Interactions of Polyethylenimines with Zwitterionic and Anionic Lipid

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Interactions of Polyethyleneimines with Zwitterionic and Anionic Lipid Membranes Urszula Kwolek, Dorota Jamróz, Ma#gorzata Janiczek, Maria Nowakowska, Pawel Wydro, and Mariusz Kepczynski Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b00490 • Publication Date (Web): 26 Apr 2016 Downloaded from http://pubs.acs.org on April 27, 2016

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Interactions of Polyethyleneimines with Zwitterionic and Anionic Lipid Membranes Urszula Kwolek, Dorota Jamróz,* Małgorzata Janiczek, Maria Nowakowska, Paweł Wydro, Mariusz Kepczynski,* Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Kraków, Poland

ABSTRACT

Interactions between polyethyleneimines (PEIs) and phospholipid membranes are of fundamental importance for various biophysical applications of these polymers such as gene delivery. Despite investigations into the nature of these interactions, their molecular basis remains poorly understood. In this paper we combined experimental methods and atomistic molecular dynamics (MD) simulations to obtain comprehensive insight into the effect of linear and branched PEIs on zwitterionic and anionic bilayers used as simple models of mammalian cellular membranes. Our results show that PEIs adsorb only partially on the surface of zwitterionic membranes, by forming hydrogen bonds to the lipid headgroups, while a large part of the polymer chains dangles freely in the aqueous phase. In contrast, PEIs readily adhere to and insert into the anionic membrane. The attraction of the polymer chains to the membrane is due to electrostatic interactions as well as hydrogen bonding between the amine groups of PEI and the

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phosphate groups of lipids. These interactions were found to induce a substantial reorganization of the bilayer in the polymer vicinity due to the reorientation of lipid molecules. The lipid headgroups were pulled towards the membrane center, which can facilitate transmembrane translocations of anionic lipids. Furthermore, the PEI-lipid interactions affect the stability of liposomal dispersions, but we did not see any evidence of disruption of the vesicular structures into small fragments at polymer concentrations typically used in gene therapy. Our results provide a detailed molecular-level description of the lipid organization in the membrane in the presence of polycations that can be useful in understanding their mechanism of in vitro and in vivo cytotoxicity.

INTRODUCTION Polycations, both natural and synthetic, are used in a variety of biophysical and biomedical applications, such as (i) delivery of genetic materials to cells in a targeted and safe manner,1,2 (ii) usage as biocides,3,4,5 or (iii) preparation of stabilized vesicles by covering liposomes with polymer films.6,7 All of these applications involve the interaction of polycations with lipid or cellular membranes. Therefore, the impact of polycations on phospholipid bilayers has been extensively studied during the past decade using various experimental techniques8 and computer simulations.9,10 Polyethyleneimines (PEIs) are important bioactive polycations that have found application in medicinal chemistry. PEIs exist in a linear (lPEI) and branched (bPEI) architecture.1,11 The linear chain has only secondary nitrogens, while the branched chain consists of primary, secondary and tertiary nitrogens (Figure 1A). PEIs are among the most versatile and frequently used non-viral vectors for DNA complexation and transfection into several cell lines and tissues.11,12 These polycations are characterized by excellent gene complexing ability


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(formation of polyplexes) and exceptional transfectant properties. Unfortunately, they show in vitro cytotoxicity, which is the major challenge for their clinical applications.13 Experimental studies on the interactions between PEIs and phospholipid bilayers or cell membranes are rather limited and they mostly concern the stability of various liposomal dispersions in the presence of bPEI. Surprisingly, the impact of lPEI, which is considered as the gold standard for polymer-assisted gene transfection, on the liposomes has not been investigated. Sikor et al. studied the effect of ionic strength on stability of the zwitterionic phosphatidylcholine (PC) vesicles in the presence of bPEI.2 It was shown that introduction of an excess of the polymer with regard to the lipid content at low salt concentration resulted in aggregation of liposomes. At high ionic strength, though, the system was stable and bPEI was able to penetrate into the bilayer. Further research on the stability of various PEI-decorated liposomes was carried out by Sabín et al.14 The interaction of PC and anionic phosphatidylglycerol (PG) vesicles with bPEI as a function of the polymer concentration, pH, temperature, and the initial size of the liposomes was studied. The results showed a remarkable dependence of the stability of zwitterionic and anionic liposomes on these parameters. Yasuhara et al. demonstrated that bPEI can induce the concentration-dependent fusion of the anionic liposomes formed from phosphatidylserine (PS).15 The fusion occurred at bPEI concentrations below the transition of vesicular ζ-potential from negative to positive value, whereas the excess of bPEI in the system caused the colloidal stability of the liposomal dispersion due to the electrostatic repulsion between positively charged vesicles. Supported lipid bilayers (SLBs) have been also used as a model of cell membranes in studies on PEI-lipid bilayer interactions. Using atomic force microscopy (AFM), Hong et al. demonstrated that bPEI caused the expansion of preexisting defects in the supported PC membrane.16 This was correlated with in vitro results showing that


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bPEI can increase permeability of living cell membranes, allowing for enzyme leakage out of cells and diffusion of small dye molecules in and out of the cells. Recently, Zhang et al. studied the effect of lPEI and bPEI on the supported PC and PG membranes.17 The authors proved that PEIs are able to induce lipid translocation across both bilayers at physiological temperature. Molecular dynamics (MD) simulations can provide insight into the polymer - bilayer interactions at the molecular level, unavailable to experimental techniques.9 Recently, Choudhury et al.10 used the MD simulations to determine how lPEI in different protonation states interacts with PC bilayers. The simulations revealed that at low pH, lPEI chains have an elongated conformation due to the electrostatic interaction between charges of the protonated nitrogens. The fully protonated chain interacts with the bilayer forming water/ion channel through the membrane. Under basic conditions, the unprotonated polymer is highly coiled and predominately settles at the bilayer-water interface. Based on the literature overview presented above, one can conclude that further studies are necessary to provide insight into the molecular aspects of the interactions between PEIs and zwitterionic or anionic membranes, and thus, to better understand the nature of these interactions. In the current work we applied an approach combining experimental methods and computer modeling to examine the effect of linear and branched PEIs on zwitterionic and negatively charged lipid membranes used as models of cellular membranes. We focused mainly on four aspects: (i) a molecular view of PEI adsorption on the membrane surface, (ii) the effect of such adsorption on stabilization/aggregation of the vesicles, (iii) the possibility of PEI penetration into the lipid bilayers and (iv) formation of hydrophilic pores or disruption of the membranes. Liposomes were prepared from 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC), 1,2dipalmitoyl-sn-glycero-3-phosphocholine

(DPPC),

or

POPC/1,2-dioleoyl-sn-glycero-3-


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phosphoric acid (DOPA). These lipid vesicles were treated with aqueous solutions of the polycations at concentrations typically used in gene therapy18 at physiological pH and several experimental techniques were employed to study the polymer-membrane interactions. Measurements of ζ potential and the size (dynamic light scattering, DLS) of the liposomes were used to confirm adsorption of the polymers on the liposome surface and to verify the possibility of vesicle aggregation. We used micro-differential scanning calorimetry (microDSC) to study the effect of PEIs on the thermotropic behavior of DPPC/DOPA liposomes. The permeability of the lipid membranes treated with the polycations was monitored by leakage experiments applying a fluorescence dye encapsulated in the phospholipid vesicles. Additionally, intermolecular interactions between lipid molecules and PEIs were studied using Langmuir monolayer measurements. Finally, atomic-scale MD simulations allowed us to gain insight into the polycation-lipid interactions at the molecular level. Based on these results, we discussed the role of the hydration of the lPEI and bPEI macromolecules and hydrogen bonding in these interactions.


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Figure 1. (A) Chemical structures of linear (lPEI) and branched (bPEI) polyethyleneimine. Primary, secondary and tertiary amine groups are marked in green, blue and red, respectively. (B) Effect of polycation concentration on the melting enthalpy of water. MATERIALS AND METHODS Experiments.

Materials.

2-Oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine

(POPC,

≥

99.0%), 1,2-dioleoyl-sn-glycero-3-phosphoric acid monosodium salt (DOPA, ≥ 98%), 1,2dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, ≥ 99.0%,), and calcein were obtained from Sigma. Branched polyethyleneimine (bPEI) with average molecular weight of ~25,000 was purchased from Sigma-Aldrich. Linear polyethyleneimine (lPEI) with average molecular weight


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of ~25,000 was received from PolySciences, Inc. Triton X-100 was purchased from POCh (Gliwice, Poland). Millipore-quality water was used for all solution preparations. Monolayer Experiments. The experiments were carried out with a KSV 2000 Langmuir trough (KSV Instruments Ltd., Helsinki, Finland) as described previously.19 Briefly, the lipids were dissolved in a mixture of chloroform/methanol (9:1 v/v) to form stock solutions. Spreading solutions, prepared from the stock solutions, were deposited onto the subphase containing different concentrations of PEI (0, 6 or 60 µg/mL). The monolayers were left for 20 min and then compressed at the barrier speed of 5 cm2/min (0.025 nm2 molecule−1 min−1). The surface pressure was measured to a resolution of ± 0.1 mN/m using a Wilhelmy plate made of filter paper (ashless Whatman Chr1) connected to an electrobalance. Preparation of Liposomes. Small unilamellar phospholipid vesicles (SUVs) were prepared by extrusion technique as described previously.20 POPC was weighed to a glass flask and dissolved in chloroform. To obtain the anionic SUVs, a chloroform solution of DOPA was added to reach the lipid ratio of 9:1. The solvent was evaporated under a gentle stream of argon to complete dryness. 1 mM NaCl solution of pH adjusted to 7.4 was added till a desired lipid concentration was attained (usually 1.0 or 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. Light Scattering and Zeta Potential Measurements. Dynamic light scattering (DLS) and ζ potential measurements were performed as described previously using a Malvern Nano ZS lightscattering apparatus (Malvern Instrument Ltd., Worcestershire, UK).21 The time-dependent autocorrelation function of the photocurrent was acquired every 10 s with 15 acquisitions for


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each run at 25 °C. The z-averaged hydrodynamic mean diameters (dz), polydispersity (PDI) and distribution profiles of the samples were calculated using the software provided by the manufacturer. Calcein-release Studies. Calcein-loaded (CL) liposomes were prepared as described above with the only difference that the lipid film was hydrated with 0.06 M aqueous solution of calcein. The measurements were performed using a Perkin-Elmer LSD50B spectrofluorimeter. Excitation and emission wavelengths were set at 496 and 520 nm, respectively. A complete release of the dye was achieved by adding a 30 µL of 15% solution of Triton X-100. The corresponding fluorescence intensity was used as 100% leakage. The amount of calcein released after time t, RF, was calculated using the following equation22 RF( t ) = 100

It − I0 in % I max − I 0

(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. Differential Scanning Calorimetry (DSC). Hydration of polymers was evaluated using a Q2000 calorimeter (TA Instruments). Samples were encapsulated in aluminum pans and scanning was conducted between –30 and 10 °C at a heating rate of 1 ºC/min. Prior to the experiment the sealed pans were warmed at 60 °C for 24 h. The melting enthalpy of water in the solutions containing PEIs was calculated by integration of the peak at around 0 °C and dividing by the weight of water in the sample. Thermotropic behavior of lipid membranes was studied using a Nano DSC calorimeter (TA Instruments) in the range of 10 – 65 °C and at a scan speed of 0.5 ºC/min. Liposomes for DSC experiments were prepared from DPPC and DOPA (9:1).


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Molecular Dynamics Simulations. Model Polymer Molecules. A chain consisting of 30 units was applied as a model for lPEI. In the case of bPEI, the main chain of the model molecule consisted of 20 units and another 20 units constituted short side chains of one, two or three units, grafted onto the backbone at random positions. 30 % amine groups were protonated, which corresponds to the experimental value of pKa for bPEI reported by Nagaya et al.23 To obtain physically feasible conformers of both oligomers in aqueous medium, the model molecules of bPEI and lPEI were put into water boxes with the appropriate number of Cl– anions to neutralize the 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/DOPA membranes. The POPC membrane doped with ca. 10 mol % of DOPA was prepared by arranging 258 POPC molecules and 30 DOPA anions onto a 12 × 12 × 2 regular grid, giving a bilayer of 129 POPC and randomly distributed 15 DOPA molecules in each leaflet. Two mixed membranes were built in this way, corresponding to two possible protonation states of DOPA: monoanionic (DOPA−) and dianionic (DOPA2−). The mixed membranes were hydrated with ~9800 water molecules (ca. 37 H2O molecules per one lipid molecule), which ensures full hydration of the lipid membrane. Appropriate number of K+ ions was added to neutralize the DOPA negative charges. After energy minimization the membranes were simulated for 100 ns at 310 K, with a semiisotropic pressure control. PEI-membrane systems. A summary of the studied systems is given in Table 1 and images of the initial configurations are shown in Figures s1 and s2 (Supporting Information). To study behavior of PEIs at the membrane surface we simulated three systems (S1, S2, and S3) containing different type of bilayer (POPC, POPC/DOPA−, and POPC/DOPA2−) and one


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oligomer chain, initially inserted shallowly in the bilayer, thus in contact mainly with the hydrophilic part of the membrane. In three additional oligomer-bilayer systems, one PEI chain was embedded deeply into the hydrophobic core of the POPC or POPC/DOPA– membrane (systems E1 and E2) and transfixing the POPC/DOPA– membrane, with both chain ends in contact with the headgroups of both leaflets (system T2). Each of the systems was build with both forms of PEI. Three selected dehydrated conformers of bPEI and lPEI were reoriented and inserted into the pre-equilibrated membranes with the g_membed utility24 that is a part of the Gromacs simulation package. In all systems containing lPEI and those with bPEI 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 K+ and Cl– ions. Prior to productive runs of 200 ns, all the systems were subjected to short simulations of 500 ps to fully relax any unfavorable contacts, which might have arisen during the insertion process.

Table 1. Summary of the simulated systems, indicating the number of molecules in the given system. System

POPC

DOPA

Water

K+/Cl-

polymer on the membrane surface; shallowly in the bilayer S1-bPEI

125

-

7783

-/12

S1-lPEI

124

-

7592

-/9

S2-bPEI

250

29 of DOPA−

9734

29/12

S2-lPEI

250

30 of DOPA−

14063

30/12


10

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S3-bPEI

250

29 of DOPA2−

9653

58/12

S3-lPEI

251

30 of DOPA2−

14063

60/9

polymer embedded deeply into the hydrophobic core E1-bPEI

123

-

4992

-/12

E1-lPEI

121

-

7925

-/9

E2-bPEI

248

29 of DOPA−

9717

28/12

E2-lPEI

253

30 of DOPA−

14063

30/9

polymer transfixing the membrane T2-bPEI

251

27 of DOPA−

9725

27/12

T2-lPEI

253

30 of DOPA−

14062

30/9

Simulation Conditions and Force Fields. The POPC molecules and DOPA acyl chains were parameterized using the united atom Berger lipid force field25 with further improvements for double bonds.26 Atomic charges for the hydrophilic headgroups of DOPA– and DOPA2– were calculated as Mulliken charges in phosphatidic acid (PA) mono- and dianions with geometry optimized at the HF/6-31G* level, according to the method applied originally to other lipids. The calculation was done with a Gaussian 09 software.27 PEI topology was build with parameters from the OPLS-AA force field. To ensure proper computation of 1-4 interactions while combining these two force fields, a method described by Chakrabarti et al.28 was applied. The simple point charge (SPC) model was used for water and the appropriate OPLS parameters were chosen for the K+ and Cl− ions. The MD simulations were performed using the GROMACS 4.6 software package.29,30 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 thermostat31,32 and the pressure was kept constant using the semi-isotropic Parrinello-Rahman


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barostat.33 The long-range electrostatic potential was calculated using the particle-mesh Ewald (PME) method with the Coulomb cut-off radius of 1.0 nm.34 The LINCS constraints algorithm was employed for all bonds,35 allowing for a 2 fs time step. Visualizations of the trajectories were made with a VMD package.36

RESULTS AND DISCUSION PEIs are weak polycations, which means that their charge density depends on the protonation state and thus on pH of the medium. Numerous studies on bPEI protonation show that the acidic dissociation constant of this polycation depends on the ionic strength and pH of the solution, the polymer structure, concentration and molecular weight.23,37,38,39 As a result, a wide variety of pKa values at the physiological pH (7.4) has been reported, ranging from ca. 6.5037 up to over 9,38 For our PEI models in MD simulations, we assumed a moderate protonation ratio of 30%, which corresponds to the value reported by Nagaya et al., who measured the degree of protonation for bPEI of similar MW to that used in this study.23 In the case of bPEI, we protonated the primary and secondary amines, which is in line with the results of the recent study showing that the tertiary N atoms are unprotonated in physiological media.39 Hydration of PEIs. Hydration of the polymer can be an important factor influencing its interactions with lipid membranes. We performed quantitative analysis of the amount of water bound to the lPEI and bPEI chains measuring the ice-to-water phase transition. Three types of hydration water that are present in an aqueous solution of polymers are defined as follows: type I – free water having the melting point at ~0 °C; type II – water weakly interacting with macromolecules and melting below 0 °C; and type III – non-freezing water, strongly bound to hydrophilic and ionic groups of the polymer.40 The phase transition in the studied range of


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temperature was observed for types I and II water (freezable water). Mixtures containing the polymer weight fractions in the range of 0 - 38% were prepared and the melting enthalpies (∆H) were determined. Figure 1B shows that the increasing polycation concentration resulted in the gradual reduction of the melting enthalpy. In the case of bPEI, the plot of ∆H versus the polymer concentration was linear and at the weight fraction of ~49% all water present in the system had non-freezing character, indicating that it was strongly bound to the polymer chain. Thus, the number of water molecules bound to a single unit of bPEI was calculated as 2.49 ± 0.15. For lPEI, we observed significant deviations from linearity of the relationship between ∆H and the polymer concentration. This is probably due to the limited solubility of lPEI in water at low temperature. It is known that lPEI is soluble in hot water and in cold water at low pH, but it is sparingly soluble in cold water at physiological pH. The reason for this is that at higher concentrations the lPEI chains are strongly associated due to hydrogen bonding. Fitting the line to the points for the lPEI concentrations 6.14 Thus, the positively charged PEIs can interact electrostatically with the zwitterionic liposomes adsorbing on their surface. Indeed, the ζ potential of POPC SUVs increased with the increasing polymer content. In the case of bPEI, it turned to the slightly positive value of 4-5 mV after treatment with relatively large amounts of the polycation (20- 30 wt% compared to the lipid content). This is clear evidence that PEIs can physically adsorb on the vesicle surface. Our computational modeling of PEIs interacting with the POPC membrane showed that PEIs were capable to form H-bonds to polar groups of zwitterionic lipids. These bonds seem to be of a similar strength as bonds to water molecules, as a result no strong affinity of PEIs to the zwitterionic lipid membrane was observed during the run time. However, the simulations of model chains showed clearly that small fragments of the oligomers interact with the POPC bilayer, when the rest of the chain dangles freely in the aqueous phase. Moving to the real polymers having a length of ca. 580 units (corresponding to 25 000 g mol-1), it can be assumed that lPEI is partially adsorbed on the liposomal surface forming long loops and tails (Figure s12,


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Supporting Information) that extend deep into surrounding medium, according to a model developed previously by Fleer et al.46 for linear homopolymers adsorbed onto the particle surfaces. In this model, the chain fragments bound to the surface are called trains. The loops have no contact with the surface and connect two trains, while the tails are non-adsorbed chain ends. Judging from a comparable number of H-bonds (Table 3), bPEI has a similar amount of contacts with the liposomal surface as the linear form, however, due to its more compact architecture the whole polymer is located closer to the surface. The results of DLS demonstrated a significant impact of the PEI adsorption on the stability of POPC SUVs; however, we observed a difference between both the polycations studied. This is schematically depicted in Figure s12 (Supporting Information). In the case of lPEI, the presence of more than 2 wt% of the polymer compared to the lipid content was sufficient to form large infinite aggregates. For bPEI, the addition of more than 17 wt% was needed to approximately double the size of structures. Thus, the mechanism of polymer-induced liposome aggregation seems to be different for both PEIs and it is connected with the arrangement of polymer chains at the liposomal surface. The effect of bPEI on the stability of zwitterionic liposomes has been examined previously.2,14 It was demonstrated that at pH 7 bPEI adsorbs on the negative surface of DOPC liposomes and at the weight fractions of bPEI varying from 10 to 1000 %, the bPEIdecorated SUVs aggregate forming stable clusters with diameters larger than 103 nm.14 Similarly, Sikor et al. have shown that at low ionic strength 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) vesicles aggregate upon addition of more than 300 wt% of bPEI, forming stable clusters, whose final size depends on the polycation concentration.2 The aggregation process was explained using a model of aggregation of nonuniformly charged particles developed by Velegol and Thwar.47 In this model, the competition between the attraction of the polymer chain to the


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particle surface and the repulsion between chains result in a nonuniform adsorption of the polymer (polymer patches). The nonuniform distribution of net electrostatic charge in these polymer-decorated colloids leads to aggregation due to an effective attraction between polymer patches on one particle and polymer-free patches on another particle. lPEI-induced aggregation of zwitterionic liposomes has not yet been investigated. Since it occurs at a much lower polycation concentration than bPEI, its possible explanation is based on consideration of physical bridging between vesicles by the polymer. The concept of the bridging mechanism was used previously to explain the flocculation in colloids in the presence of polycations.48 The partial adsorption of lPEI chains on different vesicles leads to formation of infinite vesicle clusters. Interestingly, our simulation demonstrated that the polymer molecules initially embedded in the membrane are capable to remain inside the bilayer and they can trigger a reorientation of some nearby located lipid molecules. Similar results of MD simulations have been reported for fully protonated lPEI that was oriented parallel to the bilayer normal, spanning across the water and bilayer regions.10 Thus, the MD simulations indicate that PEI can enter into the zwitterionic lipid membrane. Although, our Langmuir monolayer experiments did not confirm these findings, Sikor et al. showed that bPEI at the concentration of 1800 wt% can penetrate into the DMPC bilayer at high ionic strength.2 Concluding, at low concentrations, PEIs interact with the surface of zwitterionic membranes due to creation of H-bonds, however, under more drastic conditions (high polymer concentration and ionic strength) these polycations can enter into the membranes changing their organization, as shown by the MD simulations. PEIs and Anionic Membranes. The zeta potential results provided evidence that both PEIs can strongly interact with the POPC/DOPA membranes. The ζ potential turned to the positive


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value at very low weight fraction of both polycations (2 wt%). Similar results were obtained previously for the interaction of different strong polycations with the POPC/DOPA liposomes.7 The DLS results, in turn, demonstrated little effect of the polymer architecture on the stability of anionic vesicles. At low concentrations of bPEI (< 3 wt%) the vesicles aggregate, whereas the higher polymer content results in obtaining polymer-decorated electrostatically stabilized liposomes with hydrodynamic diameters larger than the native SUVs. The liposomes treated with lPEI, though, have hydrodynamic diameters smaller than the native SUVs and no aggregation was observed. Our results obtained for bPEI are comparable with the literature findings. Aggregation14 or fusion15 of the vesicles at low concentrations of bPEI was described and explained by nonuniform distribution of the electric charge on the surface of liposomes (the Velegol and Thwar model of aggregation). Addition of appropriately higher amount of bPEI, resulted in complete coating of the vesicles and stabilization of them, as it was observed in our experiments. Similar interactions were described also for other polycations, such as poly-Llysine (PLL).

49

This branched polymer was deposited on DPPC/DPPG/cholesterol liposomes

and different types of interactions were observed from stabilization to aggregation of lipid vesicles, depending on the concentration and molecular mass of PLL. Interactions between lPEI and anionic liposomes have not been studied so far. Our computational modeling showed that this polycation adhered tightly to the POPC/DOPA liposomes and electrostatically stabilized the vesicles against aggregation. The adhered chains increase the ionic strength at the surface of the liposomes, compressing thus the surrounding electrical double layer and reducing the measured hydrodynamic diameter. The MD simulations confirmed that both PEIs can associate strongly with or penetrate into the POPC/DOPA membrane. The presence of the anionic lipid substantially increases the polycation


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affinity to the bilayer. The polymer chains are attracted to the liposomal surface through electrostatic interactions between the oppositely charged groups of PEIs and DOPA, thus the formation of H-bonds between the protonated amine groups of PEI and the anionic phosphate groups of DOPA is strongly facilitated. Both oligomers form the growing number of H-bonds with the lipid molecules, which is accompanied by release of some PEI hydration water. Moreover, the polymer strongly anchored to the DOPA molecules becomes more prone to create H-bonds to nearby located POPC lipids. Analysis of number of H-bonds between PEI and the lipid molecules shows that protonated amine groups of PEIs are better hydrogen donors than non-protonated ones and therefore more prone to form a hydrogen bond to lipid molecules. Due to both electrostatic interactions and formation of H-bonds to lipid molecules the PEI chains are completely incorporated into the bilayer structure, as shown in Figure 6 for the model oligomers. DSC measurements revealed the lipid separation in the presence of PEIs; however, the extent of this separation depends on the polymer architecture. These findings are consistent with the results of the MD simulations, which showed that bPEI may locally accumulate DOPA in the mixed lipid membrane more effectively than lPEI. bPEI can interact with the membrane mainly by primary amino groups. It was proven previously that PA is able to form a strong H-bond with primary amines due to proton transfer from the phosphate monoanion to amine.44 Thus, bPEI attracts adjacent DOPA molecules and forms DOPA-free domains. High mobility of this polymer on the surface of liposome resulted in moving and growing of these domains (Figure 9). In contrast, lPEI is more tightly adsorbed onto the liposomes and can interact with DOPA by secondary amine groups. Protonation of the higher order amino groups is more difficult than that of primary,50 so lPEI forms a lower number of H-bonds to DOPA (Table 3). Therefore, this polymer is less efficient in accumulation of the DOPA molecules. Similar separation of the


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anionic lipid was previously described for interactions between poly(N-ethyl-4-vinylpyridinium bromide) (PEVP) and a mixed membrane composed of DPPC and cardiolipin (CL2–).51,52 The addition of the proper amount of positively charged PEVP caused a shift of Tm for the DPPC/CL2– membrane to the value characteristic of the pure DPPC. The authors suggested that the polycation induced translocation (flip-flop) movements of CL2– to the outer leaflet of the membrane and concentration of the anionic lipid in the vicinity of the PEVP. It is known that PEI’s cytotoxicity is quite complex and can involve loss of plasma membrane integrity, phosphatidylserine (PS, an anionic lipid) translocation from the inner plasma membrane to the outer cell surface, and changes in mitochondrial membrane potential.1,53 The PS translocation is a hallmark of apoptosis. Recently, the impact of lPEI and bPEI on transmembrane translocation (flip-flop) of anionic lipids in SLBs was studied.17 It was demonstrated experimentally that at near physiological temperature (39 °C), both bPEI and lPEI can induce lipid translocation in the absence of membrane proteins. The weaker effect of lPEI on lipid transbilayer movements compared to bPEI was explained by lower charge density of this polymer. However, the molecular mechanism of these translocations has not been elucidated. Although, our MD simulations did not reveal full lipid translocations, since the time scale of the calculations was too short to observe such slow phenomena, they can shed some light on the flipflop of anionic lipids triggered by PEIs. Translocation of lipids within a membrane not containing any specific membrane proteins is normally very slow (from several hours to several days), because it requires the polar headgroup of a lipid to traverse the hydrophobic core of the membrane. The central part of the membrane is a hydrophobic barrier to permeation of small polar or ionic molecules.54 Our MD simulations showed that the PEI chains can enter the anionic bilayer. This was confirmed experimentally by expansion of the POPC/DOPA monolayer treated


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with PEIs. The embedded PEI chains induced rearrangement of the bilayer structure in the normal direction within a few hundred nanoseconds, so that the polar headgroups of nearby located lipids were quickly pulled towards the membrane center. Thus, the presence of PEIs inside the bilayer facilitates overcoming the barrier constituted by the hydrophobic core of the membrane for the anionic species. This statement is supported by the results of the calcein release experiments, showing that PEIs slightly improved the lipid membrane permeability for that negatively charged compound.

CONCLUSIONS We used comprehensive experimental methods and atomistic MD simulations to investigate the impact of linear and branched PEIs on the properties and molecular organization of zwitterionic and anionic lipid membranes, and on the stability of liposomes. Our results show that this impact depends strongly on both the polycation architecture and the nature of membrane/liposome surface. PEIs are highly hydrated in aqueous solutions at physiological pH. Since both PEI-water and PEI-lipid interactions are driven by H-bonding, these interactions compete. Both, in simulations and in experiments, PEIs readily and quickly adhere to and insert into the anionic lipid membrane structures. Due to electrostatic interactions the polymer chains are attracted to the membrane and the formation of H-bonds between the amine groups of PEI and the phosphate groups of DOPA and POPC is strongly facilitated. Thus, both PEIs form significant number of H-bonds to the lipid molecules, at the expense of H-bonds to water molecules. By contrast, PEIs only partially adsorb at the lipid/water interface of the zwitterionic membranes by forming H-bonds to the lipid headgroups, while a large part of polymer chain dangles freely in the aqueous phase.


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The PEI-lipid interactions affect the stability of liposomal dispersions; however, we did not see any evidence of disruption of the vesicle structures into small fragments at concentrations typically used in gene therapy. lPEI is a very effective aggregation agent for zwitterionic liposomes via the bridging mechanism but it strongly stabilizes the anionic liposomes even at very low concentrations. bPEI, in turn, causes association of PC liposomes at higher concentrations and the anionic liposomes at lower concentration due to the nonuniform charge distribution mechanism. The introduction of higher concentrations of bPEI to anionic liposomes results in preparation of well-separated polymer-decorated lipid vesicles. The addition of lPEI resulted in obtaining polymer-decorated well-separated SUVs, even at the lower polymer concentrations. The most important conclusion of our study is that the association and incorporation of PEIs into the anionic membrane induces a significant reorganization of the bilayer due to reorientation of lipid molecules, which direct their phosphate groups towards the ammonium nitrogen atoms of the polycation. Strong attractive interaction of PEI and anionic lipid caused gradual accumulation of anionic lipids close to the polymer, which led to lateral separation of the lipids. The incorporation of PEIs, in turn, caused pulling of lipid headgroups towards the membrane center, facilitating transmembrane translocations of anionic lipids. Therefore, it can be postulated that PEIs induce disturbances in the functioning of the living cell membranes by changes in the distribution and facilitating the flip-flop of anionic lipids such as PS. This can be considered as an important issue in the mechanism of in vitro and in vivo cytotoxicity of PEIs. Further studies linking experiment and simulation for other polycations and various compositions of lipid membranes are necessary to expand our knowledge on the impact of these polymers on biomembranes. As indicated by the present results, the combined approach using comprehensive


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experimental and computer simulation methods is adequate for understanding the polymer behavior at the membrane surface and its impact on the lipid organization in the bilayer at the molecular level.

ASSOCIATED CONTENT Supporting Information. The values of area per lipid measured in monolayer experiments. The values of the mean hydrodynamic diameter (dz), polydispersity (PDI) and the zeta potential (ζ) of POPC SUVs treated with PEIs. Snapshots of initial and final configurations of PEI-POPC and PEI-POPC/DOPA systems and final PEIs configurations in water. The radial distribution functions for the atomic pairs of PEI nitrogens and O of water and lPEI nitrogen atoms and the lipids oxygen atoms of the phosphate and the ester groups. Mass density profiles of the headgroup atoms water, and Cl– anions across the membranes. Mean square displacement (MSD) of “core” water and “bulk” water in the normal and lateral directions. Trajectory of the z coordinates of the chloride anions. Scheme of interactions between PEIs and zwitterionic and anionic membranes. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *D. Jamróz. Tel: +48 12 6632263. Fax: +358 3 3115 3015. E-mail: [email protected]. *M. Kepczynski, Tel: +48 12 6632020. Fax: +48 12 6340515. E-mail: [email protected]. Author Contributions


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The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources National Science Centre Poland (Grand Number DEC-2012/07/B/ST5/00913). ACKNOWLEDGMENT The project was financed by the National Science Centre Poland on the basis of decision number DEC-2012/07/B/ST5/00913. The MD simulations were performed using the supercomputer facilities provided by ACC Cyfronet AGH, Cracow, which constitutes a part of the PL-Grid Infrastructure. The authors thank to Mr. Artur Strzelecki from TA Instruments for his assistance in the DSC measurements. REFERENCES

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rearrangements

in

the

negative

vesicular

membrane

upon

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adsorption/desorption of the polycation. Biochim. Biophys. Acta 2002, 1560, 14–24. (52) Yaroslavov, A. A.; Rakhnyanskaya, A. A.; Yaroslavova, E. G.; Efimova, A. A.; Menger, F. M. Polyelectrolyte-coated liposomes: Stabilization of the interfacial complexes. Adv. Colloid Interface Sci. 2008, 142, 43–52. (53) Moghimi, S. M.; Symonds, P.; Murray, J. C.; Hunter, A. C.; Debska, G.; Szewczyk, A. A Two-Stage Poly(ethylenimine)-Mediated Cytotoxicity: Implications for Gene Transfer/Therapy. Mol. Ther. 2005, 11, 990–995.


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(54) Subczynski, W. K.; Wisniewska, A.; Yin, J.-J.; Hyde, J. S.; Kusumi, A. Hydrophobic Barriers of Lipid Bilayer Membranes Formed by Reduction of Water Penetration by Alkyl Chain Unsaturation and Cholesterol? Biochemistry 1994, 33, 7670-7681.

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