Interactions of a Hydrophobically Modified Polycation with Zwitterionic

ARTICLE pubs.acs.org/Langmuir

Interactions of a Hydrophobically Modified Polycation with Zwitterionic Lipid Membranes Mariusz Kepczynski,*,† Dorota Jamr
oz,*,† Magdalena Wytrwal,† Jan Bednar,‡,§ Ewa Rzad,† and Maria 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 ABSTRACT: The interactions between synthetic polycations and phospholipid bilayers play an important role in some biophysical applications such as gene delivery or antibacterial usage. Despite extensive investigation into the nature of these interactions, their physical and molecular bases remain poorly understood. In this Article, we present the results of our studies on the impact of a hydrophobically modified strong polycation on the properties of a zwitterionic bilayer used as a model of the mammalian cellular membrane. The study was carried out using a set of complementary experimental methods and molecular dynamic (MD) simulations. A new polycation, poly(allyl-N,Ndimethyl-N-hexylammonium chloride) (polymer 3), was synthesized, and its interactions with liposomes composed of 2-oleoyl-1-palmitoyl-sn-glycero-3phosphocholine (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 membrane functionality 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, thus increasing considerably its permeability.

’ INTRODUCTION The interactions between polycations and lipid or cell membranes play an important role in many biophysical applications. The most important of them include (i) delivery of genetic materials to cells in a targeted and safe manner,1
3 (ii) usage as biocidal agents,4
6 and (iii) obtaining stabilized vesicles by covering the liposomes surface with multilayer films.7,8 Because of these issues, the impact of both natural and synthetic polycations on phospholipid bilayer has been extensively studied using various experimental techniques.9 The success of gene therapy largely depends on the availability of suitable delivery vehicles. Polycations, especially poly(ethylenimine) (PEI), are frequently studied as nonviral synthetic transfectants, which are characterized by excellent gene complexing ability (formation of polyplexes) and eminent transfectant properties.3 Unfortunately, they show in vitro cytotoxicity, which constitutes the major challenge for their clinical applications.3,10 Transport of molecules to and from the cell nucleus, mediated by polycations, is likely to be the result of nonspecific interactions between the polyplexes and the nuclear membrane.2 To date, research has focused rather on the r 2011 American Chemical Society

efficiency of transfecting the cell membranes. The molecular mechanisms of both the transfection and the polycation-induced cytotoxicity are yet unknown. Much empirical evidence suggests though that the important role in these processes is the ability of polycations to disrupt the nuclear membrane and to increase the membrane permeability. It is known that the transfection process occurs when small pores open in the nuclear membrane to admit genetic materials.2 It is well-known that most antimicrobial peptides disrupt the bacterial membrane via transmembrane pore formation and/or membrane destabilization.11,12 The majority of these peptides are cationic or amphipathic. Recently, polymers containing quaternary ammonium or alkyl pyridinium moieties have been commonly used as biocides.5 The quaternary amino groups in antimicrobial polymer are believed to be responsible for causing cell death by disrupting the cell membrane, thus allowing release of the intracellular contents. Received: September 24, 2011 Revised: November 8, 2011 Published: November 15, 2011 676

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Langmuir It was shown that polycations associate strongly with or penetrate into negatively charged liposomes bilayer, mostly due to the electrostatic interactions.6,9 The interactions between positively charged macromolecule and a zwitterionic membrane, commonly used as a mammalian membrane model, are more puzzling. Quemeneur et al.13 studied adsorption of chitosan (a cationic polysaccharide) and chitosan alkylated with hydrocarbon chains of different lengths on phosphatidylcholine (PC) liposomes. It was demonstrated that the alkyl chains do not interact with the lipid membrane and the chitosan adsorption is governed by electrostatic interaction mechanism. Eren et al. studied amphiphlic polyoxanorbornene functionalized with quaternary pyridinium side moieties substituted with various alkyl chains.4 The authors showed that the polymers modified with short alkyl chains (ethyl, buthyl) introduced only little disruption of PC membranes, while those with longer side chains (hexyl to decyl) were much more active toward the PC vesicles. Ding et al. studied the interactions of antimicrobial cationic conjugated polyelectrolytes (CPEs) with membranes composed of lipids with varying headgroup charge.6 Their results showed that the polymers can efficiently associate with and insert into anionic phosphatidylglycerol (PG) membranes. However, these polymers did not interact with the zwitterionic lipid membranes composed of PCs. Wang et al. studied a series of CPEs of significant structural diversity.14 They concluded that the functional groups on the side chains dominate the polymer ability for membrane perturbation and that the macromolecules with the high charge density and hydrophobic alkyl side chains have the affinity to the zwitterionic membranes. Sikor et al. studied the interactions between PEI and the zwitterionic lipid vesicles.2 It was demonstrated that at low salt concentration the introduction of the polymer leads to aggregation of vesicles with formation of stable clusters, while at high salt content there is no aggregation and PEI can penetrate into the bilayer. On the basis of the presented above literature, one can conclude that further studies giving an insight into the molecular aspects of the interactions between cationic polymers and zwitterionic membranes are necessary to better understand the nature of these interactions. Molecular dynamics (MD) simulations can provide the necessary insight into several aspects of the interaction between bilayers and various compounds and have been extensively used in the past few years to describe lipid membranes in terms of their structure and dynamics.15 MD simulation of interaction between polycations and lipid membranes has been addressed in a few studies. Lee and Larson performed coarse-grained MD simulations of polyamidoamine (PAMAM) dendrimers of different size and with different terminal groups put in contact with dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) bilayers.16
18 Their simulations show that charge on the terminal groups plays a crucial role in this interaction. The dendrimers with charged terminal groups deform and insert into the bilayer, whereas those with uncharged terminals remain on the membrane surface. The bigger size and high concentration of the dendrimer facilitate its penetration into the membrane. On the contrary, a linear polycation, poly-L-lysine (PLL), was not observed to insert into the bilayer despite its high charge density.17 The importance of electrostatics in the PAMAM
lipid interaction has been supported by all-atom MD study of PAMAM approaching a DMPC surface.19,20 Free energy calculations performed for dendrimers with three different terminal groups show that the gain in free energy on binding to the membrane surface is much larger for the charged molecules.

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This study also points out the role of lipid chain mobility in the binding of dendrimers to the bilayer surface. Membrane in the fluid phase was found to bind the dendrimer molecules stronger than while in the gel phase.20 In this Article, we examined the effect of a hydrophobically modified strong polycation on the oleoyl-1-palmitoyl-sn-glycero3-phosphocholine (POPC) bilayer. For that purpose, a new polymer, poly(allyl-N,N-dimethyl-N-hexylammonium chloride) (3), was synthesized in a two-step modification of a commercially available poly(allylamine hydrochloride) (PAH). PAH was chosen because that polymer has found a wide variety of applications in both nanotechnology and biomedical fields.7,8 The POPC vesicles were treated with aqueous solutions of polymer 3 at various concentrations, and several experimental techniques were employed to study the polymer
membrane interactions. We used dynamic light scattering (DLS) to measure the size of the lipid vesicles and verify the possibility of vesicle aggregation. Additionally, we measured zeta potentials of the liposomes to confirm adsorption of polymer 3 on the liposome surface. Cryotransmission electron microscopy (Cryo-TEM) was applied to observe the morphology of the polymer-covered liposomes. Change of the permeability of the POPC membrane induced by the presence of polymer 3 was monitored using a fluorescence method. Finally, we performed MD computer simulations to get insight into the polycation
lipid interactions at the molecular level. The MD simulations allowed one to determine the position and orientation of the studied molecule and to follow the molecular motion at a level typically not accessible to the experimental observation.

’ EXPERIMENTAL SECTION Materials. 2-Oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC, g99.0%) and calcein were obtained from Sigma. Poly(allylamine hydrochloride) (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 (99.9 atom % D) were purchased from Aldrich and used as received. NaI (g99.0%) was received from POCh (Gliwice, Poland). Benzoylated dialysis tubing (2 000 g/mol molecular weight cutoff) and Sephadex G-50 (20
50 μm) were purchased from Sigma-Aldrich. Millipore-quality water 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 in D2O/DMSO-d6 mixture (1:3 v/v) using DMSO-d6 residual peaks as internal standards. IR spectra were recorded on a Nicolet IR200FT-IR spectrophotometer with an ATR attachment. Elemental analysis was performed on a EuroEA 3000 Elemental Analyzer. Fluorescence spectra were recorded on a Perkin-Elmer LSD50B spectrofluorimeter equipped with a thermostatted cuvette holder. Synthesis of 3. Synthesis of poly(allyl-N,N-dimethyl-N-hexylammonium chloride) (3) is schematically presented in Figure 1. 0.5 g of compound 1 (5.34 mmol of the amino groups) was dissolved in 5 mL of water. One milliliter of 5% NaOH solution was added, and then the pH of the solution was adjusted to 3
4 using 1% acetic acid. After 30 min of stirring, 1.6 mL of hexanal (1.5-fold excess to the amino groups of 1) was added, and the reaction mixture was stirred for 24 h at room temperature. Next, 0.52 g of NaBH4 dissolved in water was dropwise added (1.5-fold excess to the added aldehyde), and the mixture was stirred for 12 h. The crude intermediate product was purified by dialysis against water. Finally, water was evaporated under a vacuum to give poly(allyl-N-hexyl-amine) (2) as white crystals (0.8 g, 53.1%). 677

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Figure 1. Synthesis of poly(allyl-N,N-dimethyl-N-hexylammonium chloride) (polymer 3). IR νmax (cm
1): 3395 (N
H); 2926 (C
H); 1600 (N
H); 1511, ̅ 1 H NMR (DMSO-d6:D2O (3:1 v/v)): δ (ppm) 0.76
1.03 1460 (C
C). (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 with 33% 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 was stirred for 0.5 h at room temperature. Next, 1.4 M NaOH, 6.7 mL of CH3I (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, to exchange the iodide counterions of the methylated derivative of polymer 2 for chloride ones, the reaction mixture was placed in dialyzing tube and dialyzed in the following sequence (4 days in each environment): against deionized water, 0.1 M KCl, and again deionized water. Finally, water was evaporated under a vacuum to give the desired product 3 as fine pale yellow crystals (0.77 g, 66.2% yield). IR νmax (cm
1): 3374 (N
H); 2934 (C
H); 1633 (N
H); 1481 ̅ (C
C). 1H NMR (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 was placed in a volumetric flask, and the solvent was evaporated under a gentle stream of nitrogen to complete dryness. One millimolar solution of NaCl was added until a desired lipid concentration was attained (usually 2.5 mg/mL), and the sample was vortexed for 5 min. The resulting multilamellar vesicle dispersion was subjected to five freeze
thaw cycles from the liquid nitrogen temperature to 60 °C and then extruded six times through the membrane filters with 100 nm pores using a gas-pressurized extruder. The pH value of the POPC dispersions was determined to be 6.5. Coating of Liposomes with Polymer 3. A 0.5 mL dispersion of liposomes (2.5 mg/mL) in 1 mM NaCl was placed in a sonication bath, and the appropriate volume of 0.5 mg/mL solution of the polymer was quickly added. 1 mM NaCl was next added to the final volume of sample of 1 mL. Cryo-Transmission Electron Microscopy (cryo-TEM). CryoTEM was used to visualize the morphology of liposomes as described

previously.22 This technique allows the least perturbing and direct imaging of the hydrated sample. The liposome dispersion was prepared at cPOPC = 2.5 mg/mL, and the concentrated solution of polymer 3 was added to the final concentration of 0.06 mg/mL (2.35 wt % content of 3). Three microliters of the sample solution was applied to an electron microscopy grid covered with perforated supporting film. Most of the sample was removed by blotting for approximately 1 s, and the grid was immediately plunged into liquid ethane held at
183 °C. The sample was then transferred without rewarming into a Tecnai Sphera G20 electron microscope using a Gatan 626 cryo-specimen holder. The images were recorded at 120 kV accelerating voltage and microscope magnification ranging from 5000 to 14 500 using a Gatan UltraScan 1000 slow scan CCD camera and low dose mode with the electron dose not exceeding 15 electrons per square Å. The microscopic observations were performed immediately 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 zeta potential measurements. The time-dependent autocorrelation function of the photocurrent was acquired every 10 s with 15 acquisitions for each run. The samples were illuminated by a 633 nm laser, and the intensity of light scattered at an angle of 173° was measured by an avalanche photodiode. The z-averaged hydrodynamic mean diameters (dz), polydispersity (PDI), and distribution profiles of the samples were calculated using the software provided by the manufacturer. The zeta potential of the liposomes was measured using the technique of laser Doppler velocimetry (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 with 0.06 M solution of calcein (pH 8.5). The nonencapsulated calcein was separated from the CL liposomes by size-exclusion chromatography on a Sephadex G-50 column using PBS buffer containing 1 mM NaCl (pH 7.4) as an eluent. The fluorescence intensity of the CL liposomes was measured and found to be low due to the self-quenching effect. The appropriate amount of the polymer solution was introduced, and the change in fluorescence intensity due to calcein release from the vesicles was monitored. Excitation and emission wavelengths were set at 490 and 520 nm, respectively. The experiments were conducted at 25 °C. POPC liposomes display a single phase transition centered at
3.4 °C.23 Therefore, at 25 °C, the chains of lipids are fluid and the POPC membrane is in the liquid-crystalline state. A complete release of the dye was achieved by adding 30 μL of 15% solution of Triton-X100. The corresponding fluorescence intensity was used as 100% leakage. The amount of calcein released after time t, RF, was calculated using the following equation:24 RFðtÞ ¼ 100 678

It
I0 in % Imax
I0

ð1Þ

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Simulation Conditions. The MD simulations were performed with the GROMACS 4.0.7 software package.28 The periodic boundary conditions were applied in all three directions. The simulated system was maintained at the temperature of 298 K and the pressure of 1 bar according to the NPT ensemble regime. The temperature was controlled by the velocity rescaling thermostat,29 and the pressure was kept constant using the isotropic Berendsen barostat.30 The long-range electrostatic potential was calculated using the particle-mesh Ewald (PME) method with the Coulomb cutoff radius of 1.0 nm.31 The Lennard-Jones potential was calculated with a twin range cutoff with the radii set at 1.0 and 1.4 nm and the pair list updated every 10 steps. The LINCS constraints algorithm was employed for all bonds32 allowing for a 2 fs time step. The simulations were carried out for 110 ns in total, the first 10 ns considered as system equilibration and the remaining 100 ns treated 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, a short simulation of 10 ns was performed on the equilibrated membrane of 128 POPC and 3820 water molecules. To compare the hydration state of the decamer molecules in the membrane and in bulk water, a small system of four decamer 3 molecules embedded in a box of water was simulated for 40 ns. Both supplementary simulations were carried out under the same conditions as the studied membranes.

’ RESULTS Polymer 3 Synthesis. A quaternized derivative of poly(allylamine hydrochloride) (1) with covalently attached alkyl chains was synthesized using the modified procedure reported in the literature for chitosan.34,35 Poly(allyl-N,N-dimethyl-N-hexylammonium chloride) (3) was obtained from commercially available polymer 1 in two steps as shown in Figure 1. Amino groups of 1 reacted with hexyl aldehyde to form Schiff base intermediates, which was followed by reduction with sodium borohydride. In this way, N-hexyl derivative (2) of 1 was obtained. The analysis of the 1H NMR spectrum of the product has shown that 1 was substituted by a n-hexyl group. The degree of substitution by alkyl groups was determined from elemental analysis to be ca. 33%. In the next step, the weak polyelectrolyte, 2, was transformed into a strong polycation by quaternization of the amino groups using a Hoffmann exhaustive alkylation with iodomethane in the presence of sodium hydroxide, water, and sodium iodide. The 13C NMR spectrum of the product shows the distinctive signal at ca. 54 ppm, attributed to methyl groups linked to the quaternized amino groups. In the spectrum, there is no signal at ca. 46 ppm, characteristic of tertiary amino groups. These confirmed that under our experimental conditions the Nmethylation of the amino groups of polymer 2 was effective and led to the complete substitution of the amino groups with methyl groups. The content of quaternary ammonium groups was estimated from the 1H NMR spectrum to be ca. 100%. Interaction of Polymer 3 with Liposomes. DLS Measurements and Zeta Potential. The POPC vesicles were prepared by extrusion through 100 nm filters. The intensity weighted mean sizes (dz) of the vesicles determined with DLS were found to be ca. 124.8 nm, and the polydispersity was less than 0.1. Thus, the population of the liposomes has a narrowly distributed size range as indicated by the polydispersity index. A series of samples with a constant total concentration of POPC (cPOPC = 1.25 mg/mL) and the mass fraction of 3 varying from 0 to ca. 4.0% as compared to a lipid content were prepared. The scattering measurements were carried out to determine the hydrodynamic diameters of the vesicles coated with the polymer. Figure 3 depicts the

Figure 2. The conformation of decamer 3 molecule. where I0, It, and Imax are the fluorescence intensities measured without polymer, at time t after the polymer introduction, and after the addition of Triton X-100, respectively. Molecular Dynamics Simulations. Initial Structure. A short chain consisting of 10 units (decamer 3, Figure 2) was used in the simulations as a model for polycation 3. A conformer of decamer 3 with an elongated main chain was generated, and its geometry was optimized at the HF/6-31* level of theory. Two such molecules were introduced at a vertical orientation into an empty box with their centers of mass placed along the box diagonal. The size of the box was the same as that of the simulation box of a previously equilibrated POPC bilayer, consisting of 128 POPC molecules. The molecules of decamer 3 were subsequently immersed into the equilibrated POPC membrane, after removing some of the POPC molecules to create suitable compartments to accommodate the decamer molecules. On completing the number of POPC molecules to 128 (64 in each leaflet), a layer of water was added on top of each side of the membrane. The number of water molecules corresponds to a hydration ratio of ca. 30 H2O molecules per one POPC molecule, that is, a fully hydrated lipid membrane. Two initial systems, differing in the depth of the decamer immersion into the hydrophobic region of the membrane 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 any possible unfavorable contacts, they were submitted to a preliminary constant volume simulation run of 200 ps with a time step of 1 fs. The POPC molecule was described with the commonly used Berger lipid force field.25 For the sake of compatibility, the parameters for the decamer molecule were based on the same force field. A minor change in the charge distribution in the
C
N+(CH3)2
C
fragment was introduced due to a different number of methyl substituent in the POPC and decamer ammonium moieties. The Lennard-Jones parameters for the Cl
anion were taken from Joung and Cheatham paper.26 Water was described using the simple point charge (SPC) model.27 679

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not show any tendency for aggregation and the presence of the polymer did not cause membrane destabilization. The average diameters determined from the microscopic visualizations were lower than that estimated with DLS measurements. That is because DLS method measures the mean hydrodynamic diameter, which is heavily weighted toward the largest structures in the solution.36 Thus, DLS showed a systematically larger mean vesicle size. Fluorescence Studies. Calcein-release phenomena have been utilized as an effective index to characterize the permeability properties of biomembranes and to evaluate the interactions between the bilayer lipid membrane and target materials such as proteins, peptides, and (model) biomembranes. Calcein was entrapped inside the liposomes, which were next covered with polymer 3 at various concentrations. The release of the dye from the CL liposomes, both native and polymer-coated ones, was next monitored using fluorescence measurements. The amount of released calcein, RF, was calculated, and Figure 5 shows the typical time-course of the RF values of calcein from the POPC liposome. In the case of the pure POPC liposomes, the fluorescence intensity did not change during our experiments. This can be explained assuming that the noncovered membrane constitutes a barrier for the polar calcein and its structure remained intact during the time of measurement. The addition of polymer 3 to the initial liposome dispersion led to a pronounced increase of the fluorescence intensity due to the release of dye. The leakage of calcein increased strongly immediately after the polymer addition and reached a plateau after a certain time. The observed total leakage of the calcein was strongly dependent on the amount of the introduced polymer. To quantify the calcein leakage through the lipid membrane, a permeability coefficient to calcein, Ps, was calculated (in cm s
1) by using the relationship:16 r ð2Þ Ps ¼ k 3

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

distribution profiles of the hydrodynamic diameters of the initial POPC liposomes and the liposomes treated with various amounts of polymer 3. The profile for these vesicles was invariable for several days. The introduction of polymer 3 up to the concentration of 0.05 mg/mL (3.85% mass fraction) to the liposome dispersion caused changes in the size distribution of vesicles. The distributions became wider due to the appearance of a shoulder at the larger diameter region. The values of dz and polydispersity index (PDI) are collected in Table 1. The results show that the initial size of liposomes shifted gradually to larger diameters with the 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 fixed ionic strength. The results are summarized in Table 1. The value of ζ-potential of the neat liposomes was close to zero as a result of the zwitterionic character of POPC. The addition of polymer 3 to the liposomal dispersion up to the mass fraction of ca. 3% resulted in a pronounced increase of zeta potential of the POPC vesicles. Introduction of larger quantities of the polymer led to only a slight increase in the potential value. That observation can be explained assuming the adsorption of polymer 3 onto the POPC bilayer surface. The positively charged ammonium groups were exposed to the bulk solution, increasing the surface potential of the liposomal surface. Cryo-TEM Observation. In the next step, a cryo-TEM technique was used to study the effect of polymer 3 on the morphology and stability of the liposomes. For this purpose, the lipid vesicles were covered with polymer 3, and the liposomal dispersion was analyzed with cryo-TEM after various periods of time. Figure 4A and B presents the typical cryo-TEM micrographs of the liposomes incubated for less than 1 min and 24 h with polymer 3. As can be seen, in both cases, the vesicles showed the regular spherical morphology with a distinct lipid membrane surrounding the aqueous core. Although the unilamellar structures constitute the main population, sparse multilayered vesicles can also be observed. The size distribution profiles of liposomes determined from the cryo-TEM measurements are also presented in Figure 4 (right). Both distributions are broad, but the shape and the average diameters of the vesicles are very close. This is clear evidence that the polymer-covered liposomes did

where r is the radius of liposome and k is the apparent rate constant of calcein release and can be obtained by the analysis of a first-order kinetic calcein fluorescence according to the equation: RFðtÞ ¼ RFmax ð1
e
kt Þ

ð3Þ

Fitting the results presented in Figure 5 to eq 3, the permeability coefficient values, Ps, for the calcein release from the liposome were estimated as 5.74  10
8, 1.01  10
7, and 1.69  10
7 cm s
1 for the polymer concentrations of 0.01, 0.02, and 0.03 mg/mL, respectively. The value of Ps for the pure POPC was previously reported to be (1.88 ( 0.8)  10
11 cm s
1.16 Thus, the presence of polymer 3 in the liposome system caused an increase of the bilayer permeability by several orders of magnitude. Molecular Dynamics Simulations. To get some insight into the nature of interaction of polycation 3 with lipid membrane at the molecular level, we performed molecular dynamics (MD) simulations of two model membranes, composed of 128 POPC molecules hydrated with ca. 30 water molecules per lipid, into which two decamers of allylo-N,N-dimethyl-N-hexylammonium chloride (decamer 3) were inserted. Both simulated systems became fully equilibrated within the first 10 ns of simulation, as indicated by the potential energy, density, and area per lipid converging to constant values (Figure s1, Supporting Information). Three of the four simulated decamer molecules 680

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Table 1. Values of the Mean Hydrodynamic Diameter (dz), Polydispersity (PDI), and Zeta Potential (ζ) at 298 K of POPC Liposomes 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) POPC vesicles (cPOPC = 1.25 mg/mL) with 3 (c3 = 0.02 mg/mL)

0.8 1.57

132.8 ( 3.2 132.4 ( 1.8

0.11 ( 0.02 0.12 ( 0.02

15.6 ( 0.9 22.0 ( 1.8

POPC vesicles (cPOPC = 1.25 mg/mL) with 3 (c3 = 0.03 mg/mL)

2.35

133.5 ( 1.8

0.13 ( 0.02

26.3 ( 1.2

POPC vesicles (cPOPC = 1.25 mg/mL) with 3 (c3 = 0.04 mg/mL)

3.10

134.2 ( 1.4

0.13 ( 0.03

31.4 ( 1.2

POPC vesicles (cPOPC = 1.25 mg/mL) with 3 (c3 = 0.05 mg/mL)

3.85

136.4 ( 3.2

0.11 ( 0.01

33.3 ( 1.6

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

Area per Lipid. The area per lipid was calculated as the ratio of the area of the xy plane of the simulation box to the number of POPC molecules in the monolayer (64). This parameter was practically identical for both membranes and equals 0.822 ( 0.002 nm2 (membrane I) and 0.818 ( 0.002 nm2 (membrane II). Comparison with the area per lipid of the pure POPC membrane (0.682 ( 0.002 nm2) shows that the insertion of two decamer molecules and the concomitant structural changes resulted in a considerable expansion of the membrane surface. An experimental estimate of the 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 the membrane) calculated for membrane I and the pure POPC bilayer. The curves describing the distribution of atoms constituting the polar headgroup of POPC are significantly broader for the POPC
decamer 3 system than for the pure POPC membrane, and, as opposed to the latter, they extend visibly toward the bilayer center. The presence of the polar groups in the hydrophobic region of the membrane accompanied by a nonzero water concentration in this region is in accordance with the observed reorientation of some POPC molecules and formation of hydrophilic pores in the membrane structure. The thickness of membrane I, calculated as the distance between the maxima in the distribution curves of the POPC choline nitrogen, is practically the same as for the POPC bilayer (3.45 and 3.49 nm, respectively). Radial Distribution Function. To characterize the surroundings of the decamer ammonium nitrogen atoms (NA), the radial

Figure 4. Cryo-TEM micrographs and the diameter profiles of POPC liposomes (cPOPC = 2.5 mg/mL) coated with polymer 3 (c3 = 0.06 mg/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.

(which will be referred to as A, B, C, and D) remained emerged in the hydrocarbon region of the bilayer during the whole simulation period. This result could be surprising, considering the good solubility of polycation 3 in water, which shows its undoubtedly hydrophilic character. Inspection of the trajectory reveals though that during the equilibration period the bilayer underwent a substantial molecular reorganization in the hydrophobic regions adjacent to decamers 3, which forced some of the neighboring POPC molecules to reorient, and led to creation of a hydrophilic envelope surrounding the polycation molecule. This new, energetically favorable local environment allowed the decamer molecules to remain in the bilayer core for the whole simulation period. This structural rearrangement resulted in the formation of a hydrophilic pore spanning the bilayer, which allowed water and 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, it translocated entirely into the water
bilayer interface of the POPC membrane and remained there for the rest of the simulation (Figure 6). 681

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

oriented in such a way that one or both of the nonester oxygen atoms of the phosphate group point toward the decamer ammonium group. The coordination number for the P atom could suggest there is nearly one PO4
group in the close vicinity of each decamer ammonium group. However, due to the short distance between the NA atoms in decamer 3 and a possibility of the same P atom being within the coordination radius of two NA atoms, the actual number of the phosphate groups interacting closely with the decamer is smaller. The average number of the phosphate groups inside the pores was estimated by counting the number of phosphorus atoms found in the bilayer hydrophobic core. The approximate extent of this core was defined as a region (1.0 nm from the bilayer center. The calculation over the whole trajectory with a 1 ns time window gives the average number of 14 PO4
groups engaged in the pores creation, that is, seven groups per pore. It should be mentioned 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 bilayer central 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 system consisting of four decamer molecules immersed in a water box. In both systems, the gNA
OW(r) function shows a distinct maximum at r = 0.43 nm (Figure 8). Integration of the first peak gives the average number of water molecules in the first solvation sphere of the ammonium nitrogen in decamer 3. These coordination numbers are very similar for both membranes and equal to 10.5 (membrane I) and 11.1 (membrane II). These values are only slightly smaller than the coordination number calculated for the fully hydrated decamer molecule (13.2, respectively),

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

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), and NA
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 membranes. It is worth reminding at this point that molecule D (membrane II), as opposed to the other three decamer 3 molecules, remained completely immersed in the hydrophilic region of the bilayer for most of the simulation period. The fact that its rdf curves do not differ from those calculated for the molecules partially or completely incorporated into the region of the POPC hydrocarbon chains shows that, regardless of the location of a decamer molecule inside the membrane, its closest vicinity is very much alike. The gNA
P(r) function shows a distinct peak at a distance of 0.508 nm. Integration of this peak gives the average number of P atoms per one NA nitrogen equal to 0.89. The maximum of the gNA
O(r) appears at a distance of 0.415 nm. The average number of O(P) atoms per one ammonium group was found to be equal to 1.32. These results allow one to conclude that the hydrophilic heads of POPC become 682

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

Figure 8. The gNA
X(r) radial distribution functions for the atomic pairs for membrane I (a) and membrane II (b); NA, ammonium nitrogen of decamer 3; P and O(P), phosphorus and nonester oxygen in the phosphate group of POPC; OW, water oxygen. The functions have been averaged over both decamer 3 molecules in each membrane. Figure 10. Distribution of the z-tilt angle of the C(21)
C(3) vector (see Figure 2 for the definition).

showing that the hydration state of decamer 3 in the membrane is comparable 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 is sufficient for most of the water molecules, which are present in the central region of the bilayer at the observation starting point, to drift out into the headgroup region of the membrane. Meanwhile, other water molecules, and also the Cl
anions, diffuse into the pore. To get a deeper insight into the mobility of water molecules inside the pores, a mean square displacement (MSD) was computed 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 vertical span of the pore was estimated as equal to the thickness of the water-free region in the OW distribution curve of pure POPC (see Figure 7), which gives the value of ca. 1.6 nm. However, to ensure that the MSD calculation for “inner” water will not include water molecules that leave the pore within the time period used for MSD calculation (500 ps), we limited the definition to water present in the central, cylindrical region of the pore. The length of this part of the pore has been estimated as ca. 1.2 nm, on the basis of visual inspection of simulation snapshots. 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 the Supporting Information). The “outer” water group included all of the remaining water molecules. The identity of “inner” water molecules 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 ps starting 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 that the rate of diffusion along the z direction of the “inner” water is comparable to that for the “outer” water. Diffusion coefficients, calculated from the slopes of the linear part of the appropriate MSD curves, equal (0.69 ( 0.11)  10
5 (“inner” water) and (0.72 ( 0.03)  10
5 cm2/s (“outer” water). The mobility of water molecules in the lateral direction is distinctly different for both 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 of water molecules on the membrane surface plane is rather fast. On the contrary, the Dxy for the “inner” water is 1 order of magnitude smaller (0.19 ( 0.05)  10
5 cm2/s), indicating a very poor mobility of the in-membrane water in that direction. Undoubtedly, this is caused by a limited diameter of the hydrophilic channel formed around vertically oriented decamer molecules. Location and Conformations of Decamer 3. To gain some information about the orientation of the main hydrocarbon chain of the decamer molecule and its conformational state, a few 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 683

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are centered around the angle of 100° (molecules B
D) and 110° (molecule A). This angle is pretty close to 120°, the value characteristic for a regular arrangement of the side chains around a 3-fold symmetry axis, collinear with the main axis. Such a regular arrangement of the NA atoms is clearly visible when looking at the molecule along its main axis (Figure s3, Supporting Information).

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 between the CB
NA vectors of the successive side chains (see Figure 2).

decamer main chain with respect to the POPC alkyl chains (which, on the average, are tilted ca. 31° to the membrane normal). The distribution of this tilt angle, averaged over the last 80 ns of the simulation, is shown in Figure 10. As may be concluded from this diagram, the decamer molecules incorporated into the region of the POPC alkyl chains assumed an orientation with their main axis at a rather low angle (10°
45°) with the membrane normal. The small peak at 83° in the distribution curve for the C molecule originates from the last 20 ns of the trajectory and reflects the drastic reorientation this molecule underwent at the end of the simulation. On the contrary, the molecule entirely immersed in the headgroup region of POPC (molecule D) prefers the orientation with its main axis nearly perpendicular to the z direction (parallel to the membrane 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 molecule model, equals ca. 2.24 nm in the case of the fully stretched chain (all of the dihedrals along the chain equal to 180°). In molecules A 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 main chain. The distribution for molecule D in particular shows a broad shoulder at the close distant side, which suggests a greater flexibility of the polymer main chain adsorbed at the water
lipid interface. Relative arrangement of the ammonium residues in decamer 3 was established by calculating the distribution of the angle between the vectors defined by the CB and NA atoms (Figure 2) in the successive side chains (Figure 11b). The distribution curves

’ DISCUSSION To explore the effect of a positively charged and hydrophobically modified polyelectrolyte on a neutral lipid membrane, we synthesized a strong polycation modified with short side alkyl chains (polymer 3) and studied its interactions with a zwitterionic lipid membrane using experimental methods and MD simulations. Polymer 3 has the quaternary ammonium groups in its structure; therefore, its properties, especially the charges on the backbone, are independent of pH of the environment. Cationic polymers containing pendant quaternary ammonium groups are the most promising candidates as the effective antimicrobials and biocides.38 These groups were modified by attachment of hexyl chains. As was shown previously, poly-4vinyl pyridine (PVP) with hexyl units on its backbone had the highest antibacterial activity in the series of polymers with various lengths of alkyl units.4 Such structure of polymer 3 makes it relatively well soluble in water. Unilamellar liposomes composed of lipids with zwitterionic phosphatidylcholine heads have been widely utilized as a model biomembrane system, because such lipids are the main constituents of natural membranes. Interactions between various polycations and the liposomes have been extensively studied to mimic behavior of these macromolecules in contact with cellular membranes.2,6 Taking into account the results of studies reported in the literature, one could expect various possibilities of polycation behavior at the zwitterionic membrane surface. They include (i) lack of interactions,6 (ii) adsorption on the bilayer hydrophobic surface,13 (iii) insertion into the hydrophobic core of the bilayer,2 and (iv) disruption of the bilayer with the formation of mixed polymer
lipid micelles or other aggregates.39 Therefore, several issues such as the location of polymer 3 within the lipid bilayer (at the surface or entrapped in the bilayer) or the possibility of disruption of liposomes into small fragments need to be considered in the present study. The results of DLS and zeta potential measurements provided evidence that the polymer can interact with the POPC membranes, causing a slight increase of the hydrodynamic diameter of the vesicles and a drastic increase of zeta potential. It is wellknown that the adsorption of polymers on the liposome surface or penetration of polymers into the liposome bilayer will correspondingly change the size of the vesicle.5 However, the small increase in liposome size induced by polymer 3 could be attributed to the fact that it mainly incorporates into the lipid bilayer instead of being adsorbed on the surface of the liposomes.5 The increase of vesicle diameter upon incorporation of polymer 3 can additionally affect the stability of the vesicles. As was shown, the stability of liposomes is connected with the curvature of the bilayer.40 Small unilamellar vesicles are inherently unstable due to their highly strained, curved bilayers. The increase of vesicle diameter involves a reduction of bilayer curvature improving the vesicle stability. The increase of surface charge usually increases the zeta potential of particles (the electric potential in the slipping plane) whose values are well 684

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

correlated with the stability of suspensions. The ζ-potential is a quantity well accessible experimentally, for example, by using the microelectrophoretic method as was done in this work. Upon addition of the increasing amount of polymer 3 to the POPC dispersion, a rise of the zeta potential was observed, indicating that the surface charge on the liposomes increased. This is clear evidence that polymer 3 can be also physically adsorbed at the vesicle surface probably with alkyl chains immersed into the bilayer and the ammonium groups exposed to the bulk solution. The presence of the hydrophilic polymer 3 layer at the liposome surface leads to polymer-coated, electrostatically stabilized vesicles. The appearance of positive charge at the surface of liposomes is essential for the liposome stability. Similar results were obtained previously for zwitterionic liposomes covered by chitosan or alkylated chitosan (cationic polyelectrolytes).12 It was shown that the adsorption of these macromolecules on the lipid membranes can be monitored by changes in the ζ-potential of liposomes. The chitosan
lipid bilayer interaction enhances vesicles stability with regard to various stresses (for example, pH and salt shocks). The incorporation of the polymer into the hydrophobic part of membrane should have a substantial impact on the permeability of the membrane. Indeed, the presence of polycation 3 caused a significant increase in release of hydrophilic compounds entrapped inside the POPC liposomes, as was shown using calcein as a model compound. These findings indicate that the polymer
lipid interactions can induce conformational rearrangement of the polymer and result in the insertion of the polymer

into the lipid membrane, possibly causing alternations in the membrane structure. Earlier, Eren et al. observed increased release of calcein from liposomes under the influence of polycations with different quaternary alkyl pyridinium side chains.4 To explain this phenomenon, the authors suggested that the phospholipid membrane undergoes disruption. To check such possibility, we applied the direct microscopic observation of lipid vesicles treated with polymer 3. The cryo-TEM visualization of the POPC liposomes incubated for different time with the polymer showed that the lipid vesicles maintain their integrity during interactions (Figure 4). This is strongly supported by DLS measurements showing invariable in the size profile of polymer coated liposomes with time. Therefore, the enhanced permeability of POPC membrane covered with polymer 3 is caused by a formation of pores (holes) in the bilayer structure. The formation of pores in phospholipid membranes has been demonstrated using AFM microscopy. Several AFM studies on supported lipid bilayer interacting with different polycations showed that these macromolecules disturb the structure of lipid bilayers, leading to formation of nanoscale holes.41
44 The defects in the membrane were accompanied by a significant increase of membrane permeability, as indicated by in vitro observed leakage of the cytosolic enzymes and free diffusion of fluorescent dye molecules into and out of the cells.35 Comparison of the effect exerted by different polymers led to a conclusion that the size of the polymer molecule does not affect its ability for hole formation, but the charge density on the polymer chain influences markedly the degree of membrane permeability. The defect-creating ability of 685

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different polymers was studied in a whole cell patch-clamp experiment on living cells.45 A substantial increase of the plasma membrane conductance was observed after exposing the cells to positively charged polymers (PEI, PLL, and PAMAM dendrimers), whereas neutral polymers like poly(vinyl alcohol) and poly(ethylene glycol) did not show this effect. These observations suggest that formation of the permeabilitypromoting defects in the lipid bilayers is most probably a result of strong interactions of the lipid molecules with the electrostatic potential of the polycation molecule. Such a hypothesis is supported by the well-known fact that application of an external electric field to cells or tissues leads to a drastic increase of their permeability to ions and small polar molecules. This effect is attributed to creation of transient water filled pores in the cell membranes on applying a transmembrane potential of several hundred milivolts (so-called electroporation).46,47 The incorporation of the positively charged polymer into the membrane can induce alterations in lipid packing and organization. This process was studied at the molecular level by the MD simulations of POPC membranes doped with the fragments of polymer 3. The simulations reveal an important influence of polymer 3 on the membrane integrity and functionality. The critical structural modification of the membrane was connected with the formation of hydrophilic pores around the decamer molecules incorporated in the core region of the membrane (Figure 6). Such pores were observed to form spontaneously in a time span of a few nanoseconds and remain stable within the simulation time scale (100 ns). Three of the four simulated molecules were involved in the creating of pores. The porepromoting molecules occupied positions in the middle of the membrane, with their main chain nearly straightened out and their hexyl side chains directed toward the hydrocarbon region of membrane. The fourth of the simulated molecules adopted a location above the carbonyl groups region of POPC with the backbone parallel to the membrane plane. The results of MD simulations of decamer 3 show that there are at least two energetically preferable locations of the polycation 3 chain in the lipid membrane. Therefore, we believe that the backbone of polymer 3 in contact with a lipid membrane can enter easily into the bilayer, inducing formation of pores. Some of its fragments may stay adsorbed on the membrane plane, increasing the surface potential, as shown with the zeta potential measurements. The molecular mechanism of pore formation is presented in Figure 12. The process was initiated by some water molecules drifting into the region occupied by the polycation, well below the line of the lipid hydrophilic heads (Figure 12, 0.1 ns). A significant number of water molecules around decamer 3 molecules could be observed even at the very early stage of the simulation (t < 200 ps). The formation of a water column across the membrane was soon followed by reorganization of some adjacent POPC molecules, which began to reorient in such a way that their phosphate groups become pointed toward the ammonium nitrogen in the decamer (Figure 12, 1 and 4 ns). This process was most probably driven by the strong electrostatic interaction between the positively charged ammonium moieties of decamer 3 and the negatively charged phosphate groups of POPC and was additionally facilitated by the presence of water molecules in close vicinity of the decamer. This new organization of POPC hydrophilic head groups creates a shield protecting the
N+(CH3)2
groups from unfavorable interactions with the nonpolar medium of POPC acyl chains, thus contributing to the energetic stabilization of the system. This rearrangement leads to

an important modification of membrane functionality; hydrophilic pores constitute around decamer 3 molecules. They enable migration 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 C within the first few nanoseconds of the simulation. The pores created around molecules A and B, once formed, remained stable for the whole simulation period, although their internal surface manifested significant flexibility. In particular, a considerable mobility of the PO4
groups around the decamer molecule was observed. Molecule C (membrane II) behaved identically as molecules A and B for most of the simulation time, but, within the last 20 ns of the simulation period, it underwent a substantial reorientation and assumed a position with its main axis almost perpendicular to the acyl chains of POPC. This reorientation, accompanied by a rearrangement of neighboring POPC molecules, led to a considerable enlargement of the hydrophilic channel (Figure 6b). To estimate the lateral size of the pores, a water density xy map was computed for the bilayer core region for both systems. The average diameter of circular blotches marking the region of high water density around molecules A, B, and C (the latter calculated from the first 80 ns) equals ∼1.7 nm. The map for the membrane II, computed for the last 20 ns of the trajectory, shows a region of high water density with ellipsoidal shape, with the long and the short axes equal to ∼2.7 and ∼1.7 nm, respectively (Figures s4 and s5, Supporting Information). The structural modification described above resulted in a considerable increase of the membrane surface. The area per lipid for the POPC membrane doped with decamer 3 is by ca. 21% larger than for the pure POPC membrane. This result is corroborated by the experimentally observed increase of the vesicles diameter after addition of polymer 3 to the liposome dispersion. Assuming a spherical geometry of the vesicles, the growth of the diameter from ca. 125 to 136 nm corresponds to the increase of its surface by some 18%. The process of hydrophilic pore formation induced by different stimuli has been addressed in numerous MD studies. Various lipid bilayers have been simulated under the effect of a uniform, transmembrane electric field (electroporation)48
52 or a field created as a result of an imbalance of ion concentration on both leaflets of the bilayer.53
56 Many of these works report a common scenario of pore formation, regardless of the origin of the electric field applied. The process is triggered off by some water molecules penetrating the lipid/water interface and intruding into the hydrocarbon region of the bilayer. Such water defects present on both sides of the bilayer tend to grow and merge until the water wires/columns spanning the whole bilayer is created. Formation of a new water/lipid interface is soon followed by a reorientation of lipids, which line the inner surface of the pore and stabilize it. The entire pore formation process takes no more than 4
5 ns. The mechanism of pore formation as observed in our simulations is in agreement with the results of many MD studies on pore formation due to transmembrane potential. It seems justified though to propose that the polycation-mediated poration of the cellular membrane is basically of the same molecular nature as the electroporation and it is driven by some water defects arousing as a result of a response of the water dipols to the applied or local electric field. The intensity of the electric field around decamer 3 molecule may be roughly estimated by assuming a cylindrical shape of the molecule. Taking a cylinder 686

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of the height of 1.7 nm and the radius of 0.75 nm (approximate longitudinal and lateral dimensions of the molecule), which encloses a charge of 10e (i.e., 1.602  10
18 C), gives the electric field of the magnitude of ∼2 V/nm. The actual field will be weaker by some 30
40% due to partial neutralizing of the positive charge by some Cl
anions remaining within the cylinder space. Still, its magnitude is of the same order as the transmembrane potential applied in the electroporation.

(4) Eren, T.; Som, A.; Rennie, J. R.; Nelson, C. F.; Urgina, Y.; Nusslein, K.; Coughlin, E. B.; Tew, G. N. Macromol. Chem. Phys. 2008, 209, 516–524. (5) Ding, L.; Chi, E. Y.; Chemburu, S.; Ji, E.; Schanze, K. S.; Lopez, G. P.; Whitten, D. G. Langmuir 2009, 25, 13742–13751. (6) Ding, L.; Chi, E. Y.; Schanze, K. S.; Lopez, G. P.; Whitten, D. G. Langmuir 2010, 26, 5544–5550. (7) Pereira da Silva Gomes, J. F.; Rank, A.; Kronenberger, A.; Fritz, J.; Winterhalter, M.; Ramaye, Y. Langmuir 2009, 25, 6793–6799. (8) Stadler, B.; Chandrawati, R.; Goldie, K.; Caruso, F. Langmuir 2009, 25, 6725–6732. (9) Tribet, C.; Vial, F. Soft Matter 2008, 4, 68–81. (10) Fischer, D.; Li, Y.; Ahlemeyer, B.; Krieglstein, J.; Kissel, T. Biomaterials 2003, 24, 1121–1131. (11) Epand, R. M.; Vogel, H. J. Biochim. Biophys. Acta 1999, 1462, 11–28. (12) Shai, Y. Biopolymers 2002, 66, 236–248. (13) Quemeneur, F.; Rinaudo, M.; Pepin-Donat, B. Biomacromolecules 2008, 9, 2237–2243. (14) Wang, Y.; Tang, Y.; Zhou, Z.; Ji, E.; Lopez, G. P.; Chi, E. Y.; Schanze, K. S.; Whitten, D. G. Langmuir 2010, 26, 12509–12514. (15) Kepczynski, M.; Kumorek, M.; Stepniewski, M.; R
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’ CONCLUSIONS Our results show that the hydrophobically modified polycations can readily and quickly associate with and insert into the zwitterionic lipid membrane structures. The vesicle structures after the incorporation of the polymers are retained instead of being disrupted into small fragments. The polymer-covered liposomes have no tendency to form aggregates and maintain their integrity for long time. This process may be driven by a combination of the electrostatic interactions between cationic polymer and the anionic phosphate groups of lipid and hydrophobic interactions between hexyl moieties of polycation 3 and the lipid acyl chains. The association and incorporation of polymer 3 in the lipid membrane cause alternation of packing and organization of membrane lipids, which directed their phosphate groups toward the ammonium N atoms of the polycation. The hydrophilic pore thus stabilized facilitates diffusion of water molecules and Cl
anions across the bilayer. ’ ASSOCIATED CONTENT

bS

Supporting Information. The value of ζ-potential of the objects formed in a 0.03 mg/mL solution of polymer 3. The time profile of the potential energy, system density, and area per surfactant molecule for the first 20 ns of the simulation. Definition of the “inner” water. Conformation of decamer 3 (molecule A) after 50 ns of the simulation. Water number density xy maps. 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 Education for financial support in the form of Grant No. N N209 118937. J.B. acknowledges the support of the Czech Grants LC535, MSM0021620806, and AV0Z50110509. The MD simulations were performed on the HP Cluster Platform 3000 BL 2  220 available at the Academic Computer Centre CYFRONET AGH, Krak
ow, Poland. ’ REFERENCES (1) Parhamifar, L.; Larsen, A. K.; Hunter, C.; Andresen, T. L.; Moghimi, S. M. Soft Matter 2010, 6, 4001–4009. (2) Sikor, M.; Sabin, J.; Keyvanloo, A.; Schneider, M. F.; Thewalt, J. L.; Bailey, A. E.; Frisken, B. J. Langmuir 2010, 26, 4095–4102. (3) Luten, J.; Nostrum, C. F.; van; De Smedt, S. C.; Hennink, W. E. J. Controlled Release 2008, 126, 97–110. 687

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