hexyl aldehyde to form Schiff baseintermediates, which was followed by reduction with sodiumborohydride. In this way, N-hexyl derivative (2) of 1 wasobtained. The analysis of the 1H NMR spectrum of the producthas shown that 1 was substituted by a n-hexyl group. The degreeof substitution by alkyl groups was determined from elementalanalysis to be ca. 33%. In the next step, the weak polyelectrolyte,2, was transformed into a strong polycation by quaternization ofthe amino groups using a Hoffmann exhaustive alkylation withiodomethane in the presence of sodium hydroxide, water, andsodium iodide. The 13CNMR spectrum of the product shows thedistinctive signal at ca. 54 ppm, attributed to methyl groupslinked to the quaternized amino groups. In the spectrum, there isno signal at ca. 46 ppm, characteristic of tertiary amino groups.These confirmed that under our experimental conditions the N-methylation of the amino groups of polymer 2 was effective andled to the complete substitution of the amino groups with methylgroups. The content of quaternary ammonium groups wasestimated from the 1H NMR spectrum to be ca. 100%.Interaction of Polymer 3 with Liposomes. DLS Measure-

ments and Zeta Potential. The POPC vesicles were prepared byextrusion through 100 nm filters. The intensity weighted meansizes (dz) of the vesicles determined with DLS were found to beca. 124.8 nm, and the polydispersity was less than 0.1. Thus, thepopulation of the liposomes has a narrowly distributed size rangeas indicated by the polydispersity index. A series of samples with aconstant total concentration of POPC (cPOPC = 1.25 mg/mL)and the mass fraction of 3 varying from 0 to ca. 4.0% as comparedto a lipid content were prepared. The scattering measurementswere carried out to determine the hydrodynamic diametersof the vesicles coated with the polymer. Figure 3 depicts the

Figure 2. The conformation of decamer 3 molecule.


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distribution profiles of the hydrodynamic diameters of the initialPOPC liposomes and the liposomes treated with various amountsof polymer 3. The profile for these vesicles was invariable forseveral days. The introduction of polymer 3 up to the concentra-tion of 0.05 mg/mL (3.85% mass fraction) to the liposomedispersion caused changes in the size distribution of vesicles. Thedistributions becamewider due to the appearance of a shoulder atthe larger diameter region. The values of dz and polydispersityindex (PDI) are collected in Table 1. The results show that theinitial size of liposomes shifted gradually to larger diameters withthe increasing content of polymer 3.In the next step, the zeta potential (ζ-potential) of POPC

liposomes with and without polymer 3 was measured for a fixedionic strength. The results are summarized in Table 1. The valueof ζ-potential of the neat liposomes was close to zero as a result ofthe zwitterionic character of POPC. The addition of polymer 3 tothe liposomal dispersion up to the mass fraction of ca. 3%resulted in a pronounced increase of zeta potential of the POPCvesicles. Introduction of larger quantities of the polymer led toonly a slight increase in the potential value. That observation canbe explained assuming the adsorption of polymer 3 onto thePOPC bilayer surface. The positively charged ammonium groupswere exposed to the bulk solution, increasing the surfacepotential of the liposomal surface.Cryo-TEM Observation. In the next step, a cryo-TEM tech-

nique was used to study the effect of polymer 3 on themorphology and stability of the liposomes. For this purpose,the lipid vesicles were covered with polymer 3, and the liposomaldispersion was analyzed with cryo-TEM after various periods oftime. Figure 4A and B presents the typical cryo-TEM micro-graphs of the liposomes incubated for less than 1 min and 24 hwith polymer 3. As can be seen, in both cases, the vesicles showedthe regular spherical morphology with a distinct lipid membranesurrounding the aqueous core. Although the unilamellar struc-tures constitute the main population, sparse multilayered vesiclescan also be observed. The size distribution profiles of liposomesdetermined from the cryo-TEM measurements are also pre-sented in Figure 4 (right). Both distributions are broad, but theshape and the average diameters of the vesicles are very close.This is clear evidence that the polymer-covered liposomes did

not show any tendency for aggregation and the presence of thepolymer did not cause membrane destabilization. The averagediameters determined from the microscopic visualizations werelower than that estimated with DLS measurements. That isbecause DLS method measures the mean hydrodynamic dia-meter, which is heavily weighted toward the largest structures inthe solution.36 Thus, DLS showed a systematically larger meanvesicle size.Fluorescence Studies. Calcein-release phenomena have been

utilized as an effective index to characterize the permeabilityproperties of biomembranes and to evaluate the interactionsbetween the bilayer lipid membrane and target materials such asproteins, peptides, and (model) biomembranes. Calcein wasentrapped inside the liposomes, which were next covered withpolymer 3 at various concentrations. The release of the dye fromthe CL liposomes, both native and polymer-coated ones, wasnext monitored using fluorescence measurements. The amountof released calcein, RF, was calculated, and Figure 5 shows thetypical time-course of the RF values of calcein from the POPCliposome. In the case of the pure POPC liposomes, the fluores-cence intensity did not change during our experiments. This canbe explained assuming that the noncovered membrane consti-tutes a barrier for the polar calcein and its structure remainedintact during the time of measurement. The addition of polymer3 to the initial liposome dispersion led to a pronounced increaseof the fluorescence intensity due to the release of dye. Theleakage of calcein increased strongly immediately after thepolymer addition and reached a plateau after a certain time.The observed total leakage of the calcein was strongly dependenton the amount of the introduced polymer. To quantify thecalcein leakage through the lipid membrane, a permeabilitycoefficient to calcein, Ps, was calculated (in cm s�1) by usingthe relationship:16

Ps ¼ r3k ð2Þ

where r is the radius of liposome and k is the apparent rateconstant of calcein release and can be obtained by the analysis ofa first-order kinetic calcein fluorescence according to the equa-tion:

RFðtÞ ¼ RFmaxð1� e�ktÞ ð3ÞFitting the results presented in Figure 5 to eq 3, the perme-

ability coefficient values, Ps, for the calcein release from theliposome were estimated as 5.74 � 10�8, 1.01 � 10�7, and1.69� 10�7 cm s�1 for the polymer concentrations of 0.01, 0.02,and 0.03mg/mL, respectively. The value of Ps for the pure POPCwas previously reported to be (1.88 ( 0.8) � 10�11 cm s�1.16

Thus, the presence of polymer 3 in the liposome system causedan increase of the bilayer permeability by several orders ofmagnitude.Molecular Dynamics Simulations. To get some insight into

the nature of interaction of polycation 3 with lipid membrane atthe molecular level, we performed molecular dynamics (MD)simulations of two model membranes, composed of 128 POPCmolecules hydrated with ca. 30 water molecules per lipid, intowhich two decamers of allylo-N,N-dimethyl-N-hexylammoniumchloride (decamer 3) were inserted. Both simulated systemsbecame fully equilibrated within the first 10 ns of simulation, asindicated by the potential energy, density, and area per lipidconverging to constant values (Figure s1, Supporting In-formation). Three of the four simulated decamer molecules

Figure 3. Distribution profiles of the hydrodynamic diameters mea-sured by light scattering experiments for the POPC liposomes (cPOPC =1.25 mg/mL) before and after addition of polymer 3 to the indicatedconcentrations.


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(which will be referred to as A, B, C, andD) remained emerged inthe hydrocarbon region of the bilayer during the whole simula-tion period. This result could be surprising, considering the goodsolubility of polycation 3 in water, which shows its undoubtedlyhydrophilic character. Inspection of the trajectory reveals thoughthat during the equilibration period the bilayer underwent asubstantial molecular reorganization in the hydrophobic regionsadjacent to decamers 3, which forced some of the neighboringPOPCmolecules to reorient, and led to creation of a hydrophilicenvelope surrounding the polycation molecule. This new, en-ergetically favorable local environment allowed the decamermolecules to remain in the bilayer core for the whole simulationperiod. This structural rearrangement resulted in the formationof a hydrophilic pore spanning the bilayer, which allowed waterand ions to flow across the membrane.The fate of the fourth molecule (D, membrane II) was

different; within the first few nanoseconds of the simulation, ittranslocated entirely into the water�bilayer interface of thePOPC membrane and remained there for the rest of thesimulation (Figure 6).

Area per Lipid. The area per lipid was calculated as the ratio ofthe area of the xyplane of the simulation box to the number of POPCmolecules in the monolayer (64). This parameter was practicallyidentical for both membranes and equals 0.822 ( 0.002 nm2

(membrane I) and 0.818( 0.002 nm2 (membrane II). Comparisonwith the area per lipid of the pure POPC membrane (0.682 (0.002 nm2) shows that the insertion of two decamer molecules andthe concomitant structural changes resulted in a considerableexpansion of the membrane surface. An experimental estimate ofthe area per lipid for fully hydrated POPC is 0.64 ( 0.01 nm2,37

which is in very good agreement with the simulated value.Density Distribution. Figure 7 presents a comparison of

number density profiles along the z direction (normal to themembrane) calculated for membrane I and the pure POPCbilayer. The curves describing the distribution of atoms consti-tuting the polar headgroup of POPC are significantly broader forthe POPC�decamer 3 system than for the pure POPC mem-brane, and, as opposed to the latter, they extend visibly towardthe bilayer center. The presence of the polar groups in thehydrophobic region of the membrane accompanied by a nonzerowater concentration in this region is in accordance with theobserved reorientation of some POPC molecules and formationof hydrophilic pores in the membrane structure. The thickness ofmembrane I, calculated as the distance between the maxima inthe distribution curves of the POPC choline nitrogen, is practi-cally the same as for the POPC bilayer (3.45 and 3.49 nm,respectively).Radial Distribution Function. To characterize the surround-

ings of the decamer ammonium nitrogen atoms (NA), the radial

Table 1. Values of the Mean Hydrodynamic Diameter (dz), Polydispersity (PDI), and Zeta Potential (ζ) at 298 K of POPCLiposomes Dispersed in 1 mM NaCl Solution Treated with Polymer 3 (Values Are the Mean ( Standard Deviation)

system content of 3 [%] dz [nm] PDI ζ (mV)

POPC vesicles (cPOPC = 1.25 mg/mL) 0 124.6 ( 0.8 0.09 ( 0.01 2.3 ( 1.0

POPC vesicles (cPOPC = 1.25 mg/mL) with 3 (c3 = 0.01 mg/mL) 0.8 132.8 ( 3.2 0.11 ( 0.02 15.6 ( 0.9





Figure 4. Cryo-TEM micrographs and the diameter profiles of POPCliposomes (cPOPC = 2.5 mg/mL) coated with polymer 3 (c3 = 0.06mg/mL, 2.35 wt% content of 3). Specimens were vitrified immediately (A)and 24 h (B) after the polymer addition. Scale bars represent 200 nm.

Figure 5. Time-course of calcein leakage from POPC liposomes causedby different concentrations of polymer 3.


Langmuir ARTICLE

pair distribution function (rdf) values for atomic pairs NA�P(phosphor atom in lipid phosphate group), NA�O(P)(nonester oxygen atoms in the lipid phosphate group), andNA�OW (water oxygen) were calculated (Figure 8).The shape and the maxima locations of the rdf curves are very

similar for all four decamer molecules in both simulated mem-branes. It is worth reminding at this point that molecule D(membrane II), as opposed to the other three decamer 3molecules, remained completely immersed in the hydrophilicregion of the bilayer for most of the simulation period. The factthat its rdf curves do not differ from those calculated for themolecules partially or completely incorporated into the region ofthe POPC hydrocarbon chains shows that, regardless of thelocation of a decamer molecule inside the membrane, its closestvicinity is very much alike. The gNA�P(r) function shows adistinct peak at a distance of 0.508 nm. Integration of this peakgives the average number of P atoms per one NA nitrogen equalto 0.89. The maximum of the gNA�O(r) appears at a distance of0.415 nm. The average number of O(P) atoms per one ammo-nium group was found to be equal to 1.32. These results allowone to conclude that the hydrophilic heads of POPC become

oriented in such a way that one or both of the nonester oxygenatoms of the phosphate group point toward the decamerammonium group. The coordination number for the P atomcould suggest there is nearly one PO4

� group in the close vicinityof each decamer ammonium group. However, due to the shortdistance between the NA atoms in decamer 3 and a possibility ofthe same P atom being within the coordination radius of two NAatoms, the actual number of the phosphate groups interactingclosely with the decamer is smaller. The average number of thephosphate groups inside the pores was estimated by counting thenumber of phosphorus atoms found in the bilayer hydrophobiccore. The approximate extent of this core was defined as a region(1.0 nm from the bilayer center. The calculation over the wholetrajectory with a 1 ns time window gives the average number of14 PO4

� groups engaged in the pores creation, that is, sevengroups per pore. It should bementioned that most of these PO4

�

groups occupy the “entrance” regions of a pore. Hardly any PO4�

group could be found within the region(0.2 nm from the bilayercentral line.The rdf for the NA�OW pair has been compared to the rdf

calculated from a trajectory of a 40 ns simulation run of a systemconsisting of four decamermolecules immersed in a water box. Inboth systems, the gNA�OW(r) function shows a distinct max-imum at r = 0.43 nm (Figure 8). Integration of the first peak givesthe average number of water molecules in the first solvationsphere of the ammonium nitrogen in decamer 3. These coordi-nation numbers are very similar for bothmembranes and equal to10.5 (membrane I) and 11.1 (membrane II). These values areonly slightly smaller than the coordination number calculatedfor the fully hydrated decamer molecule (13.2, respectively),

Figure 6. Snapshots of membrane I (a) and membrane II (b) at t = 100ns taken in the yz plane. Molecules A, B (membrane I), and C(membrane II) are engaged in the hydrophilic pore formation. Mole-cules of decamer 3 are shown as sticks (violet, main chain; yellow, sidechains) and small blue spheres for ammonium nitrogens. P atoms(orange) and nonester O atoms (red) in phosphate groups of POPClipids are shown as spheres. Gray sticks represent alkyl chains of POPC.The water molecules are displayed as blue sticks.

Figure 7. Symmetrized number density profiles of water (OW) and theatoms of the POPC hydrophilic head, for membrane I (a) and purePOPC (b). The density of water (OWatoms) has been attenuated with afactor of 0.25 to obtain a suitable common intensity scale.


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showing that the hydration state of decamer 3 in themembrane iscomparable to that in bulk water.Water Diffusion through the Pores. A visual inspection of the

trajectory evolution shows that a time period of ca. 6 ns issufficient for most of the water molecules, which are present inthe central region of the bilayer at the observation starting point,to drift out into the headgroup region of the membrane. Mean-while, other water molecules, and also the Cl� anions, diffuseinto the pore.To get a deeper insight into the mobility of water molecules

inside the pores, a mean square displacement (MSD) wascomputed for “inner” and “outer” water molecules. By “inner”water, we mean the water molecules enclosed in the pores(present in the hydrophobic part of the membrane). The verticalspan of the pore was estimated as equal to the thickness of thewater-free region in the OW distribution curve of pure POPC(see Figure 7), which gives the value of ca. 1.6 nm. However, toensure that the MSD calculation for “inner” water will notinclude water molecules that leave the pore within the timeperiod used for MSD calculation (500 ps), we limited thedefinition to water present in the central, cylindrical region ofthe pore. The length of this part of the pore has been estimated asca. 1.2 nm, on the basis of visual inspection of simulationsnapshots. Accordingly, a water molecule was counted as “inner”if the z coordinate of its OW atom was found at a distance(0.6 nm from the membrane center (see Figure s2 in theSupporting Information). The “outer” water group included allof the remaining water molecules. The identity of “inner” watermolecules was established for the trajectory frames at 40, 50, 60,

70, 80, and 90 ns, and the MSD’s for both the “inner” and the“outer” molecular groups were calculated for a period of 500 psstarting from the corresponding initial trajectory frames.Figure 9 presents averaged MSD curves for the displacement

along the z and the xy (lateral) directions. This plot shows thatthe rate of diffusion along the z direction of the “inner” water iscomparable to that for the “outer” water. Diffusion coefficients,calculated from the slopes of the linear part of the appropriateMSD curves, equal (0.69 ( 0.11) � 10�5 (“inner” water) and(0.72 ( 0.03) � 10�5 cm2/s (“outer” water). The mobility ofwater molecules in the lateral direction is distinctly different forboth water groups. The steep slope of the MSD curve for the“outer” water and the high value of its Dxy diffusion coefficient((1.95 ( 0.05) � 10�5 cm2/s) show that the lateral motion ofwater molecules on themembrane surface plane is rather fast. Onthe contrary, theDxy for the “inner”water is 1 order of magnitudesmaller (0.19 ( 0.05) � 10�5 cm2/s), indicating a very poormobility of the in-membrane water in that direction. Undoubt-edly, this is caused by a limited diameter of the hydrophilicchannel formed around vertically oriented decamer molecules.Location and Conformations of Decamer 3. To gain some

information about the orientation of the main hydrocarbonchain of the decamer molecule and its conformational state, afew geometric parameters have been analyzed. The z-tilt angle,defined as the angle between the C(21)�C(3) vector (Figure 2)and the bilayer normal, informs about the orientation of the

Figure 9. The mean square displacement (MSD) of “inner” (red lines)and “outer” (blue lines) water calculated along the z direction (solidlines) and in the xy plane (dashed lines).

Figure 10. Distribution of the z-tilt angle of the C(21)�C(3) vector(see Figure 2 for the definition).

Figure 8. The gNA�X(r) radial distribution functions for the atomicpairs for membrane I (a) and membrane II (b); NA, ammoniumnitrogen of decamer 3; P and O(P), phosphorus and nonester oxygenin the phosphate group of POPC; OW, water oxygen. The functionshave been averaged over both decamer 3 molecules in each membrane.


Langmuir ARTICLE

decamer main chain with respect to the POPC alkyl chains(which, on the average, are tilted ca. 31� to the membranenormal). The distribution of this tilt angle, averaged over the last80 ns of the simulation, is shown in Figure 10. As may beconcluded from this diagram, the decamer molecules incorpo-rated into the region of the POPC alkyl chains assumed anorientation with their main axis at a rather low angle (10��45�)with the membrane normal. The small peak at 83� in thedistribution curve for the C molecule originates from the last20 ns of the trajectory and reflects the drastic reorientation thismolecule underwent at the end of the simulation. On thecontrary, the molecule entirely immersed in the headgroupregion of POPC (molecule D) prefers the orientation with itsmain axis nearly perpendicular to the z direction (parallel to themembrane plane).The length of the C(21)�C(3) vector (Figure 2) is an

indicator of the degree of bending or coiling of the main chain.This vector length, as measured on the optimized moleculemodel, equals ca. 2.24 nm in the case of the fully stretched chain(all of the dihedrals along the chain equal to 180�). In moleculesA and B, the average C(21)�C(3) distance equals ca. 1.55�1.65 nm (Figure 11a), which indicates a slight bent of the mainchain. The distribution for molecule D in particular shows abroad shoulder at the close distant side, which suggests a greaterflexibility of the polymer main chain adsorbed at the water�lipidinterface.Relative arrangement of the ammonium residues in decamer

3 was established by calculating the distribution of the anglebetween the vectors defined by the CB and NA atoms (Figure 2)in the successive side chains (Figure 11b). The distribution curves

are centered around the angle of 100� (molecules B�D) and110� (molecule A). This angle is pretty close to 120�, the valuecharacteristic for a regular arrangement of the side chains arounda 3-fold symmetry axis, collinear with the main axis. Such aregular arrangement of the NA atoms is clearly visible whenlooking at themolecule along its main axis (Figure s3, SupportingInformation).

’DISCUSSION

To explore the effect of a positively charged and hydropho-bically modified polyelectrolyte on a neutral lipid membrane, wesynthesized a strong polycation modified with short side alkylchains (polymer 3) and studied its interactions with a zwitter-ionic lipid membrane using experimental methods and MDsimulations. Polymer 3 has the quaternary ammonium groupsin its structure; therefore, its properties, especially the charges onthe backbone, are independent of pH of the environment.Cationic polymers containing pendant quaternary ammoniumgroups are the most promising candidates as the effectiveantimicrobials and biocides.38 These groups were modified byattachment of hexyl chains. As was shown previously, poly-4-vinyl pyridine (PVP) with hexyl units on its backbone had thehighest antibacterial activity in the series of polymers with variouslengths of alkyl units.4 Such structure of polymer 3 makes itrelatively well soluble in water.

Unilamellar liposomes composed of lipids with zwitterionicphosphatidylcholine heads have been widely utilized as a modelbiomembrane system, because such lipids are the main consti-tuents of natural membranes. Interactions between variouspolycations and the liposomes have been extensively studied tomimic behavior of these macromolecules in contact with cellularmembranes.2,6 Taking into account the results of studies re-ported in the literature, one could expect various possibilities ofpolycation behavior at the zwitterionic membrane surface. Theyinclude (i) lack of interactions,6 (ii) adsorption on the bilayerhydrophobic surface,13 (iii) insertion into the hydrophobic coreof the bilayer,2 and (iv) disruption of the bilayer with the forma-tion of mixed polymer�lipid micelles or other aggregates.39

Therefore, several issues such as the location of polymer 3withinthe lipid bilayer (at the surface or entrapped in the bilayer) or thepossibility of disruption of liposomes into small fragments needto be considered in the present study.

The results of DLS and zeta potential measurements providedevidence that the polymer can interact with the POPC mem-branes, causing a slight increase of the hydrodynamic diameter ofthe vesicles and a drastic increase of zeta potential. It is well-known that the adsorption of polymers on the liposome surfaceor penetration of polymers into the liposome bilayer willcorrespondingly change the size of the vesicle.5 However, thesmall increase in liposome size induced by polymer 3 could beattributed to the fact that it mainly incorporates into the lipidbilayer instead of being adsorbed on the surface of theliposomes.5 The increase of vesicle diameter upon incorporationof polymer 3 can additionally affect the stability of the vesicles. Aswas shown, the stability of liposomes is connected with thecurvature of the bilayer.40 Small unilamellar vesicles are inher-ently unstable due to their highly strained, curved bilayers. Theincrease of vesicle diameter involves a reduction of bilayercurvature improving the vesicle stability. The increase of surfacecharge usually increases the zeta potential of particles (theelectric potential in the slipping plane) whose values are well

Figure 11. (a) Distribution of the length of the C(21)�C(3) vector(see Figure 2 for the definition). (b) Distribution of the angle betweenthe CB�NA vectors of the successive side chains (see Figure 2).


Langmuir ARTICLE

correlated with the stability of suspensions. The ζ-potential is aquantity well accessible experimentally, for example, by using themicroelectrophoretic method as was done in this work. Uponaddition of the increasing amount of polymer 3 to the POPCdispersion, a rise of the zeta potential was observed, indicatingthat the surface charge on the liposomes increased. This is clearevidence that polymer 3 can be also physically adsorbed at thevesicle surface probably with alkyl chains immersed into thebilayer and the ammonium groups exposed to the bulk solution.The presence of the hydrophilic polymer 3 layer at the liposomesurface leads to polymer-coated, electrostatically stabilized vesi-cles. The appearance of positive charge at the surface ofliposomes is essential for the liposome stability. Similar resultswere obtained previously for zwitterionic liposomes covered bychitosan or alkylated chitosan (cationic polyelectrolytes).12 Itwas shown that the adsorption of these macromolecules on thelipid membranes can be monitored by changes in the ζ-potentialof liposomes. The chitosan�lipid bilayer interaction enhancesvesicles stability with regard to various stresses (for example, pHand salt shocks).

The incorporation of the polymer into the hydrophobic part ofmembrane should have a substantial impact on the permeabilityof the membrane. Indeed, the presence of polycation 3 caused asignificant increase in release of hydrophilic compounds en-trapped inside the POPC liposomes, as was shown using calceinas a model compound. These findings indicate that the poly-mer�lipid interactions can induce conformational rearrange-ment of the polymer and result in the insertion of the polymer

into the lipid membrane, possibly causing alternations in themembrane structure. Earlier, Eren et al. observed increasedrelease of calcein from liposomes under the influence of polycat-ions with different quaternary alkyl pyridinium side chains.4 Toexplain this phenomenon, the authors suggested that the phos-pholipid membrane undergoes disruption. To check such possi-bility, we applied the direct microscopic observation of lipidvesicles treated with polymer 3. The cryo-TEM visualization ofthe POPC liposomes incubated for different time with thepolymer showed that the lipid vesicles maintain their integrityduring interactions (Figure 4). This is strongly supported byDLSmeasurements showing invariable in the size profile of polymercoated liposomes with time. Therefore, the enhanced perme-ability of POPCmembrane covered with polymer 3 is caused by aformation of pores (holes) in the bilayer structure. The forma-tion of pores in phospholipid membranes has been demonstratedusing AFM microscopy. Several AFM studies on supported lipidbilayer interacting with different polycations showed that thesemacromolecules disturb the structure of lipid bilayers, leading toformation of nanoscale holes.41�44 The defects in the membranewere accompanied by a significant increase of membrane perme-ability, as indicated by in vitro observed leakage of the cytosolicenzymes and free diffusion of fluorescent dye molecules into andout of the cells.35 Comparison of the effect exerted by differentpolymers led to a conclusion that the size of the polymermolecule does not affect its ability for hole formation, but thecharge density on the polymer chain influences markedly thedegree of membrane permeability. The defect-creating ability of

Figure 12. Snapshots taken after different times of simulation showing the pore formation in membrane I. Penetration of water molecules into thecavities occupied by decamer 3 is followed by a progressive reorientation of the POPC hydrophilic heads. Twomolecules of decamer 3 are show as sticks(violet, main chain; yellow, side chains) and small blue spheres for ammoniumN. P atoms (orange) and nonester O atoms (red) in phosphate groups ofPOPC lipids are shown as spheres. The water molecules are displayed as blue sticks.


Langmuir ARTICLE

different polymers was studied in a whole cell patch-clampexperiment on living cells.45 A substantial increase of the plasmamembrane conductance was observed after exposing the cellsto positively charged polymers (PEI, PLL, and PAMAMdendrimers), whereas neutral polymers like poly(vinyl alcohol)and poly(ethylene glycol) did not show this effect. Theseobservations suggest that formation of the permeability-promoting defects in the lipid bilayers is most probably a resultof strong interactions of the lipid molecules with the electrostaticpotential of the polycation molecule. Such a hypothesis issupported by the well-known fact that application of an externalelectric field to cells or tissues leads to a drastic increase of theirpermeability to ions and small polar molecules. This effect isattributed to creation of transient water filled pores in the cellmembranes on applying a transmembrane potential of severalhundred milivolts (so-called electroporation).46,47

The incorporation of the positively charged polymer into themembrane can induce alterations in lipid packing and organiza-tion. This process was studied at the molecular level by the MDsimulations of POPC membranes doped with the fragments ofpolymer 3. The simulations reveal an important influence ofpolymer 3 on the membrane integrity and functionality. Thecritical structural modification of the membrane was connectedwith the formation of hydrophilic pores around the decamermolecules incorporated in the core region of the membrane(Figure 6). Such pores were observed to form spontaneously in atime span of a few nanoseconds and remain stable within thesimulation time scale (100 ns). Three of the four simulatedmolecules were involved in the creating of pores. The pore-promoting molecules occupied positions in the middle of themembrane, with their main chain nearly straightened out andtheir hexyl side chains directed toward the hydrocarbon region ofmembrane. The fourth of the simulated molecules adopted alocation above the carbonyl groups region of POPC with thebackbone parallel to the membrane plane. The results of MDsimulations of decamer 3 show that there are at least twoenergetically preferable locations of the polycation 3 chain inthe lipid membrane. Therefore, we believe that the backbone ofpolymer 3 in contact with a lipid membrane can enter easily intothe bilayer, inducing formation of pores. Some of its fragmentsmay stay adsorbed on themembrane plane, increasing the surfacepotential, as shown with the zeta potential measurements.

The molecular mechanism of pore formation is presented inFigure 12. The process was initiated by some water moleculesdrifting into the region occupied by the polycation, well belowthe line of the lipid hydrophilic heads (Figure 12, 0.1 ns). Asignificant number of water molecules around decamer 3 mole-cules could be observed even at the very early stage of thesimulation (t < 200 ps). The formation of a water column acrossthe membrane was soon followed by reorganization of someadjacent POPCmolecules, which began to reorient in such a waythat their phosphate groups become pointed toward the ammo-nium nitrogen in the decamer (Figure 12, 1 and 4 ns). Thisprocess was most probably driven by the strong electrostaticinteraction between the positively charged ammonium moietiesof decamer 3 and the negatively charged phosphate groups ofPOPC and was additionally facilitated by the presence of watermolecules in close vicinity of the decamer. This new organizationof POPC hydrophilic head groups creates a shield protecting the�N+(CH3)2� groups from unfavorable interactions with thenonpolar medium of POPC acyl chains, thus contributing to theenergetic stabilization of the system. This rearrangement leads to

an important modification of membrane functionality; hydro-philic pores constitute around decamer 3molecules. They enablemigration of water molecules and Cl� ions across the membrane,thus increasing considerably its permeability (Figure 12, 8 ns).Such pores were observed to form around molecules A, B, and Cwithin the first few nanoseconds of the simulation. The porescreated around molecules A and B, once formed, remained stablefor the whole simulation period, although their internal surfacemanifested significant flexibility. In particular, a considerablemobility of the PO4

� groups around the decamer moleculewas observed. Molecule C (membrane II) behaved identically asmolecules A and B for most of the simulation time, but, withinthe last 20 ns of the simulation period, it underwent a substantialreorientation and assumed a position with its main axis almostperpendicular to the acyl chains of POPC. This reorientation,accompanied by a rearrangement of neighboring POPC mol-ecules, led to a considerable enlargement of the hydrophilicchannel (Figure 6b). To estimate the lateral size of the pores, awater density xy map was computed for the bilayer core regionfor both systems. The average diameter of circular blotchesmarking the region of high water density around molecules A,B, and C (the latter calculated from the first 80 ns) equals∼1.7 nm. Themap for the membrane II, computed for the last 20ns of the trajectory, shows a region of high water density withellipsoidal shape, with the long and the short axes equal to ∼2.7and ∼1.7 nm, respectively (Figures s4 and s5, SupportingInformation).

The structural modification described above resulted in aconsiderable increase of the membrane surface. The area perlipid for the POPC membrane doped with decamer 3 is by ca.21% larger than for the pure POPC membrane. This result iscorroborated by the experimentally observed increase of thevesicles diameter after addition of polymer 3 to the liposomedispersion. Assuming a spherical geometry of the vesicles, thegrowth of the diameter from ca. 125 to 136 nm corresponds tothe increase of its surface by some 18%.

The process of hydrophilic pore formation induced by differ-ent stimuli has been addressed in numerous MD studies. Variouslipid bilayers have been simulated under the effect of a uniform,transmembrane electric field (electroporation)48�52 or a fieldcreated as a result of an imbalance of ion concentration on bothleaflets of the bilayer.53�56 Many of these works report acommon scenario of pore formation, regardless of the origin ofthe electric field applied. The process is triggered off by somewater molecules penetrating the lipid/water interface and in-truding into the hydrocarbon region of the bilayer. Such waterdefects present on both sides of the bilayer tend to grow andmerge until the water wires/columns spanning the whole bilayeris created. Formation of a new water/lipid interface is soonfollowed by a reorientation of lipids, which line the inner surfaceof the pore and stabilize it. The entire pore formation processtakes no more than 4�5 ns.

The mechanism of pore formation as observed in our simula-tions is in agreement with the results of many MD studies onpore formation due to transmembrane potential. It seemsjustified though to propose that the polycation-mediatedporation of the cellular membrane is basically of the same mole-cular nature as the electroporation and it is driven by some waterdefects arousing as a result of a response of the water dipols to theapplied or local electric field. The intensity of the electric fieldaround decamer 3 molecule may be roughly estimated byassuming a cylindrical shape of the molecule. Taking a cylinder


Langmuir ARTICLE

of the height of 1.7 nm and the radius of 0.75 nm (approximatelongitudinal and lateral dimensions of the molecule), whichencloses a charge of 10e (i.e., 1.602� 10�18 C), gives the electricfield of the magnitude of ∼2 V/nm. The actual field will beweaker by some 30�40% due to partial neutralizing of thepositive charge by some Cl� anions remaining within the cylinderspace. Still, its magnitude is of the same order as the transmembranepotential applied in the electroporation.

’CONCLUSIONS

Our results show that the hydrophobically modified polycat-ions can readily and quickly associate with and insert into thezwitterionic lipid membrane structures. The vesicle structuresafter the incorporation of the polymers are retained instead ofbeing disrupted into small fragments. The polymer-coveredliposomes have no tendency to form aggregates and maintaintheir integrity for long time. This process may be driven by acombination of the electrostatic interactions between cationicpolymer and the anionic phosphate groups of lipid and hydro-phobic interactions between hexyl moieties of polycation 3 andthe lipid acyl chains. The association and incorporation ofpolymer 3 in the lipid membrane cause alternation of packingand organization of membrane lipids, which directed theirphosphate groups toward the ammonium N atoms of thepolycation. The hydrophilic pore thus stabilized facilitates diffu-sion of water molecules and Cl� anions across the bilayer.

’ASSOCIATED CONTENT

bS Supporting Information. The value of ζ-potential of theobjects formed in a 0.03 mg/mL solution of polymer 3. The timeprofile of the potential energy, system density, and area persurfactant molecule for the first 20 ns of the simulation. Defini-tion of the “inner” water. Conformation of decamer 3 (moleculeA) after 50 ns of the simulation. Water number density xymaps.This material is available free of charge via the Internet at http://pubs.acs.org.

’AUTHOR INFORMATION

Corresponding Author*Phone: +48 12 6632020 (M.K.); +48 12 6632263 (D.J.). Fax:+48 12 6340515. E-mail: [email protected] (M.K.);[email protected] (D.J.).

’ACKNOWLEDGMENT

We thank the Polish Ministry of Science and Higher Educa-tion for financial support in the form of Grant No. N N209118937. J.B. acknowledges the support of the Czech GrantsLC535, MSM0021620806, and AV0Z50110509. The MD simu-lations were performed on the HPCluster Platform 3000 BL 2�220 available at the Academic Computer Centre CYFRONETAGH, Krak�ow, Poland.

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Interactions of a Hydrophobically Modified Polycation with Zwitterionic Lipid Membranes

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