Effect of Polycation Structure on Interaction with ... - ACS Publications

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Effect of Polycation Structure on Interaction with Lipid Membranes Natalia Wilkosz,† Dorota Jamróz,*,† Wojciech Kopeć,†,⊥ Keita Nakai,‡ Shin-ichi Yusa,‡ Magdalena Wytrwal-Sarna,† Jan Bednar,§,∥ Maria Nowakowska,† and Mariusz Kepczynski*,† †

Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-348 Kraków, Poland Department of Applied Chemistry, University of Hyogo 2167 Shosha, Himeji, Hyogo 671-2280, Japan § Université de Grenoble Alpes/CNRS, Institut Albert Bonniot, UMR 5309, 38042 Grenoble Cedex 9, France ∥ Charles University in Prague, first Faculty of Medicine, Laboratory of Biology and Pathology of the Eye, Institute of Inherited Metabolic Disorders, KeKarlovu 2, 12800 Prague 2, Czech Republic ‡

S Supporting Information *

ABSTRACT: Interaction of polycations with lipid membranes is a very important issue in many biological and medical applications such as gene delivery or antibacterial usage. In this work, we address the influence of hydrophobic substitution of strong polycations containing quaternary ammonium groups on the polymer−zwitterionic membrane interactions. In particular, we focus on the polymer tendency to adsorb on or/and incorporate into the membrane. We used complementary experimental and computational methods to enhance our understanding of the mechanism of the polycation−membrane interactions. Polycation adsorption on liposomes was assessed using dynamic light scattering (DLS) and zeta potential measurements. The ability of the polymers to form hydrophilic pores in the membrane was evaluated using a calcein-release method. The polymer− membrane interaction at the molecular scale was explored by performing atomistic molecular dynamics (MD) simulations. Our results show that the length of the alkyl side groups plays an essential role in the polycation adhesion on the zwitterionic surface, while the degree of substitution affects the polycation ability to incorporate into the membrane. Both the experimental and computational results show that the membrane permeability can be dramatically affected by the amount of alkyl side groups attached to the polycation main chain.

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INTRODUCTION Interactions between polyelectrolytes and lipid or cell membranes play an important role in many biophysical applications. Polycations are particularly interesting in that regard because they can find application as drug/gene delivery systems and as biocidal agents.1 Therefore, there has been considerable interest in experimental studies on the interactions between positively charged polymers and lipid membranes. The literature dealing with this issue has been recently reviewed by several authors.2−4 Computer simulations can be used to obtain a complementary view of soft matter systems, revealing a level of detail that is very difficult to assess experimentally, mostly due to the spatial and temporal resolution limits of current experimental techniques, coupled with inherently chaotic behavior of such systems. Application of molecular dynamics (MD) simulations to investigate the interaction of polycations with lipid bilayers has been reviewed by Rossi and Monticelli1,5 and Ramezanpour et al.6 We have found from these reports that studies of systems containing linear polycations and lipid membranes using the atomic-scale MD simulations are rather limited. Hill et al. performed atomistic MD simulations of a system containing cationic phenylene ethynylene oligomers, cationic biocides, and © 2017 American Chemical Society

a model bacterial membrane composed of zwitterionic (DOPE) and negatively charged (DOPG) phospholipids.7,8 The atomistic MD simulations probing the interaction between polyethylenimine (PEI), a frequently used nonviral transfectant, and zwitterionic or anionic lipid membrane were performed by Choudhury et al.9 and recently by Kwolek et al.10 Kostritskii et al. used the MD simulations to explore adsorption of several linear polycations (polyallylamine (PAA), PEI, polyvinylamine (PVA), and poly-L-lysine (PLL)) on model bacterial membranes composed of zwitterionic phosphatidylethanolamine and anionic phosphatidylglycerol (PG).11 In this work, we focus on the interaction of hydrophobically modified linear polycations with zwitterionic bilayers. Such interactions have been previously investigated, and discrepancies in the influence of alkyl side chains on the adsorption of polymers have been reported. Quemeneur et al. studied the adsorption of chitosan (a cationic polysaccharide) and its alkylated derivatives (a degree of alkylation with chains of C6, C10, and C12 length was 5%) with phosphatidylcholine (PC) Received: May 30, 2017 Revised: June 30, 2017 Published: July 5, 2017 7318

DOI: 10.1021/acs.jpcb.7b05248 J. Phys. Chem. B 2017, 121, 7318−7326

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The Journal of Physical Chemistry B

or channels in the bilayer was assessed using fluorescence methods. Finally, we performed the MD simulations of systems containing the polycations placed initially on the surface or embedded into the POPC bilayer to gain a molecular view of the organization of the polycation−membrane systems.

liposomes.12 The authors showed that the alkyl chains did not interact with the lipid membrane, and the adsorption of chitosan occurred via electrostatic interactions. Eren et al. studied a series of polynorbornenes functionalized with different alkyl chains.13 They reported that the polymers modified with short alkyl groups (C2, C4) caused only minor disturbance of the PC liposomes, while those with longer side chains (from C6 to C10) were much more active in the interaction with PC vesicles. Wang et al. studied geminized amphiphilic cationic homopolymers, containing both double hydrophilic groups and two hydrophobic octyl chains in each structural unit.14 They found that the polymers exhibited significant activity against PC membrane causing the disintegration of the liposomes. Ivanov et al. used MD simulations to study the impact of a series of antimicrobial polymethacrylate copolymers with short alkyl side chains (from C2 to C6) and different ratio of hydrophobic and cationic units on the DOPC bilayer.15 The authors observed partial insertion of the polymers into the bilayer during the nanosecond time scale of MD simulations. Insertion occurred with the polymer in an almost vertical orientation, despite the fact that originally it was set up in a fully extended conformation parallel to the bilayer surface. In our previous paper, we studied the interaction between a strong polycation with hexyl side groups (a degree of alkylation was 33%) and the POPC membrane using MD simulations and experiments.16 This polymer adsorbed strongly to the lipid membrane, causing considerable perturbation of membrane structure and pore formation. The presence of hydrophobic side chains in the polymer structure appeared to play an important role in the polymer−membrane interactions. Although the studies presented above provide important findings about the systems investigated, it is difficult to build a consistent overall picture of the nature of the polymer− membrane interactions. This is mainly due to the incompatibility of results presented in various papers. Usually, experiments and simulations were carried out separately and it is difficult to compare the results obtained. Therefore, in our study we use complementary experimental and computational approaches to gain insight into the nature of polycation−lipid membrane interactions. We are particularly interested in enhancing our understanding of the molecular mechanism of interactions between the hydrophobically modified polymers and biomembranes. This study involves observation of the effect of alkyl chains (their length and the degree of substitution) grafted on a strong polycation on the mechanism of its adsorption on/incorporation into zwitterionic membranes. A series of new polycations were obtained by modification of commercially available poly(allylamine hydrochloride) (PAH). PAH was chosen because this polymer has found a variety of applications in both biomedical fields and nanotechnology.17,18 2-Oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC) liposomes were used as a model of biomembrane. The liposomes were treated with aqueous solutions of the polymers at various concentrations, and the polymer−membrane interactions were studied using several experimental techniques. Dynamic light scattering (DLS) and zeta potential measurements were employed to confirm polycation adsorption on the POPC vesicles and to evaluate the tendency of liposome aggregation. To check the integrity of the polymer-covered liposomes, their morphology was visualized directly using cryo-transmission electron microscopy (cryo-TEM). Next, the ability of the polycations to form pores

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MATERIALS AND METHODS Materials. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC, ≥99.0%) and calcein were obtained from Sigma. Poly(allylamine hydrochloride) (PAH) with an average molecular weight of 15 000, dodecanal, DMSO-d6 (99.9 atom % D), and D2O (99.9 atom % D) were purchased from Sigma− Aldrich and used as received. Millipore-quality water was used in all experiments. Strong Polycations. Poly(allyltrimethylammonium chloride) (1) was synthesized according to the procedure described previously.19 Poly(allyltrimethylammonium chloride-co-allylN,N-dimethyl-N-dodecylammonium chloride) (2) was prepared according to the modified procedure.16 Briefly, PAH (1 g, 10.7 mmol of the amino groups) was dissolved in water (5 mL). 5% NaOH solution (1 mL) was added, and the pH of the solution was adjusted to 3−4 using 1% acetic acid. After 30 min of stirring, dodecanal (0.37 mL for 2a and 3.0 mL for 2b) was added. The reaction mixture was stirred for 24 h at room temperature. Next, NaBH4 (1.5-fold excess to the added aldehyde), dissolved in water, was dropwise added, and the mixture was stirred for 12 h. The crude intermediate product was purified by dialysis against water. TLC was performed using n-hexane/chloroform (4/1 v/v) as an eluent to confirm the absence of unreacted dodecanal. The product was transformed to the strong polycation by an exhaustive methylation of PAH amino groups, as described previously.16,19 Poly(allyltrimethylammonium chloride-co-allyl-N-stearoylallylamine) (3) was obtained in two steps, as shown in Figure S1. The details of the syntheses are described in the Supporting Information. Apparatus. 1H and 13C NMR spectra were recorded on a Bruker AMX 500 Hz instrument. The NMR spectra were taken at 80 °C in DMSO-d6/D2O (3/1, v/v) using DMSO-d6 residual peaks as internal standards. Elemental analysis was performed using a EuroEA 3000 Elemental Analyzer. Fluorescence spectra were recorded on a PerkinElmer LSD50B spectrofluorimeter equipped with a thermostated cuvette holder. A Malvern Nano ZS light-scattering apparatus (Malvern Instrument Ltd., Worcestershire, UK) was used for dynamic light scattering (DLS) and zeta potential measurements.20 The z-averaged hydrodynamic mean diameters (dz), dispersity index (DI), and distribution profiles of the samples were calculated using the software provided by Malvern. Preparation of Liposomes. Small unilamellar phospholipid vesicles (SUVs) were prepared by an extrusion technique as described previously.21 Briefly, POPC (12.5 mg) was dissolved in chloroform (300 μL) in a volumetric flask. The solvent was evaporated under a gentle stream of nitrogen to form a dry lipid film. A 1 mM solution of NaCl at pH 7.4 was added until a desired lipid concentration was attained (usually 2.5 mg/mL), and the sample was vortex mixed 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. Covering with Polycations. 0.5 mL of the zwitterionic SUV dispersion was placed in a sonication bath, and the appropriate volume of a 0.5 mg/mL solution of the polycation 7319


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The Journal of Physical Chemistry B was quickly added. Next, 1 mM NaCl was added to the final volume of 1 mL. Cryo-transmission Electron Microscopy (cryo-TEM). Cryo-TEM was used to visualize the morphology of liposomes. The liposome dispersion was prepared at cPOPC = 2.5 mg/mL, and the concentrated solution of a polymer was added to obtain the desired concentration. First, the vitrification apparatus was made ready for immediate use. The C-flat CF-4/2-4C EM grid was glow discharged for 10 s at 8 mA current at 2.0 × 10−1 mbar pressure in a Balzers MED 020 coating system with a glow discharge attachment, then fixed in tweezers, and placed into the vitrification apparatus. A 3 μL aliquot of the mixture was immediately applied to the electron microscopy grid, excess liquid was blotted away with Whatman No. 1 filter paper, and the grid was immediately plunged into liquid ethane held at −183 °C. The grid was then transferred without rewarming into a Tecnai Sphera G20 electron microscope using a Gatan 626 cryo-specimen holder. Calcein-Release Studies. Calcein-release experiments were performed as described previously.10 Briefly, the calceinloaded SUVs (CL-SUVs) were prepared, and the appropriate amount of the polymer solution was promptly introduced to CL-SUVs. The change in fluorescence intensity due to calcein release from the vesicles was monitored at 25 °C. Complete release of the dye was achieved by adding 30 μL of a 15% solution of Triton X-100. The corresponding fluorescence intensity was used as 100% leakage. The calcein release, RF(t), was calculated using the following equation. RF(t ) = 100[(It − I0)/(Imax − I0)]

one oligomer chain embedded into the hydrophobic core of the POPC membrane (systems E-o2a, E-o2b, and E-o3b). The oligomer−bilayer systems were generated by inserting a selected conformer of the oligomer into the equilibrated POPC membrane, using a g_membed utility,22 which is a part of the Gromacs simulation package. In all systems containing oligomers on the membrane surface, a layer of water above the upper leaflet of the bilayer was thickened to ensure full accommodation of the oligomer molecule within the simulation box and to avoid its interaction with the membrane periodical image in the normal direction. The systems were electrically neutralized by adding the appropriate number of Cl− ions. A summary of the studied systems is given in Table 1, and images Table 1. Summary of the Simulated Systems, Indicating the Number of Molecules in the Given System System

POPC

Water

polymer on the membrane surface; shallowly in the bilayer S-o1 288 35 161 S-o2a 288 35 202 S-o2b 288 35 153 S-o3b 288 34 938 polymer embedded deeply into the hydrophobic core E-o2a 279 22 668 E-o2b 277 22 605 E-o3b 281 22 662

Cl− 30 30 30 28 30 30 28

of the initial configurations are shown in Figures S5 and S6 (Supporting Information). Prior to productive runs of 300 or 400 ns, all the systems were subjected to short simulations of 10 ns to fully relax any unfavorable contacts, which might have arisen during the insertion process and to pre-equilibrate the systems. Simulation Conditions and Force Fields. The POPC molecules were parametrized using the united atom Berger lipid force field23 with further improvements for double bonds.24 The oligomer topology was built based on the same force field; in particular, the quaternary ammonium group was described with the same parameters as those used for the −(CH2)N+(CH3)3 moiety of the choline group in POPC, with the atomic charges slightly modified in the case of the alkyl substituted groups of o2. This modification was based on Mulliken charges obtained from the HF/6-31G* calculations (the method for attributing partial charges used in the Berger force field). The parameters (including the partial charges) for the amide bond in the side chain of o3 were adapted from the united atom force field developed for sphingomyelin.25 The partial charges in the three types of oligomer units are shown in Figure S4 (the Supporting Information). The simple point charge (SPC) model was used for water, and the appropriate OPLS parameters were used for the Cl− ions.26 The MD simulations were performed using the GROMACS 4.6 software package.27,28 Periodic boundary conditions were applied in all three directions. The simulated systems were maintained at the temperature of 310 K and under the pressure of 1 bar according to the NPT ensemble regime. The temperature was controlled by the Nosé−Hoover thermostat,29,30 and the pressure was kept constant using the semi-isotropic Parrinello−Rahman barostat.31 The long-range electrostatic potential was calculated using the particle-mesh Ewald (PME) method with the Coulomb cutoff radius of 1.0 nm.32 The LINCS constraints algorithm was employed for all bonds,33 allowing for a 2 fs time

(1)

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. The experiments were performed at least three times for each polymer. MD Simulations. Model Polymer Molecules. A chain consisting of 30 units was applied as a model of polymer 1 (oligomer o1). To mimic polymers 2a and 2b, the main chain of the model molecule was substituted with 2 (oligomer o2a) and 10 (o2b) dodecyl side chains, respectively. The polymer 3b was modeled by grafting 2 stearoyl moieties onto the backbone at random positions (oligomer o3b). To obtain physically feasible conformers of the oligomers in the aqueous medium, the model molecules were put into water boxes with the appropriate number of Cl− anions to neutralize the quaternary ammonium groups, and the systems were simulated for 50 ns at 298 K. Several conformers with shapes suitable to place in the membrane at various positions were picked up from the last 10 ns of the trajectories. POPC Membrane. The POPC membrane was prepared by arranging 288 POPC molecules onto a 12 × 12 × 2 regular grid. The membrane was hydrated with ∼9800 water molecules (ca. 37 H2O molecules per one lipid molecule), which ensures full hydration of the lipid membrane. After energy minimization, the membrane was simulated for 100 ns at 310 K, with semiisotropic pressure control. Oligomer−Membrane Systems. To study the behavior of the oligomers at the membrane surface we simulated four systems (S-o1, S-o2a, S-o2a, S-o2b, and S-o3b) containing the POPC bilayer and one oligomer chain initially put in close contact with the bilayer surface. As only the substituted oligomers (2a, 2b, and 3b) showed a marked affinity to the membrane, to further probe the phase space of those systems, we simulated three additional oligomer−bilayer systems, with 7320


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potential of POPC liposomes was about −3.2 mV which is in agreement with the previously reported values.10 The changes in ζ for the POPC liposomes treated with polymer 1 were not significant. The addition of 8 wt % of polymer 1 relative to the lipid content changed the surface potential to a slightly positive value (Table S1, Supporting Information). In contrast, the ζ potential of the POPC SUVs strongly increased after the addition of each hydrophobically modified polycation. This observation confirmed the adsorption of the hydrophobically modified polymers on the liposomal surface. The −N(CH3)3+ groups of the PAH derivatives with long alkyl chains were exposed to the bulk solution, thereby increasing the surface potential of the liposomes. However, the increase of ζ was different for each polymer (Figure 2). Several conclusions regarding the adsorption of the polycations can be drawn from the zeta potential results: (i) a comparison of polymers 2a to 2b and 3a and 3b shows that the macromolecules with a higher degree of alkyl substitution were more strongly adsorbed to the liposome surface; and (ii) the derivatives with the stearic moieties exhibited a higher ability to deposit on the liposomes compared to that with the dodecyl groups. The largest increase in the ζ potential of the polymer-decorated vesicles was observed for polymer 3b. The size of the POPC SUVs was around 115 nm, and the dispersity was less than 0.1, indicating a narrow size distribution of the liposome population. The effect of the polymers on liposome sizes was strongly dependent on the polymer architecture. The hydrodynamic diameter increased after the introduction of different mass fractions of the polycations into the POPC liposomes, indicating the adsorption of the polycations. In the case of 2a and 3a, the addition resulted in a slight increase in the sample dispersity, indicating the possibility of slight vesicle aggregation, whereas for 3b there was no concentration range, in which the vesicle aggregation occurred. For polymer 1, the increase in the hydrodynamic diameter is accompanied by a substantial increase in DI. Thus, the presence of 1 caused greater aggregation of the zwitterionic vesicles compared to that of the hydrophobically modified PAH derivatives. Cryo-TEM Observation. The morphology of the polymercovered liposomes was visualized using the cryo-TEM technique. Typical cryo-TEM micrographs of the POPC SUVs incubated with polymer 2b and 3b are shown in Figure 3. As can be seen, in both cases SUVs that have spherical shapes and distinct membranes surrounding the aqueous core are visible. The unilamellar vesicles constitute the main liposome

step. Visualizations of the trajectories were made with a VMD package.34

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RESULTS Strong polycations were synthesized by modifying commercially available PAH. The chemical structures of these polyelectrolytes are shown in Figure 1. All these polymers

Figure 1. Molecular structures of the synthesized polymers: poly(allyltrimethylammonium chloride) (1), poly(allyltrimethylammonium chloride-co-allyl-N,N-dimethyl-N-dodecylammonium chloride) (2), and poly(allyltrimethylammonium chloride-co-allyl-Nstearoylamine) (3).

have quaternary ammonium groups along polymer chains. In addition, polymers 2 and 3 have long hydrophobic side chains in their structures. However, the polymers differ in the way of the attachment of these chains. In the case of polymer 2, the alkyl chains are substituents at the quaternary ammonium groups, whereas in polymer 3 the side chains are attached to the main polymer chain by amide linkages. The degrees of substitution with the hydrophobic groups were determined from the NMR spectra, and they are listed in Table 2. Table 2. Degree of Substitution (x, see Figure 1) Polymer

x [%]

1 2a 2b 3a 3b

0.0 2.1 21.0 1.5 3.4

DLS Measurements and Zeta Potential. A series of samples containing POPC SUVs and various weight fractions of the polycations with regard to the lipid content were prepared. The hydrodynamic diameter (dz), the dispersity index (DI), and the zeta potential (ζ) of the vesicles were measured, and the results are collected in Table S1. The dependence of ζ versus the polymer content is plotted in Figure 2. The ζ

Figure 3. Cryo-TEM micrographs of POPC liposomes (cPOPC = 2.5 mg/mL) coated with polymer 2b (A, c2 = 0.06 mg/mL, 5.6 wt % content of 2b) and polymer 3b (B, c3 = 0.06 mg/mL, 5.6 wt % content of 3b). Scale bars represent 100 nm.

Figure 2. Effect of polycation content on the zeta potential of POPC SUVs. 7321


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The Journal of Physical Chemistry B population. This is clear evidence that the polymer-covered liposomes did not tend to merge and the presence of the polymers did not destabilize the membrane and did not disintegrate the vesicles. Fluorescence Studies. Calcein-release phenomenon has been utilized as an effective measure of the permeability properties of biomembranes, allowing us to evaluate interactions between polymers and lipid membranes. Calcein was encapsulated inside the POPC SUVs at the concentration causing the dye to self-quench its fluorescence. The vesicles were then coated with the polycations (5.6 wt % of the polymer relative to the lipid content). Changes in the fluorescence intensity induced by calcein release from the CL-SUVs were monitored, and the relative amount of released dye, RF, was calculated according to eq 1. The results are presented in Figure 4.

Figure 5. Final configurations of the oligomer-POPC systems after 400 ns simulations: S-o1 (a), S-o2a (b), S-o2b (c), S-o3b (d). The oligomer molecules are drawn in yellow, with the N atoms marked in blue. Lipids are shown as maroon sticks, and their phosphate groups are shown as maroon spheres. Cl− anions are represented by green spheres.

affinity for the lipid membrane (Figure 5a). Already within the first 10 ns, it moved entirely to the water phase, where it remained until the end of the simulation. This different behavior is well illustrated by the time evolution of the zcoordinate (along the bilayer normal) of the centers of mass (COM) of the oligomer main chain (Figure 6).

Figure 4. Time course of calcein leakage from the CL-SUVs treated with the polycations.

The observed total leakage of the calcein was strongly dependent on the type of polycation. For the POPC liposomes treated with polymer 1, the fluorescence intensity did not change with time. The addition of polymers 2a, 3a, and 3b to the CL-SUVs resulted in a slight increase (less than 3%) in the calcein leakage. These results suggest that polymer 1 had no effect and the other polymers exerted a rather small impact on the permeability of the zwitterionic membrane. In contrast, the presence of polymer 2b in the liposome dispersion led to a pronounced increase in the fluorescence intensity due to the dye release. The leakage of calcein increased strongly immediately after the polymer addition, and it reached a constant value of about 36% after a certain period of time. MD Simulations. To gain molecular insight into the polymer−bilayer interaction, we performed MD simulations of 30-unit oligomers used as models of the polyelectrolytes. The oligomers were inserted at two different positions: on the POPC membrane surface (the S systems) and embedded inside the lipid bilayer (the E systems). Oligomers at the Membrane Surface. Selected snapshots of the S-o1, S-o2a, S-o2b, and S-o3b systems, taken at the end of the simulations and illustrating the location and conformation of the oligomers, are shown in Figure 5. A fundamental difference characterizes the behavior of oligomer o1, in contrast to the other three molecules. While the oligomers with the hydrophobic side chains remained in close contact with the membrane and progressively tightened the binding to the membrane during the simulation, oligomer o1 exhibited no

Figure 6. Drift in the membrane normal (z) direction of COM of the oligomer main chain in the S-o1, S-o2a, S-o2b, and S-o3b systems. The orange lines mark averaged position of the P atoms of the upper lipid layer and the periodic image of the lower layer.

Figure 5b and 5d clearly show that a large portion of polyionic chains of oligomers o2a and o3b, initially placed at the POPC membrane surface, remained in the aqueous phase without contact with the POPC headgroups, when the hydrophobic groups were immersed in the membrane core. In contrast, oligomer o2b exhibited a stronger affinity for the POPC membrane. This is particularly evident in Figure 5c, showing that all parts of the main chain, initially located in the aqueous phase, strongly adhered to the membrane surface. This 7322


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interactions between the alkyl side chains and the membrane core. The electrostatic attraction alone is obviously too weak to hold the polycation close to the zwitterionic bilayer surface, as evidenced by the behavior of oligomer o1. We also considered a possible contribution of hydrogen bonding (H-bonds) between the amide linker of oligomer o3b and the nonester oxygens in the POPC phosphate groups to the polymer−membrane interaction. However, those linkers turned out to be exclusively associated with water molecules, and we did not find any bonding to the phosphate group over the whole simulation run. Oligomers Embedded into the Membrane. Snapshots showing the final configurations of the simulated systems containing the o2a, o2b, and o3b oligomers are presented in Figure 8. In all three systems, a hydrophilic pore transfixing the

polymer incorporated partially into the hydrophilic region of the membrane, but no full internalization was observed during the simulation time. To further visualize the behavior of the polycation main chains, we calculated oligomer mass density profiles across the POPC membrane (Figure S9). The profile for o2b has a maximum at ∼2.2 nm and is strongly overlapped by the lipid phosphates. In the case of the sparsely substituted oligomers, the profiles have maxima located in the aqueous phase (approximately 2.5 and 2.7 nm for o3b and o2a, respectively), indicating the tendency of the main backbones of these polycations to remain in the surrounding medium. Anchoring the substituted polycations to the membrane surface due to the incorporation of alkyl side groups into the hydrophobic region of the membrane can promote electrostatic attraction between the positively charged quaternary ammonium groups of the polymers and the negatively charged oxygen atoms of POPC. To gain quantitative information about these interactions, we calculated the radial distribution functions (rdf) for the N−O(P) and N−O(OC) atomic pairs, including the last 200 ns of the trajectories. The rdf functions of the N−O(P) (Figure S7, Supporting Information) show a distinct and narrow maximum located at 0.45 nm, which evidences a strong coordination between these two atoms. In contrast, the rdf functions for the N−O(OC) pair do not show any maximum at a distance shorter than 1.5 nm, which excludes any direct interaction between the oligomer N atoms and the glycerol groups of the POPC. A time evolution of the number of contacts between the oligomer N atoms and the phosphate O(P) atoms were then calculated (Figure 7), assuming a limiting contact distance of 0.55 nm, which corresponds to the first minimum on the appropriate rdf.

Figure 8. Final configurations of the oligomer−POPC systems: S-o2a (a), S-o2b (b), and S-o3b (c). The oligomer molecules are drawn in yellow, with the N atoms marked in blue. Lipids are shown as maroon sticks, and their phosphate groups are shown as maroon spheres. Cl− anions are represented by green spheres.

membrane was formed in the close vicinity of the oligomer molecule. A visual analysis of the trajectory revealed that the pore formation was a very rapid process. In its primary step (within the first nanoseconds), we observed the reorientation of some lipid molecules in the closest oligomer vicinity, leading to the internalization of several lipid headgroups in the hydrophobic core. Already a small number (∼5) of the polar moieties present around the oligomer molecule was enough to trigger a substantial flow of water into the hydrophobic bilayer core, which in turn stimulated the reorientation of further lipid molecules. The average number of the “core” phosphate groups, where the core was defined as a rectangular region spanning ±0.8 nm from the membrane center, reached a final value of 11−14 in a fully constituted channel. The average number of water molecules found in this region exceeds 130 for all three systems. Although all three oligomers, embedded into the POPC membrane, showed the ability to reorient the lipid molecules and create hydrophilic pores, they differ considerably in their behavior once the pore was formed. Figure 9 shows how the oligomers move in the E systems along the membrane normal. The densely substituted oligomer (o2b) resided inside the pore until the end of the simulation and did not display any tendency to translocate to the bulk water phase. Instead, it was clearly pulled down toward the membrane center. In addition, its presence stabilized the formed hydrophilic channel, ensuring the free flow of water molecules and chloride ions. In contrast, two sparsely substituted oligomers (o2a and o3a) progressively evacuated from the pore until they settled at the membrane surface in close contact with the pore.

Figure 7. Number of contacts between the oligomer N atoms and the O phosphate atoms of POPC.

Figure 7 shows that the number of the N−O(P) contacts systematically grows over the first 200 ns to reach the values of 32 ± 7, 55 ± 7, and 37 ± 9 (averaged over the last 200 ns) for the S-o2a, S-o2b, and S-o3b systems, respectively, which in each case corresponds to more than double the initial value. This increase correlates very well with the observed gradual penetration of the second (in the case of S-o2a and S-o3b) or further (S-o2b) side chains into the membrane. The marked supremacy of the densely substituted S-o2b over the other two oligomers as to the number of contacts clearly indicates that this electrostatic attraction is driven by the hydrophobic 7323


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affinity for the POPC membrane. However, as a strong polycation, it can interact electrostatically with the slightly negative liposomes, adsorbing on their surface. The addition of a slight amount of polymer 1 was enough to increase the size of the liposomes. Moreover, the dispersity of the objects increased significantly, so polymer-induced liposome aggregation occurred. The effect of polycations on the aggregation of zwitterionic liposomes is well-known and described in the literature.10,37,38 Our computational modeling of the polycations interacting with the POPC bilayer confirmed the adsorption of the hydrophobically modified polycations on the POPC membrane. The alkyl chains were buried in the hydrophobic region of the bilayer, and the main backbones were located at the water−lipid headgroup interface (Figure 5). However, the simulations revealed clearly that the charged backbones are located at different depths and adopt different conformations. Oligomer 2b firmly binds to the bilayer, whereas oligomers 2a and 3b are partially adsorbed on the liposomal surface, forming small loops and tails that protrude into the surrounding medium. Considering the real polymers that have one alkyl chain per 30 (x = 3.4) to 66 (x = 1.5) cationic units, one can expect formation of much longer loops and tails dangling in the aqueous phase. These pictures are consistent with the experimental findings that loosely anchored polycations prevent liposome fusion processes.35 Indeed, for the polymers sparsely grafted with alkyl chains we did not observe a significant increase in the DI value (Table S1). Another aspect of polycation−membrane interaction is the possibility of incorporating the polymer chain into a lipid bilayer, which can lead to changes in molecular organization and permeability for polar substances of the bilayer. The calcein leakage experiments clearly indicate that the degree of substitution is an important factor governing the partitioning of polycations into membrane interior. The results show that the substitution of the flexible polycation with 21% of the dodecyl groups is enough to induce pore formation through the bilayer. In contrast, the polymers with the low degree of substitution were not effective in releasing the hydrophilic dye from the liposomes. It has been reported that hydrophobically modified polycations can affect membrane permeability. We have previously shown that the strong polycation modified by attaching 33% of hexyl side groups, poly(allyltrimethylammonium chloride-co-allyl-N,N-dimethyl-N-hexylammonium chloride), induces a significant leakage of calcein from the POPC/DOPA (ca. 30%) and POPC (ca. 80%) liposomes.16,39 Eren et al. performed the leakage experiments form PC liposomes triggered by polycations with different alkyl side groups (C2−C10) and the degree of substitution of 100%.13 They found that polymers modified with short alkyl chains were much less active then those containing hexyl or longer chains, when almost 100% of calcein was released. Also, Wang et al. demonstrated that flexible polymers containing one or two octyl moieties attached to each cationic unit (x = 100%) were very effective and they caused almost 80% efflux of calcein form PC liposomes.14 The MD simulations of the E systems starting from the oligomers embedded into the POPC membrane should be analyzed with necessary care. As noted by Kostritskii et al., such calculations start from an essentially nonequilibrium state, when a strongly charged polymer is artificially introduced into the hydrophobic bilayer interior.11 This leads to fast reorientation of lipid headgroups toward the charged polymer groups and

Figure 9. Trajectories of the COM of the oligomer main chain along the membrane normal. The horizontal orange line marks the average position of the POPC phosphate groups.

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DISCUSSION We studied the interactions of several strong linear polycations with zwitterionic lipid membranes using experimental methods and atomic-scale MD simulations. All the polyelectrolytes have the same main chain with the quaternary ammonium groups, but they differ in the amount (the degree of substitution) and length (C12 and C17) of the attached alkyl side units. Moreover, in polymers 3 the alkyl chains were attached to the main backbone via the amide linker (see Figure 1). Thus, the impact of the strong polycation structure on its ability to adhere to or incorporate into zwitterionic lipid membranes was recognized in this study. We used the DLS and zeta potential measurements to assess adsorption of the polymers on the POPC membrane. The bare POPC SUVs had a slightly negative ζ potential, which is consistent with the previous observations,35 while the zeta potential of liposomes treated with polymers 2 and 3 significantly increased with increasing polymer content in the system. This is clear proof that the hydrophobically modified polycations were deposited on the surface of POPC liposomes. However, the observed increase was dependent on the polycation structure. Since the polycations have the same charge density, it can be concluded that the length of the alkyl substituents has a greater effect on the affinity of the polycation for the liposome surface than the degree of substitution. This is clearly visible when comparing data for polymers 2b and 3a (see Figure 2). Although the x value for polymer 2b is 14 times higher than that for polymer 3a, both polycations showed a similar dependence of ζ versus the mass fraction of polymer. The DLS results revealed that the size of liposomes slightly increased after the polycation addition, further confirming the deposition of the hydrophobically modified polycations on the liposomes. In the case of polymer 2b, we observed an increase in the DI value, which could point toward aggregation processes. However, the direct visualization of the polymer decorated SUVs using cryo-TEM microscopy demonstrated the presence of well separated vesicles in the system. Another explanation of the DI increase is fusion of the lipid vesicles. It was demonstrated previously that that polycations forming a planar thin layer on the liposome surface can induce fusion of anionic liposomes, where looping and disorganization among adsorbed cationic polymers physically prevent fusion.36 In the case of polymer 1, which is devoid of hydrophobic side groups, the zeta potential turned toward the slightly positive value after the addition of relatively large amounts of the polycation. This indicates that this polymer did not show high 7324


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*E-mail: [email protected]. Tel.: +48 12 6632263 (D.J.).

flux of water molecules into the vicinity of the polymer. Thus, the water pore is formed in the membrane. However, in these simulations we focused on the behavior of the oligomers when the system settled down after the pore formation processes. The results show that the sparsely grafted oligomers gradually escaped from the pore, and they are finally located at the bilayer surface, as is depicted in Figure 5. In contrast, oligomer o2b remained inside the water pore until the end of the simulations. Therefore, we concluded that polymer 2b is able to form hydrophilic pores or channels through lipid membranes and release polar compounds from the liposomes, as was observed experimentally.

ORCID

Shin-ichi Yusa: 0000-0002-2838-5200 Mariusz Kepczynski: 0000-0002-7304-6881 Present Address ⊥

Computational Biomolecular Dynamics Group, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany. E-mail:[email protected] (W.K.).

Notes

The authors declare no competing financial interest. Complete topology files for the oligomers are available on email request from [email protected].

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CONCLUSIONS In this study, experimental methods and atomistic MD simulations have been used to investigate the effect of structure of strong polycations on their interaction with zwitterionic lipid membranes. Since the polycations containing the quaternary ammonium groups are not able to form H-bonds with the zwitterionic POPC headgroups, the presence of hydrophobic side groups is very important for their adsorption on the surface of zwitterionic membranes. In both simulations and experiments, the hydrophobically modified polycations were shown to readily adsorb on the lipid bilayer. Our experimental results suggest that the length of the alkyl side groups is more important for the polycation adhesion to the liposome surface, while the ability of the polymers to penetrate the membrane is governed by the degree of substitution with alkyl chains. The addition of sparsely alkyl substituted polycations resulted in obtaining polymer-coated, well-separated liposomes, even at the lower polymer concentrations. The hydrophobic groups penetrate the POPC membrane, while a large part of the polymer main chain dangles freely in the aqueous phase, stabilizing the vesicles against aggregation and fusion processes. In contrast, the more densely substituted polycations adhere firmly to the membrane. Their main chains are anchored to the membrane by the alkyl chains, and the formation of contacts between the N(CH3)3 groups of polycation and the lipid phosphate groups is facilitated. The calcein-release experiments indicate that the densely substituted polycations can penetrate into the hydrophobic core of the bilayer to form water pores, thus affecting membrane permeability for polar compounds. Further studies using comprehensive experimental and computer simulation methods for other hydrophobically modified strong polycations (e.g., differing in stiffness of the main chain) are necessary to expand our understanding of the polymer behavior at the membrane surface and its impact on the lipid organization in the bilayer.

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ACKNOWLEDGMENTS This project was supported by the National Science Centre Poland on the basis of the decision number DEC-2012/07/B/ ST5/00913, by JSPS KAKENHI (Grant Numbers JP25288101 and JP16K14008) and by the Cooperative Research Program “Network Joint Research Center for Materials and Devices” (No. 20164026). J.B. appreciates the use of the EM facility at the Institute of Cellular Biology and Pathology, first Faculty of Medicine of Charles University in Prague, supported by funding from Grant Agency of the Czech Republic (302/12/ G157) and by Charles University in Prague (PRVOUK P27/ LF1 and UNCE 204022). The MD simulations were performed using the supercomputer facilities provided by ACC Cyfronet AGH, Cracow, which constitutes a part of the PL-Grid Infrastructure.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.7b05248. Description of polymers 3a and 3b synthesis, NMR spectra of polymers 3a and 3b, DLS and zeta potential data, additional figures (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel.: +48 12 6632020. Fax: +48 12 6340515 (M.K.). 7325


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