Interactions of PAMAM Dendrimers with Negatively Charged Model

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On the Interactions of PAMAM Dendrimers with Negatively Charged Model Biomembranes Marianna Yanez Arteta, Marie-Louise Ainalem, Lionel Porcar, Anne Martel, Helena Coker, Dan Lundberg, Debby Pei-Shan Chang, Olaf Soltwedel, Robert David Barker, and Tommy Nylander J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/jp506510s • Publication Date (Web): 13 Oct 2014 Downloaded from http://pubs.acs.org on October 20, 2014

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The Journal of Physical Chemistry

On the Interactions of PAMAM Dendrimers with Negatively Charged Model Biomembranes Marianna Yanez Arteta, ‡ Marie-Louise Ainalem,* ,†Lionel Porcar, ∥ Anne Martel, ∥ Helena Coker,∥ Dan Lundberg, ‡, § Debby P. Chang,

‡

Olaf Soltwedel,⊥ Robert Barker ∥ and Tommy

Nylander*,‡ ‡

Physical Chemistry, Center for Chemistry and Chemical Engineering, Lund University, SE-221

00 Lund, Sweden; † European Spallation Source ESS AB, SE-221 00 Lund, Sweden; ∥ Institut Laue-Langevin, 71 avenue des Martyrs, CS 20156, 38042 Grenoble Cedex 9, France; § CR Competence AB, SE-221 00 Lund, Sweden; ⊥ Max-Planck-Institute for Solid State Research, Outstation at MLZ, Lichtenbergstr. 1, 85747 Garching, Germany *Corresponding authors: Marie-Louise Ainalem. E-mail: [email protected]; Tommy Nylander. E-mail: [email protected]

Keywords: PAMAM, supported bilayer, droplet interface bilayer, PC, PS

ACS Paragon Plus Environment

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Abstract

We have investigated the interactions of cationic poly(amidoamine) (PAMAM) dendrimers of generation 4 (G4), a potential gene transfection vector, with net-anionic model biomembranes composed of different ratios of zwitterionic phosphocholine (PC) and anionic phospho-L-serine (PS) phospholipids. Two types of model membranes were used: solid supported bilayers, prepared with lipids carrying palmitoyl-oleoyl (PO) and diphytanoyl (DPh) acyl chains, and free standing bilayers, formed at the interface between two aqueous droplets in oil (Droplet Interface Bilayers, DIBs) using the DPh-based lipids. G4 dendrimers were found to translocate through POPC:POPS bilayers deposited on silica surfaces. The bilayer charge density affects translocation, which is reduced when the ionic strength increases. This shows that the dendrimerbilayers interactions are largely controlled by their electrostatic attraction. The structure of the solid-supported bilayers remains intact upon translocation of the dendrimer. However, the amount of lipids in the bilayer decreases and dendrimer/lipid aggregates are formed in bulk solution, which can be deposited on the interfacial layers upon dilution of the system with dendrimer-free solvent. Electrophysiology measurements on DIBs confirm that G4 dendrimers cross the lipid membranes containing PS, which then become more permeable to ions. The obtained results have implications for PAMAM dendrimers as delivery vehicles to cells.

Introduction The synthesis of cationic poly(amidoamine) (PAMAM) dendrimers was reported almost three decades ago and since then many studies have shown their large potential for use as vehicles for drug delivery and gene therapy delivery vehicles.1, 2, 3, 4 The low polydispersity of dendrimers, as


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well as the tunable size and the possibility of modifying the functional groups, are of benefit for their reproducible pharmacokinetic behavior.5 Dendrimers are hyperbranched macromolecules with the level of branching being described by the generation Gn. PAMAM dendrimers of generation n carry 2n+2 primary amine functional groups on the surface of an ethylenediamine core. Even though PAMAM dendrimers are the most studied type of dendrimer, the mechanism by which they are transported across membranes is still debated. This is true also for simple models of biological membranes, i. e. lipid bilayers. The colloidal and interfacial properties of these dendrimers are complex even in well-defined media and these are the factors that govern their potential as delivery vehicles. Rational development of systems for therapeutic applications requires fundamental understanding of the mechanisms and the consequences of the interactions of the drug delivery vehicles, e. g. dendrimers, with the biological membranes. Such knowledge will help to clarify the route of internalization of vehicle into cells as well as the mechanisms underlying cytotoxicity. This requires more advanced model membrane studies, allowing e.g. investigating the effect of membrane composition on the interactions with the vehicle. An ideal drug delivery vehicle should be non-toxic, non-hemolytic, non-immunogenic and biodegradable.6 Preliminary work has revealed that the toxicity of PAMAM dendrimers increases with the generation, but no general indications on immunogenicity was observed.7 Active transport via endocytosis is believed to be an important pathway for internalization of PAMAM dendrimers into cells.8, 9, 10 However, it has been found that PAMAM dendrimers can also induce the formation of pores in model membranes, which suggests that dendrimers may also be taken up by cells through passive transport mechanisms.11, 12 A Raman spectroscopy and DSC study by Gardikis et al. showed that the incorporation of PAMAM dendrimers into


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dipalmitoylphosphatidylcholine (DPPC) bilayers increased the acyl chain fluidity.13 This was concluded from a shift in the relevant Raman intensity ratio as well as a decrease in the enthalpy of the chain melting transition. These observations imply that the dendrimers can cause changes in the properties of a biological membrane.13 Parimi et al. showed a qualitative correlation between dendrimer-induced cytotoxicity and leakage of liposomes, which increase as a function of dendrimer concentration and generation.14 We have previously shown that PAMAM dendrimers readily translocate across model biomembranes composed of zwitterionic phospholipid bilayers of POPC.15 Using neutron reflectometry (NR) and ellipsometry it was demonstrated that dendrimers of generations 2 and 4 penetrated the model biomembranes without permanently affecting the bilayer integrity, while the larger generation 6 dendrimer induced partial bilayer destruction. All dendrimers studied were found to elevate the deposited lipid bilayers from the substrate surface and floating bilayers were formed upon membrane penetration by the smaller dendrimers. Our previous study only concerned the interactions of dendrimers with zwitterionic membranes. However, anionic lipids are abundant in cellular membranes and believed to take part in the translocation of proteins across the membrane.16 The objective of the present study was to reveal the effects of the presence of anionic PS lipids in the bilayer as well as the solution ionic strength, which in turn disclose the role of electrostatic interactions between PAMAM dendrimers and model membranes. PS was selected because it is the most abundant anionic phospholipid in eukaryotic cell membranes, with a content amounting to about 10% of the lipids, mainly localized in the inner leaflet of the bilayers.17, 18 The PAMAM dendrimers selected for this study were of generation 4 (G4) with 64 primary amine functional groups, which are


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protonated at neutral pH.19 This gives the dendrimer a cationic charge, and a hydrodynamic radius r of 24.5 Å as previously determined by dynamic light scattering (DLS) measurements.20 The first part of this study reports on data recorded using model systems of lipid membranes composed of single bilayers of the zwitterionic Lα 1-palmitoyl-2-oleoyl-sn-glycero-3phosphocholine (POPC) and the negatively charged Lα 1-palmitoyl-2-oleoyl-sn-glycero-3phospho-L-serine (POPS) deposited on a solid support. In order to assess the importance of the electrostatics in the interactions between the dendrimer and the anionic lipid bilayers, the measurements were performed at two different salt concentrations, 10 mM or 100 mM NaCl, and for the lower salt concentration at two POPC:POPS mole ratios, 9:1 and 8:2. The combination of the three complementary surface sensitive techniques, namely quartz crystal microbalance with dissipation monitoring (QCM-D), ellipsometry and NR, allows investigating the dynamics of adsorption as well as the structure and composition when dendrimers are added to the solid supported bilayers. In situ ellipsometry gives kinetic data on the amount of material deposited on the surface, QCM-D gives the corresponding mass, including the coupled water as well as the viscoelastic properties of the layers, while NR gives compositional and structural data of the layer as a function of the distance from the interface. On the other hand, electrophysiology measurements using droplet interface bilayers (DIBs), which are free standing model membranes,21 are useful both to assess the influence of the substrate and to monitor the extent of membrane leakage d ue to the interactions with the dendrimers. The electrophysiology measurements were performed at the higher salt concentration (100 mM KCl) and with diphytanoyl-based lipids in order to avoid chemical changes, e.g. oxidation of the lipids in the electric field. The DIBs were formed specifically with 1,2-diphytanoyl-sn-glycero-3-phosphocholine

(DPhPC)

and

1,2-diphytanoyl-sn-glycero-3-


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phosphoserine (DPhPS) at DPhPC:DPhPS mole ratios of 9:1 and 99:1. The corresponding QCMD measurements on solid-supported DPhPC:DPhPS bilayers were done to identify the possible effect of the exchange of the diphytanoyl chains compared to the palmitoyl-oleoyl chains on the interactions with the dendrimer. The present study provides important information to better understand the functional and structural consequences of the interactions between dendrimers and cell membranes. The results obtained are of relevance for the design of dendrimer-based drug and gene delivery vehicles.

Experimental Section Materials. Standard fully hydrogenated POPC and POPS were used for ellipsometry, QCM-D and NR, d31-POPC and d31-POPS (i.e. with one perdeuterated acyl chain) for NR measurements, and DPhPC and DPhPS for electrophysiology and QCM-D measurements (all obtained from Avanti Polar Lipids). PAMAM dendrimers G4 with ethylenediamine core were purchased from Sigma (10% wt. in methanol). The samples were prepared after removal of the methanol under reduced pressure in a vacuum oven for 1 day. For the surface sensitive techniques, aqueous solutions containing 0.06 mg mL-1, 0.15 mg mL-1 or 0.30 mg mL-1 G4 dendrimer, which correspond to concentrations of positive charge 1.6x1020 mL-1, 4.1x1020 mL-1 and 8.4x1020 mL-1, respectively, were prepared. For the electrophysiology measurements, the dendrimer solutions had a concentration of 10 mg mL-1 (2.7x1022 positive charges per mL) and the pH was adjusted to 7.0 or 10.6 with small aliquots of 1 M HCl or KOH. All samples were prepared with added background electrolyte (NaCl or KCl) in deionized water that was passed through a purification


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system (Milli-Q, resistivity = 18.2 mΩ.cm, organic content = 4ppb) and/or in D2O (Euriso-top, C. E. Saclay, France). Quartz Crystal Microbalance with Dissipation Monitoring. QCM-D measurements were performed on a Q-sense E4 system (Q-Sense, Gothenburg, Sweden). The principle of the technique is described in detail elsewhere.22 The experimental setup consists of four flow cell modules allowing four adsorption experiments to be carried out in parallel. Each module, which contains a separate sensor, is temperature controlled (the temperature was set at 25 °C) and has a sample volume of 0.25 mL including the inlet (40 µl volume above the crystal). The solutions were flowed through the modules with a peristaltic pump (Ismatec IPC-N 4, Zürich, Switzerland) at a flow rate of 0.7 mL min-1 for approximately 5 min (14 times the internal sample volume), while the vesicles were injected for 2 min. The quartz crystals used are coated with a SiO2 layer (QSX 303, Q-Sense) and have a fundamental frequency of 4.95 MHz. The crystals were rinsed prior to and after their use with Milli-Q water and ethanol, blow-dried with nitrogen and plasma cleaned for 5-10 min (Harrick Scientific Corp, model PDC-3XG, New York, USA) and stored in 2% SDS solution after use. The crystals were mounted in the flow modules immediately after plasma-cleaning, and then equilibrated by flowing a salt solution through the cells. The fundamental frequencies (f) and corresponding energy dissipation factors (D) of the odd overtones 1 to 13 were measured for each crystal before each experiment. A stable baseline was ensured before depositing the lipid bilayers onto the silica surface. A change in the adsorbed amount of material (∆m) is identified by a shift of the resonance frequency of the crystal (∆f). The Sauerbrey equation gives the linear relation between the change in mass and the frequency shift:22 ∆m = −

C ∆f n


(1)

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where C is a proportionality constant (C is approximately 17.7 ng s cm-2 for a 5 MHz crystal) and n is the overtone number. This expression is valid for a rigid layer, evenly distributed and with a low weight compared to the weight of the crystal. In a QCM-D experiment, the solvent associated with the adsorbed layer also contributes to the frequency change; therefore the quantity ∆m is usually referred to as the ‘wet mass’. The linear relation between the adsorbed mass and the frequency shift is not necessarily valid if the density or viscosity of the bulk liquid or the adsorbed layer changes. For viscoelastic films, the additional energy dissipation and frequency overtone-dependent changes can be modeled employing the so-called Voigt-based representation.23,

24

In this model, the adsorbed film is represented by a frequency-dependent

complex shear modulus G:

G = G'+iG' ' = µ f (1 + i 2πfτ f

)

(2)

where µf is the elastic shear modulus, ηf the shear viscosity and τf the characteristic relaxation time of the film. The frequency and dissipation changes are related to the viscoelastic properties of the adsorbed film:  β ∆f = Im  2πt ρ q q 

   

(3)

 β ∆D = − Re  πt ρ  q q

   

(4)

and

where β is a function of the thickness and density of the adsorbed film and of the density and viscosity of the bulk liquid. The parameters employed in the fitting and the representation of β are presented in the Supporting Information (Table S1 and equations S1, S2, S3 and S4).


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The QCM-D data were analyzed using the Sauerbrey expression as well as the Voigt-based representation using the QTools software (Q-Sense, Gothenburg, Sweden), employing experimental data from the 3rd, 5th, 7th and 9th overtone. These overtones were selected because energy trapping is insufficient on lower harmonics, and the side bands interfere with the main resonance on higher harmonics.25 Both models give similar values when ∆D is lower than 1×10-6 (in agreement with literature26) but the Sauerbrey expression becomes insufficient at higher values and for frequency overtone-dependent changes. Ellipsometry. In situ null ellipsometry was performed using an automated Rudolph Research thin-film ellipsometer, type 43603-200E equipped with high precision stepper motors and operating at a wavelength of 4015 Å with an angle of incidence, φ, of 67.86°.27, 28 Silicon wafers with thermally grown oxide layers (~300 Å) purchased from SWI (Semiconductor Wafer Inc., Taiwan) were used as supports for the bilayers. The presence of the thick oxide layer increases the sensitivity of the ellipsometry parameters for an adsorbed layer. The silicon wafers were cleaned at 80°C using a mixture of 25% NH4OH, 30% H2O2 and H2O (1 : 1 : 5 by volume), followed by a mixture of 32% HCl, 30% H2O2 and H2O (1 : 1 : 5 by volume), and stored in ethanol until use. Before use, they were dried under reduced pressure (0.02 mbar) and treated in an air-plasma cleaner (Harrick Scientific Corp., model PDC-3XG) for 5 min prior to the start of an experiment. The measuring cell was a ∼5 mL trapezoidal cuvette of optical glass in which the silica surface was mounted vertically. The sample solution was stirred by a magnetic stirrer at 400 rpm. The temperature was set to 25.0 ± 0.1°C and a peristaltic pump (Ismatec ISM832C, Zürich, Switzerland) allowed for a ∼5 mL min-1 flow of aqueous salt solution through the cuvette.


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Ellipsometry measures the changes in the state of polarization of light, i.e. the relative phase shift, ∆, and amplitude change, Ψ, upon reflection, which can be used for determination of film thickness, optical constants, and composition of thin films. The optical properties of a bare silica surface were determined from the measured ∆ and Ψ in two ambient media with different refractive index, i.e. air and water.27 A calibration using four-zone averaging was carried out to correct for imperfections in the optical components and thus minimize instrumental errors.29 The recorded ellipsometric angles, ∆ and Ψ, were evaluated using an optical model of the adsorbed layer based on the assumption of isotropic media and planar interfaces within the framework of an optical four-layer model, Si-SiO2-X-solvent, where X corresponds to the bilayer in the presence or absence of dendrimers.27 The surface excess, Γ, was determined using de Feijters formula for which the mean refractive index, nf, and the mean ellipsometric thickness, df, were calculated assuming layer uniformity:29 Γ=

(n

f

− n 0 )d f dn dc

(5)

where n0 = 1.342 is the refractive index of the used bulk solution and dn/dc is the refractive index increment as a function of the bulk concentration. The dn/dc value at λ = 4015 Å used for the POPC bilayer was 0.148 g cm-3.30 The dn/dc value (independent of generation) has been reported to be 0.200 g cm-3 at λ = 5893 Å for dendrimers, but is not dependent on the wavelength in this range.31 As ellipsometry does not allow determination of the layer composition, we have taken the approach to fix the dn/dc value to a value in between the phospholipid and the dendrimer, 0.174 g cm-3, after the addition of dendrimers. It is important to note that both surface excess Γ and ellipsometry thickness df, extracted from the model are assuming an homogenous adsorbed layer. The thickness obtained therefore represents the optical average thickness, which does not necessarily represent the physical thickness of the adsorbed layer in particular at low


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surface coverage or if the layer has non-uniform density profile. However, it has been shown that the value of the adsorbed amount as calculated from the refractive index and the thickness of the layer is not strongly dependent on the optical model, as validated by other methods.32 Neutron Reflectometry. NR measurements were performed on the reﬂectometer D17 at Institut Laue-Langevin, Grenoble, France,33 and NREX operated by the Max Planck Institut at Forschungs-Neutronenquelle Heinz Maier-Leibnitz, Garching, Germany. The specular reflectivity off a flat surface is measured using a collimated neutron beam as a function of the momentum transfer (Q):

Q = (4π sin θ ) λ

(6)

where θ is the glancing angle of incidence and λ is the wavelength.34 The D17 reflectometer was used in time-of-ﬂight mode, using a wavelength band of 2 to 30 Å and two angles of incidence (0.8 and 3.2°), while NREX is an angle-dispersive fixed-wavelength instrument with a default wavelength of 4.3 Å. The neutron reflectivity profiles presented in this paper show the ratio between the intensity of the specular reflection of neutrons, corrected for background scattering, to that of the incident beam (determined for each incident angle) versus Q. This study exploits the method of contrast variation and we optimize the structural information acquired by using different solvent contrasts, i.e. D2O, H2O and H2O/D2O mixture, in 62/38 ratio by volume, which matched the scattering length density of silicon (cmSi). This is possible due to the fact that chemically identical materials that contain atoms of different isotopes, e.g. hydrogen and deuterium, scatter neutrons significantly different as neutrons interact with the nuclei of atoms. The scattering length density, ρ, of a specific material is expressed in Eq. 7:

ρ = ∑ n i bi

(7)

i


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where n is the number of nuclei in a given volume and b is the coherent scattering length.35 More details regarding NR and its use to solve biomedical problems is found in a review by Nylander et al.36 A freshly polished (Siltronix, France) single silicon (Si) crystal (8 cm × 5 cm × 1 cm) was used with the (111) crystal face on one of the two large surfaces and a thin oxide layer. The substrate was thoroughly cleaned at 80 °C for 15 min using a dilute Piranha solution composed of water, sulfuric acid (98%) and hydrogen peroxide (27.5% solution in water) of a 5:4:1 volume ratio to preserve the low roughness of the oxide layer. A liquid flow-cell of ∼2 mL was used and placed so that the silicon crystal had a vertical arrangement for the experiments using D17. For the measurements performed using NREX, the PEEK trough, where the liquid sample is located, was placed below the silicon crystal. A flow of 20 mL (i.e., 10× the cell volume) of solution at a rate of 2 mL min-1 through the sample cell ensured the efficient exchange of solution during rinsing. The temperature was set to 25.0 ± 0.1 in both setups °C. Table 1 displays the scattering length densities for some materials relevant to this work including the three solvent contrasts used: D2O, H2O and cmSi.

Table 1. The scattering length densities (ρ) of silicon crystals, silicon oxide layers and the three isotopic solvent contrasts useda Si SiO2 D2O H2O cmSi ρ × 10 (Å ) 2.07 3.41 6.34 -0.56 2.07 a SiO2 denotes the surface oxide layer on the silicon crystal and cmSi denotes the water contrast -6

-2

matched to silicon. The neutron reflectivity profiles were simulated using the Motofit computer package,37 which uses the Abeles matrix model to calculate the reflectivity of thin layers and enables simultaneous


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least squares fitting of the model to the recorded data sets of different isotopic compositions.38 The fitting parameters for each layer are the thickness (d), the interfacial roughness (δ) that is related to the preceding layer in the model (the layer closer to the Si crystal), and either ρ or the solvent volume fraction (φ) when the layer is composed of a single component and solvent. Prior to deposition of the bilayers, the silicon oxide layers of the silicon substrates were characterized in two solvent contrasts (D2O and H2O). The silica layer was modeled by simultaneously fitting a three-layer model (Si-SiO2-solvent) to the reflectivity data. The errors given are provided from the variance-covariance matrix calculated using Levenberg-Marquardt optimization.39 Formation of substrate supported model membranes. Simple model biomembranes composed of lipids carrying palmitoyl-oleoyl (PO) chains were used for the NR, QCM-D and in situ null ellipsometry experiments. The acyl chains in these lipid bilayers are in the liquid disordered state under the conditions employed.40 To be able to compare directly with the electrophysiology measurements of the free standing DIBs, the corresponding diphytanoyl (DPh) lipid membranes were studied with QCM-D measurements. The studied bilayers had a net negative charge with two different charge densities achieved by mixing the zwitterionic phosphatidylcholine (PC) and anionic phosphatidylserine (PS). Supported bilayers were formed by deposition from dispersions of small unilamellar phospholipid vesicles (SUVs). The SUVs were formed by tip-sonicating aqueous POPC:POPS (9:1 and 8:2 mole ratio) or DPhPC:DPhPS (9:1 mole ratio) coarse dispersions until a clear dispersion was obtained. Before deposition, the dispersions were diluted to a final concentration of 0.5 mg mL-1. In order to ensure good surface coverage, a vesicular dispersion containing a high salt concentration (100 or 150 mM NaCl or 100 mM KCl) was used for deposition of the net negatively charged lipid mixture. The high ionic strength reduces silica-vesicle and vesicle-vesicle electrostatic repulsions and therefore


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promotes vesicle fusion and spreading into a bilayer with high coverage. Available phase diagrams for mixtures of PC and PS show high miscibility of the lipids above the melting temperature.41,

42

The bilayers are therefore expected to be laterally uniform. The lipid

composition is the same in the outer and inner leaflet of the surface deposited bilayers as reported by Hellstrand et al.43, or by Grey et al.44 in giant unilamellar vesicles of similar lipid compositions. The vesicle deposition was monitored in situ in both ellipsometry and QCM-D experiments. The measuring cells with surface deposited lipid bilayers were rinsed with salt solutions when adsorption reached steady state (plateau values) after a few minutes. The NR data were obtained approximately 2 h after each injection and are therefore expected to be recorded under steady state conditions. Dynamic Light Scattering. The size distributions of the dendrimer/lipid aggregates collected from the QCM-D experiments during rinsing were obtained by means of dynamic light scattering (DLS). The measurements were performed using a zetasizer Nano ZS (Malvern Instruments Ltd., Worshestershire, UK). This instrument measures, using Non Invasive Back-Scatter (NIBS) technology, the fluctuation in the intensity of the scattered light at a set angle of 173° and constructs an intensity autocorrelation function of the scattered intensity. The laser used is a 4mW He-Ne laser (632.8 nm) and the detection unit comprises of one avalanche photodiode. In a DLS experiment, the intensity fluctuations measured are used to produce the time correlation function. The Siegert equation expresses the relation between the normalized time correlation function of the scattered intensity, g(2)(t), and the normalized correlation function of the electric field, g(1)(t): g ( 2 ) (t ) − 1 = α g (1) (t )

2


(8)

14

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where t is the lagtime and α(≤1) is a coherence factor accounting for deviations from an ideal correlation and the experimental geometry. For polydispersed particle sizes, g(1)(t) is given by a Laplace transform: ∞

g (t ) = ∫ τA(τ ) exp(− 1 τ )d ln τ (1)

(9)

0

where τ is the relaxation time and A(τ) is the relaxation time distribution. The measured autocorrelation functions were analyzed using the inverse Laplace transformation algorithm, which was provided by the instrument software, in order to obtain intensity-averaged size distributions in terms of the hydrodynamic diameter (DH). The diameter is related to the collective diffusion coefficient via Stokes-Einstein relation, which in turn is calculated from the relaxation time of the autocorrelation function. 1.5 mL of sample from two QCM-D modules was taken from the outlet of the sample cell when flushing through dendrimer solution and after the final rinse with dendrimer free salt solution. The solutions were collected when a change in the QCM-D signal was observed to ensure that the prior solution had been exchanged from the cell. All the measurements were performed at 25° C in low volume (1 mL) poly(methyl methacrylate) (PMMA) cuvettes. Electrophysiology on Droplet Interface Bilayers. The electrophysiology measurements were performed on DIBs following the procedure described by Syeda et al.45 This method enables to measure the bilayer capacitance (Cm)46 as well as the leakage of the membrane.21 Figure 1 shows a schematic representation of the setup, which consists of two drops of saline solution hanging at the tip of electrodes in a 5 mL bath of hexadecane in a 50 mm diameter and 10 mm deep glass Petri dish. The temperature was controlled by means of a supporting hollow plate heated by thermostat water bath. The glass surface was hydrophobized by silanization to maintain the shape of the droplets in contact with the surface.


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Lipid vesicles of DPhPC:DPhPS (9:1 and 99:1 mole ratio) were made by first freeze-thawing 5 mg mL-1 aqueous coarse lipid dispersions in 100 mM KCl pH 7. SUVs were then formed by extrusion through 0.1 µm, followed by 0.03 µm, polycarbonate films. KCl was used for these experiment, rather than NaCl since K+ and Cl- ions have almost the same size while Na+ ions are larger. This ensures similar non-specific membrane permeability of these positive and negative ions. The measurements were done only at high salt concentrations to avoid the low conductivity at low salt concentrations. The electrodes are standard electrophysiology electrodes: two 250 µm silver wires where the tips were chloride coated by immersion overnight in 5% sodium hypochlorite solution, and then coated with 5% agarose gel. The obtained hydrophilic tip of each electrode is immersed in the hexadecane bath and two droplets of 450 nL of the vesicle dispersion are deposited on them with a micropipette. After about 5 min, the lipids have spontaneously formed a dense monolayer at the oil/water interface. One electrode is manually translated to bring the two droplets in contact and a bilayer is formed. This bilayer separates the two aqueous droplets and the current flow across this bilayer is measured with help of two electrodes, one in each droplet. The electrodes are connected to an amplifier (A-M system, model 2400) that applies a potential (Vm, in mV) between them and the current (I, in pA) necessary to keep this potential constant (voltage clamp mode) is measured. The current flow through the bilayer as a flow of ions and translates into an electrical current using the redox couple of Ag (metal) and AgCl salt of the electrodes extremity. The potential applied via the amplifier is controlled by an arbitrary function generator (Agilent 33522A). This allows us to ramp the voltage at arbitrary frequencies as well as to apply a stable potential between the electrodes. A nanoinjector (Drummond Nanojet II) is used to inject 50 nL


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of a solution of 10 mg mL-1 of PAMAM G4 pH 7.0 or 10.6, 0.1 M KCl, into the drops hanging on the mobile electrode. By convention, the fixed electrode is the grounded one.

Figure 1. (a) Circuit diagram describing how the electrodes in the two droplets separated by a bilayer are connected. A potential, Vm, is applied between the electrodes and controlled by an arbitrary function generator. The changes in current and voltage as a function of time are related to the capacitance, Cm, of the membrane given in pF. The resistance of the membrane, Rm, can be calculated according to Ohm’s law. (b) Schematic of the two DIBs containing the same concentration of K+ (red dots) and Cl- (blue dots) ions. G4 (green circles) is injected in one of the drops. The injection of dendrimer in the droplet with mobile electrode always causes a dendrimer concentration gradient in one direction from the mobile to the fixed electrode droplet, while the direction of the potential gradient depends on the signed of the applied potential.

Results Interactions of PAMAM dendrimers with supported membranes Quartz Crystal Microbalance with Dissipation. Figure 2 and 3 show QCM-D data for surfacesupported POPC:POPS (9:1) bilayers in aqueous solutions with low salt concentration (10 mM NaCl) and high salt concentration (100 mM NaCl), respectively, before and after the addition of


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G4 dendrimers. Time zero corresponds to the injection of the lipid vesicle dispersion to the measuring cell. The fast shifts in frequency and dissipation that are observed upon injection of lipids and dendrimers suggest that the adsorption rate is within the time frame of the mixing process in the flow-cell. Tables 2 and 3 display the adsorbed amount (∆m) obtained from modeling of the collected data using the Voigt model. QCM-D measurements were also done with POPC:POPS 8:2 bilayers in 10 mM NaCl and DPhPC:DPhPS 9:1 bilayers in 100 mM KCl and the resulting adsorbed amounts are also displayed in tables 2 and 3. The changes in frequency and dissipation as well, as the calculated mass as a function of time from these measurements, can be found in the Supporting Information (Figures S1 and S2). The adsorbed amount, as determined by QCM-D, was 4.5 mg m-2 for the deposited POPC:POPS bilayer at high salt concentration and it was found to be independent of the bilayer charge density, i.e. whether the bilayer was formed from vesicles containing 10 or 20% POPS. This value corresponds to an average area per lipid molecule of 56 Å2, which is in good agreement with reported molecular areas for POPC (62.7 Å2), and POPS (55 Å2), presented by Kučerka et al.47 and Mukhopadhyay et al,48 respectively. The adsorbed amounts calculated from data obtained after rinsing with 10 mM NaCl appear to be slightly lower. However, QCM-D is very sensitive to slight changes in the density and/or viscosity of the solvent. The viscosity of a 86 mM NaCl solution is 1.011 mPa s at 20° C.49 If we assume that the viscosity of a 10 mM NaCl solution is closer to that of pure water at 20° C (1.002 mPa s) and correct for the viscosity changes in the Voigt model, the bilayer adsorbed amount before rinsing with 10 mM NaCl is 4.2 ± 0.1 mg m2 and after rinsing are identical, i.e. 4.3 ± 0.1 mg m-2. Under the assumption that full surface coverage is obtained, and using a density of the lipid bilayer of 1 g cm-3,50 this value corresponds to a phospholipid bilayer thickness of 43 Å. Åkesson et al.51 studied POPC bilayers


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formed by vesicle fusion using a combination of QCM-D and NR without background electrolyte. Their QCM-D results showed that at 25 °C the adsorbed amount was 4.4 ± 0.4 mg m2

and the thickness measured with NR was 46 ± 2 Å, which agrees with our data. Upon addition of 0.06 mg mL-1 G4 in 10 mM NaCl, the adsorbed amount increases by

approximately 4 mg m-2, including the coupled solvent, and this value appears to be independent of the bilayer charge density. Following the addition of 0.30 mg mL-1 G4, the adsorbed amount corresponding to dendrimer and coupled solvent is slightly above 6 mg m-2. For the QCM-D measurements performed in 100 mM NaCl, the wet mass increases upon addition of 0.06 or 0.30 mg mL-1 G4 to POPC:POPS 9:1 bilayers by 2.9 and 3.3 mg m-2, respectively (for the Voigt model). In the final rinsing step with dendrimer-free salt solution, the frequency decreases even further and becomes dependent on the overtone number and the dissipation increases independently of the salt concentration. This shows that the adsorbed film now is viscoelastic (see Experimental Section).24 The surface excess increases in a similar manner on addition of G4 dendrimers to DPhPC:DPhPS 9:1 supported bilayers, in 100 mM KCl. The increase was approximately 4 mg m-2 which is 25% higher than the corresponding value obtained with the bilayers composed of lipids carrying PO chains. However, in both systems the adsorbed amounts increase upon dendrimer addition in 100 mM of simple salt is independent of the concentration of G4 in the investigated range and it is lower compared to the corresponding values obtained in 10 mM of simple salt.


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Figure 2. (a) Frequency (∆f, closed blue symbols) and dissipation (∆D, open red symbols) change as a function of time for the addition of G4 dendrimers to a pre-adsorbed POPC:POPS 9:1 bilayer on silica using QCM-D. The overtones displayed are 5 (circles), 7 (squares) and 9 (diamonds) and the corresponding fit of the Voigt model (lines between the points) to the experimental data is shown. (b) Adsorbed amount including coupled solvent (∆m) obtained from data modeling using the Voigt model. Time zero corresponds to the injection of the lipid vesicle solution to the cuvette. The dashed lines correspond to injections of: (i) NaCl 100 mM, (ii) NaCl 10 mM, (iii) G4 0.06 mg mL-1, (iv) G4 0.15 mg mL-1, (v) G4 0.30 mg mL-1, and (vi) NaCl 10 mM. Table 2. Adsorbed amount including coupled solvent (∆m) obtained by QCM-D for the addition of G4 dendrimers to POPC:POPS (9:1 and 8:2) bilayers supported on silica in10 mM NaCla Process ∆m (mg m-2) Process ∆m (mg m-2) Lipid bilayer formation POPC:POPS 9:1 4.5 ± 0.1 POPC:POPS 8:2 4.4 ± 0.1 NaCl 100 mM 4.5 ± 0.1 NaCl 150 mM 4.5 ± 0.1 NaCl 10 mM 4.2 ± 0.1 NaCl 10 mM 4.0 ± 0.1 Adsorption of G4 to the lipid bilayer G4 0.06 mg mL-1 8.5 ± 0.6 G4 0.06 mg mL-1 8.0 ± 0.6 -1 -1 G4 0.15 mg mL 10.2 ± 0.4 G4 0.15 mg mL 9.2 ± 0.4


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G4 0.30 mg mL-1 10.7 ± 0.5 G4 0.30 mg mL-1 10.1 ± 0.2 NaCl 10 mM 25.3 ± 0.1 NaCl 10 mM 19 ± 2 a The adsorbed amount values are calculated using the Voigt representation. The data given are the average ± standard deviation between 5 and 3 experiments for PC:PS 9:1 and 8:2 mole ratio, respectively.

Figure 3. (a) Frequency (∆f, closed symbols) and dissipation (∆D, open red symbols) change as a function of time for the addition of G4 dendrimers to a pre-adsorbed POPC:POPS 9:1 bilayer on silica using QCM-D at high salt concentration. The overtones displayed are 5 (circles), 7 (squares) and 9 (diamonds), and the corresponding fit of the Voigt model (lines between the points) to the experimental data is shown. (b) Adsorbed amount including coupled solvent (∆m) obtained from data modeling using the Voigt model. Time zero corresponds to the injection of the lipid vesicle solution to the cuvette. The dashed lines correspond to injections of: (i) NaCl 100 mM, (ii) G4 0.06 mg mL-1, (iii) G4 0.15 mg mL-1, (iv) G4 0.30 mg mL-1, (v) NaCl 100 mM.


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Table 3. Adsorbed amount including coupled solvent (∆m) obtained by QCM-D for the addition of G4 dendrimers to POPC:POPS (9:1) and DPhPC:DPhPS (9:1) bilayers supported on silica at high salt concentrationa Process ∆m (mg m-2) Process ∆m (mg m-2) Lipid bilayer formation POPC:POPS 9:1 4.5 ± 0.2 DPhPC:DPhPS 9:1 5±1 NaCl 100 mM 4.5 ± 0.1 KCl 100 mM 5±1 Adsorption of G4 to the lipid bilayer 7.4 ± 0.6 G4 0.06 mg mL-1 9±1 G4 0.06 mg mL-1 -1 -1 G4 0.15 mg mL 7.6 ± 0.4 G4 0.15 mg mL 9±1 -1 -1 G4 0.30 mg mL 7.8 ± 0.5 G4 0.30 mg mL 8.9 ± 0.8 NaCl 100 mM 16 ± 2 KCl 100 mM 24 ± 5 a The adsorbed amount values are calculated using Voigt representation. The data given are the average ± standard deviation of 6 and 2 experiments for POPC:POPS and DPhPC:DPhPS bilayers, respectively. In situ null ellipsometry. Figure 4 shows the in situ null ellipsometry data corresponding to the QCM-D data for the deposition of POPC:POPS 9:1 lipid bilayers and sequential addition of G4 dendrimers at low and high salt conditions. The data show the evolution of the film thickness over time, d, and the adsorbed amount, Γ, while the corresponding raw data (the ellipsometer angles Ψ and ∆) can be found in the electronic supporting information (Figure S3). Time t = 0 indicates the point of vesicle injection into the cuvette. Table 4 lists the steady-state values of d and Γ recorded during the sequential injections of lipids, replacement of solvent (“rinsing”) with NaCl solutions, and injection of G4 dendrimers. The amount of water coupled (msolvent) to the layer as estimated from the difference between the “wet” mass obtained with the QCM-D measurements and the “dry” mass determined by ellipsometry. For the POPC:POPS (9:1) bilayer, the thickness before and after rinsing with 100 mM NaCl was found to be between 46 and 48 Å and the surface excess was 4.52 ± 0.02 mg m-2, which


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corresponds to an average area per lipid molecule of 56 Å2. These values are in good agreement with those obtained using QCM-D. Rinsing with NaCl 10 mM causes very minor changes of the bilayer, i.e. the layer thickness increases 50 ± 2 Å and the surface excess increases to 4.72 ± 0.02 mg m-2. This corresponds to an average area per lipid molecule of 54 Å2, which is very close to the value observed by QCM-D. The addition of 0.06 mg mL-1 G4 from 10 and 100 mM NaCl to surface-supported POPC:POPS bilayers resulted in increases in thicknesses of 25 Å and 47 Å , respectively. The increase in film thickness in 100 mM NaCl is in good agreement with the G4 hydrodynamic diameter (2RH = 49 Å)20. This indicates that the dendrimers do not change conformation on the surface and suggests the adsorption of a monolayer of dendrimers. The corresponding increase in adsorbed amount upon addition of 0.06 mg mL-1 G4 dendrimers from 10 mM NaCl is 0.14 ± 0.02 mg m-2, but there is no significant change in the surface excess in 100 mM NaCl. The addition of 0.30 mg mL-1 G4 dendrimers to the POPC:POPS bilayers from 10 mM NaCl leads to an increase in the surface excess and the thickness by 0.88 ± 0.02 mg m-2 and 53 Å, respectively, compared to the neat lipid bilayer. The extent of thickness increase again indicates that the added dendrimers adsorb as a monolayer. Following addition of 0.30 mg mL-1 G4 from 100 mM NaCl, the film is 57 Å thicker compared to the deposited bilayer in the absence of dendrimers. In this case, the recorded dendrimer surface excess increases slightly by 0.08 mg m-2. It is also important to note that the amount of solvent coupled to the layer is relatively large after the addition of the dendrimer, particularly for the measurements performed in 10 mM NaCl. For both ionic strengths, the adsorbed amount (excluding solvent) decreases during the final rinsing step showing removal of material from the surface. However, it should be noted that this is accompanied by an increase in the film thickness as well as in the amount of water coupled to the


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layer, i.e. swelling of the layer to some extent, and we therefore conclude that the density of the layer decreases significantly.

Figure

4.

Ellipsometry data for the time evolution of the thickness (d, closed black circles) and surface excess (Γ, open red squares) for the interactions of G4 dendrimers with a POPC:POPS (9:1) bilayer supported on silica in (a) 10 mM NaCl and (b) 100 mM NaCl. Time zero corresponds to the addition of the vesicle solution to the cuvette. The dashed lines correspond to replacement of solvent (“rinsing”) (a) with: (i) NaCl 100 mM, (ii) NaCl 10 mM, (iii) G4 0.06 mg mL-1, (iv) G4 0.15 mg mL-1, (v) G4 0.30 mg mL-1, and (vi) NaCl 10 mM; and in (b) with: (i) NaCl 100 mM, (ii) G4 0.06 mg mL-1, (iii) G4 0.15 mg mL-1, (iv) G4 0.30 mg mL-1, (v) NaCl 100 mM.

Table 4. Layer thickness and surface excess, obtained by in situ null ellipsometry, and calculated amount of coupled water for the addition of G4 dendrimers to POPC:POPS (9:1) bilayers supported on silica at low salt concentration (10 mM NaCl) and high salt concentration (100 mM NaCl).a

Process

d (Å)

10 mM NaCl Γ msolvent (mg m-2) (mg m-2)

d (Å)


100 mM NaCl Γ msolvent (mg m-2) (mg m-2)

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Lipid bilayer formation POPC:POPS 47 ± 2 4.52 ± 0.02 ---44 ± 1 NaCl 100 mM 48 ± 2 4.52 ± 0.03 ---46 ± 1 NaCl 10 mM 50 ± 1 4.72 ± 0.02 ------Adsorption of G4 to lipid bilayer G4 0.06 mg mL-1 75 ± 1 4.86 ± 0.01 3.6 ± 0.6 93 ± 4 G4 0.15 mg mL-1 98 ± 1 5.45 ± 0.01 4.8 ± 0.4 100 ± 1 G4 0.30 mg mL-1 103 ± 1 5.60 ± 0.01 5.1 ± 0.6 103 ± 1 Rinse with NaCl 315 ± 2 3.14 ± 0.01 22.2 ± 0.1 182 ± 1 a The layer thickness is described by the thickness, d, and the

4.52 ± 0.02 4.54 ± 0.02 ----

----------

4.53 ± 0.01 2.9 ± 0.6 4.59 ± 0.01 3.0 ± 0.4 4.62 ± 0.01 3.2 ± 0.2 3.57 ± 0.01 12.4 ± 2 surface excess, Γ. msolvent

represents the amount of solvent coupled to the layer calculated by the difference between the adsorbed mass obtained from the QCM-D measurements and the surface excess obtained from ellipsometry.

Neutron Reflectometry. NR was used to gain detailed information regarding the positioning of the dendrimer following addition to a supported lipid bilayer. In particular, we wanted to know whether the dendrimers translocates across the lipid bilayer or not. Figure 5a displays the reflectivity profiles in three different solvent contrasts (D2O, H2O and cmSi) to which a model describing the lipid bilayer as three layers (lipids heads-tails-heads) was fitted to the data recorded for the hydrogenated POPC:POPS 9:1 lipid bilayer. The corresponding values of the parameters obtained for the POPC:POPS (9:1) bilayer fits after rinses are displayed in Table 5. The interactions between G4 and POPC:POPS bilayers were also investigated for bilayers with a higher negative charge density (POPC:POPS 8:2 mole ratio). Phospholipids with deuterated palmitoyl chains and two solvent contrasts (D2O and H2O) were used for this experiment. Figure 6a shows the NR profiles for the POPC:POPS (8:2) bilayer with the corresponding fits and Table 7 summarizes the parameters. Average scattering length densities of the POPC:POPS lipid mixtures were estimated using values reported by Fragneto et al. and Wacklin et al.; ρh-PO chains = -0.41 × 10-6 Å2, ρd-PO chains = 3.3 × 10-6 Å2, ρPC head = 1.75 × 10-6 Å2 and ρPS head = 2.63 × 10-6 Å2.52,


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For the data displayed in Figure 5a, the total thickness and the surface coverage were 46 ± 1 Å

and 94 %, respectively. To calculate the mean molecular areas from the fits, the molecular volumes (VM) of heads and tails were taken as: VM PO chains= 934 Å3, VM PC head = 344 Å3 and VM PS head

= 321 Å3.15,

52

For the POPC:POPS 9:1 bilayer, the mean lipid molecular area was

estimated to be 59 ± 1 Å2 and the average surface excess (Γ) was 4.3 mg m-2, which is in agreement with the values obtained with QCM-D and ellipsometry. Furthermore, the bilayer profiles obtained from data recorded before and after rinsing with 10 mM NaCl were the same, which shows that the effects of rinsing on the bilayer surface coverage were insignificant. The thickness and the surface coverage of the POPC:POPS 8:2 bilayer were 46 ± 1 Å and 98 %, respectively. The mean lipid molecular area was calculated to be 56 ± 1 Å2 and the average surface excess (Γ) was 4.5 mg m-2. Figures 5b and 6b show the neutron reflectivity profiles and corresponding fits for surfacesupported POPC:POPS (9:1 and 8:2 mole ratio, respectively) bilayers in the presence of 0.30 mg mL-1 G4 dendrimers recorded after rinsing using three solvent contrasts (D2O, H2O and cmSi). The scattering length density used for G4 dendrimers was 2.2 × 10-6 Å-2 in D2O,54 which is the value applied in a previous study of dendrimer adsorption onto bare substrates (in the absence of a bilayer). However, small angle neutron scattering measurements by Li et al.55 showed that PAMAM G4, G5 and G6 in the bulk solution were contrast-matched in a mixture of D2O and H2O in a 0.36 D2O volume fraction, which corresponds to a scattering length density of 1.93 × 10-6 Å-2. Nevertheless, the value used in this work gave the best fits at the solid-liquid interface and it is valid under the assumption that exchangeable hydrogen, i.e. the hydrogen in the outermost shell (the primary amine groups), have been replaced by deuterium.


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It is noteworthy that the corresponding NR data were reported by Ainalem et al. for addition of 0.30 mg mL-1 G4 to a zwitterionic d31-POPC bilayers.15 In that study, three different positions of dendrimers with respect to the bilayer were considered: (1) beneath the bilayer (i.e. penetration through the membrane to adsorb on the silica surface), (2) spanning the bilayer (i.e. incorporation into the bilayer) and (3) on top of the bilayer (i.e. adsorption to the exposed headgroups). In this work, excellent fits of the model and experimental data for both bilayers with different charge density were obtained with model 1, which shows that G4 dendrimers are able to translocate across anionic bilayers containing PS lipids (see for example Figure 6b). Furthermore, the fits improved slightly if an additional dendrimer layer, with a lower surface coverage, is located on top of the bilayer. The neutron reflectivity profiles in this study were therefore modeled using an eight-layer model (Si-SiO2-G4-lipid head-tails-head-G4-solvent) and the corresponding parameters obtained for the fits are displayed in Table 6 and 8. The total surface excesses of G4 dendrimers adsorbed to the bilayers of low and high charge densities were calculated to be 0.49 and 0.71 mg m-2, respectively, using the reported dendrimer molecular volume of 18000 Å3.55 The values of the adsorbed amounts of G4 obtained from NR are lower than the increase in surface excess estimated from the ellipsometry measurements after the addition of 0.30 mg mL-1 G4. However, this difference is attributed to the fact that the surface excess obtained from the ellipsometry data was calculated using the average refractive index increment between the dendrimer and the lipid bilayer since ellipsometry does not allow to resolve the composition of the interfacial layers as NR does. The total thicknesses of the films were ~ 93 and 104 Å for the low and high charge density bilayers, respectively, which agree with the thicknesses of the layers that were measured using ellipsometry.


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Significant decreases in bilayer coverage as well as thickness were observed for the POPC:POPS 9:1 bilayers, when the cell was flushed with D2O after adsorption of the G4 dendrimers. In this case, the surface coverage of the bilayer decreased to 46%, which corresponds to a mean molecular area of approximately 150 Å2 and a decrease in the adsorbed amount of lipid to 1.78 mg m-2. The observed decrease in bilayer thickness is most likely a result of the lower lipid surface coverage. To ensure that simultaneous data fitting using multiple contrasts was possible, the NR measurement after rinsing using D2O was repeated following a second rinse of the sample cell. The neutron reflectivity profiles recorded did not change after the subsequent rinses, which verify that the volume used per rinse was sufficient to reach steadystate in terms of adsorbed amount and layer thickness. For the higher charge density bilayer, no changes in the NR profiles were observed when the cell was flushed with D2O after adsorption of G4 dendrimers. The results are overall consistent with the data presented for bilayer formed by POPC:POPS 9:1 vesicles, although the bilayer coverage is significantly higher after rinsing, i.e. 79% for POPC:POPS 8:2 compared with 46% for POPC:POPS 9:1. The surface excess of the POPC:POPS 8:2 bilayer after G4 adsorption decreases to 3.11 mg m-2, which corresponds to a mean molecular area of approximately 85 Å2. The total surface excesses (G4 and lipids) for the low and high charge density bilayers were 2.3 mg m-2 and 3.8 mg m-2, which is lower than the bilayers surface excess before dendrimer addition. The NR data confirms that the dendrimers translocate across the bilayer. However, due to possible defects on the solid supported membrane (its surface coverage is between 94 % and 98 %), one may speculate that this might be a result of the electrostatic attraction to the SiO2 substrate. Thus, complementary measurements on free-standing membranes are essential to reveal the G4 interactions with the anionic model biomembranes.


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Figure 5. Neutron reflectivity (plotted as log10R) as a function of momentum transfer (Q), recorded using D17, for surface-deposited POPC:POPS (9:1) bilayers in the (a) absence and (b) presence of 0.30 mg mL-1 G4 dendrimers in 10 mM NaCl. Data correspond to the steady state interfacial parameters observed using ellipsometry and QCM-D in Figure 2 and 4a. Three contrasts were used to characterize the layers, D2O (red circles), H2O (green squares) and cmSi (blue triangles). Lines correspond to the neutron reflectivity profiles calculated from multilayer model fits to the experimental data. The insets correspond to the volume fraction (v) profiles as a function of the distance (d) to the silica-water interface of G4 (blue - · -), lipid headgroups (red ―) and lipid tails (black - -) obtained from the modelling of the reflectivity profiles.

Table 5. Values of the parameters obtained for fits to neutron reflectivity profiles recorded for a POPC:POPS (9:1) bilayer deposited on a silicon substrate with a thin oxide surface layer in D2O, cmSi and H2O, where each row denotes a distinct layera Layer

106 ρ (Å-2)

d (Å)

ϕ (%)


δ (Å)

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SiO2 POPC:POPS heads POPC:POPS tails POPC:POPS heads

3.47 1.8 -0.41 1.8

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9±1 19 ± 1 0.5 ± 0.5 8.5 ± 0.3 30 ± 2 2±1 30.1 ± 0.2 5.7 ± 0.7 4.8 ± 0.3 7.8 ± 0.4 31 ± 2 2.8 ± 0.4

a Data correspond to the steady state interface for a hydrogenated POPC:POPS bilayer adsorbed on silica. The neutron reflectivity profiles were fitted using a six-layer modeled (SiSiO2-POPC:POPS (lipids heads-tails heads)-solvent). The layer thickness is d, and the interfacial roughness is described by δ. ρ is the scattering length density of the component, and the solvent content in the layer equals ϕ. The roughness between the outer heads and the bulk is 7.8 ± 0.4 Å. Table 6. Values of the parameters obtained for fits to NR profiles recorded after the addition of 0.3 mg mL-1 G4 dendrimers to a POPC:POPS (9:1) bilayer in D2O, cmSi and H2O, where each row denotes a distinct layera ϕ (%) Layer 106 ρ (Å-2) d (Å) SiO2 3.47 9±1 19 ± 1 G4 2.2 24 ± 1 91 ± 1 POPC:POPS heads 1.8 5±1 57 ± 3 POPC:POPS tails -0.41 29 ± 1 56.6 ± 0.5 POPC:POPS heads 1.8 5±1 47 ± 4 G4 2.2 30 ± 4 95 ± 1

δ (Å) 0.5 ± 0.5 5.7 ± 0.2 2±1 8.7 ± 0.8 9.3 ± 0.7 5.9 ± 0.7

a Data correspond to the steady state interface for a POPC:POPS bilayer adsorbed on silica in the presence of 0.30 mg mL-1 G4 dendrimers (observed using QCM-D and ellipsometry in Figure 2 and 4a). The neutron reflectivity profiles were fitted using an eight layer model (Si-SiO2-G4POPC:POPS (lipids heads-tails-heads)-G4-solvent). The layer thickness is d, and the interfacial roughness is described by δ. ρ is the scattering length density of the component, and the solvent content in the layer equals ϕ. The roughness between the outer heads and the bulk is 5 ± 6 Å.


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Figure 6. Neutron reflectivity (plotted as log10R) as a function of momentum transfer (Q), recorded using NREX, for a surface-deposited d31-POPC:d31-POPS (8:2) bilayer in the (a) absence and (b) presence of 0.30 mg mL-1 G4 dendrimers in 10 mM NaCl. Data corresponds to the steady state interfacial parameters observed using QCM-D in Figure S1. The layer has been characterized in two contrasts, D2O (red circles) and H2O (green squares). The solid lines correspond to the neutron reflectivity profiles calculated from multilayer model fits to the experimental data. The insets correspond to the volume fraction (v) profiles as a function of the distance (d) to the silica-water interface of G4 (blue - · -), lipid headgroups (red ―) and lipid tails (black - -) obtained from the modelling of the reflectivity profiles. In (b), the dashed lines correspond to the calculated multilayer models to the D2O contrast were the dendrimer is (− · · −) only on top of or (- -) spanning the membrane. Table 7. Values of the parameters obtained for fits to neutron reflectivity profiles recorded for a d31-POPC:d31-POPS (8:2) bilayer deposited on a silicon substrate with a thin oxide surface layer in D2O and H2O, where each row denotes a distinct layera Layer SiO2

106 ρ (Å-2) 3.47

d (Å) 7±1

ϕ (%) δ (Å) 17 ± 2 3.8 ± 0.2


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POPC:POPS heads POPC:POPS tails POPC:POPS heads

1.9 3.3 1.9

7.4 ± 0.6 24 ± 4 31.3 ± 0.6 2 ± 2 7.4 ± 0.5 19 ± 4

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5±1 6±2 4±2

a Data correspond to the steady state interface for a d31-POPC:d31-POPS bilayer adsorbed on silica. The neutron reflectivity profiles were fitted using a six layer model (Si-SiO2- POPC: POPS (lipids heads-tails-heads)-solvent). The layer thickness is d, and the interfacial roughness is described by δ. ρ is the scattering length density of the component, and the solvent content in the layer equals ϕ. The roughness between the outer lipid headgroups and the bulk is 4.3 ± 0.4 Å.

Table 8. Values of the parameters obtained for fits to NR profiles recorded after the addition of 0.3 mg mL-1 PAMAM-G4 dendrimers to a d31-POPC:d31-POPS (8:2) bilayer in D2O and H2O, where each row denotes a distinct layera Layer 106 ρ (Å-2) d (Å) ϕ (%) SiO2 3.47 7 ± 1 17 ± 2 G4 2.2 34 ± 1 90 ± 2 POPC:POPS heads 1.9 5 ± 1 26 ± 5 POPC:POPS tails 3.3 30 ± 1 18 ± 1 POPC:POPS heads 1.9 5 ± 1 20 ± 4 G4 2.2 30 ± 7 93 ± 2

δ (Å) 3.8 ± 0.2 9.7 ± 0.6 2.7 ± 0.8 4±1 5±5 3.2 ± 0.7

a Data correspond to the steady state interface for a d31-POPC:d31-POPS bilayer adsorbed on silica in the presence of 0.3 mg mL-1 G4 dendrimers. The neutron reflectivity profiles were fitted using an eightlayer model (Si-SiO2-G4- POPC: POPS (lipid heads-tails-heads)-G4-solvent). The layer thickness is d, and the interfacial roughness is described by δ. ρ is the scattering length density of the component, and the solvent content in the layer equals ϕ. The roughness between the outer heads and the bulk is 10 ± 7 Å. Dynamic Light Scattering. Ellipsometry and QCM-D data showed a large increase in the thickness of the film and the ‘wet mass’ upon rinsing with salt solution after the dendrimer injection, which was not observed with NR. One possible explanation to the large increase in thickness from ellipsometry and surface excess from QCM-D is the adsorption of discrete aggregates on the lipid layers. A low concentration of aggregates present on the surface of the bilayer might not be detected by NR as this technique is not as sensitive for this type of layer structures as e.g. QCM-D. To assess whether dendrimer/lipid aggregates may be present in


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solution during the QCM-D measurements, DLS experiments were performed on the solutions collected from the rinsing step in these experiments. The DLS size-averaged intensity distributions are shown in Figure 7 and the corresponding correlation functions can be found in the Supporting Information (Figure S4). We here note that POPC:POPS 9:1 SUVs in a dispersion with a lipid concentration of 0.5 mg mL-1, showed a hydrodynamic diameter of 62 ± 2 nm and a size polydispersity index (PDI, width of the size distribution) of 0.22 ± 0.01. Data on samples collected from the QCM-D rinses show that the vesicle size does not change much during the rinsing. G4 was injected after formation of the lipid bilayer on the silica substrate. Under these conditions, the bulk vesicles dispersion should have been replaced with 100 mM NaCl solution and the bulk should be lipid free. However, the samples obtained after rinsing with 0.06 mg mL-1 G4 dendrimer solution showed the presence of polydisperse particles with a larger average hydrodynamic diameter (700 ± 100 nm) and a PDI of 0.42 ± 0.08. This particle size cannot correspond to free SUVs (~ 62 nm) or dendrimers (~ 5 nm). From the NR measurements, partial removal of lipids is observed upon dendrimer injection. Thus, it can be concluded that dendrimer/lipid aggregates are formed in bulk solution. This most likely due to the association of dendrimer in solution to the lipids removed from the bilayer upon translocation of G4. The size of the aggregates formed decreased and the PDI increased as the dendrimer concentration of the solution passed through the sample cell increased. Upon the final rinsing with a dendrimer-free salt solution, two main diffusion modes are observed, which correspond to particle sizes of 300 ± 40 nm and 5 ± 1 nm. The smaller particle sizes can be attributed to free dendrimer while the larger particles are likely to be aggregates. However, it cannot be ruled out that the latter mode is due to inter-particle repulsive interactions due to the high charge of the dendrimers, i.e. the particle diffusion in solution becomes coupled.56 In any case, the number of aggregates should be


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very low as the correlation function intercept is low (low number of scattering particles, see Supporting Information) and the free dendrimer diffusion is observed.

Figure 7. DLS intensity-averaged size distribution for (a) a vesicle solution of POPC:POPS 9:1 with a concentration of 0.5 mg mL-1, (b) the samples collected when flushing through 0.06 mg mL-1 G4 over the POPC:POPS 9:1 supported solid bilayers and (c) the final salt solution rinse. The measurements were done in 100 mM NaCl. Interactions of PAMAM dendrimers with free standing bilayers Electrophysiology. The DIBs composed of DPhPC:DPhPS mole ratios of 1:0, 99:1 and 9:1 were first characterized, as described in previous work46, by applying potential ramps and measuring the resulting current after injection of dendrimer-free buffer. The corresponding current response was square-shaped (data not shown), which verifies the capacitive behavior of the bilayer. This confirms the bilayer integrity corresponding to (nearly) complete coverage as observed with the surface-sensitive methods. A specific capacitance of 0.6 pF cm-2 was calculated for all the three bilayer compositions based on estimations of the bilayer area based on images from optical microscope. This is close to the theoretical value of 0.55 pF cm-2.57


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The effect of PAMAM dendrimers on the permeability of the three different bilayers was then monitored under constant electric potentials. Figure 8 shows the current through membranes containing different DPhPC:DPhPS ratios after the injection of G4 to a final total concentration of 1 mg mL-1 while applying an electrical potential of +10 mV. The permeability of the bilayer did not change in the absence of negatively charged lipids, i.e. no charged species seem to go through it. However, in the presence of anionic lipids, the bilayer is permeabilized and charges flow through it under the combined forces of the concentration gradient (applying to the dendrimers) and the electrical gradient (applying to all charged particles) as shown in Figure 1. This permeabilization is more dramatic for a high DPhPS content (10%) than for a low one (1%). This measurement shows that G4 is able to permeabilize lipid bilayers upon interaction with DPhPS, and potentially with any other PS phospholipid.

Figure 8. Current flow (I) through DPhPC:DPhPS membranes of different mole ratios while applying a constant potential of +10 mV, before (closed symbols) and after injection of G4 to a final concentration of 1 mg mL-1 (open symbols). The mole ratios were: 1:0 (red circles), 99:1 (blue triangles) and 9:1 (green squares).


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In order to determine whether the observed current results from the transport of K+ and Cl- ions only or if the dendrimers actually cross the bilayer, the current across the DPhPC:DPhPS 99:1 bilayer was measured while applying +10 mV and -10 mV potentials (Figure 9). The two droplets contained the same concentration of KCl and the transport of K+ and Cl- ions is therefore driven by the electrical potential only, i.e. the amplitude and the direction of the current are determined by Vm value and sign, respectively. However, the dendrimers carry 64 positive charges per molecule and are injected only in one of the droplets. Therefore, they are subject both to the electrical gradient imposed by Vm, and to a chemical gradient resulting from the difference of their concentration between the two droplets. The direction of the concentration gradient is always from the mobile droplet (where the dendrimers are injected) to the fixed droplet (Figure 1). The current recorded at a potential of +10 mV, when the chemical and the electrical gradients of G4 have the same direction, is higher, in absolute value, than at -10 mV, when they have opposite direction. Thus, the observed current reponse can be attributed to dendrimers also crossing the bilayers between the droplets and not only to the transport of small ions. The current also has an opposite sign at a potential of - 10 mV than at + 10 mV. This means that the electrical gradient developed under a -10 mV potential exerts a higher force than the chemical one and the resulting current is much lower and changes direction. In this situation, dendrimers on average do not migrate to the fixed droplet, as they appeared to be trapped in the compartment with the negative potential. The observed current originate from the motion of K+ ions crossing from the grounded to the negative drops and Cl- ions moving in the opposite direction. Therefore, the results show that G4 is not only able to cross the bilayer, but they are also able to make the membrane permeable for other (smaller) ions.


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Figure 9. Current flow (I) through a DPhPC:DPhPS (99:1) membrane after injection of G4 to a final concentration of 1 mg mL-1 while applying a constant potential of + 10 mV (black filled circles) or - 10 mV (red open squares). Similar results were observed for bilayers containing 10% mole of DPhPS (Figure 10) at + 10 mV, but here the effect is so large that the membrane is destabilized and the droplets fuse immediately. However, by varying the applied potential from + 1 mV to - 10 mV, we observe how the current gradually decreases. At - 3 mV, the electrical and chemical gradients are almost equilibrated, resulting in a very weak current.


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Figure 10. Current flow (I) through a DPhPC:DPhPS (9:1) membrane after injection of G4 to a final concentration of 1 mg mL-1 under a tension of + 1 mV (light blue inverted triangles), 1 mV (dark blue triangles), - 2 mV (green squares), - 3 mV (black diamond) and - 10 mV (red circles). In the presence of the anionic lipid, dendrimer/lipid aggregates are immediately formed upon injection of dendrimer in the grounded droplet (Figure 11). The aggregates can be observed both through the microscope and as well with the naked eye and they are probably formed through electrostatic interactions between the DPhPC:DPhPS vesicles and the dendrimers in the droplet. There is approximately a DPhPC:DPhPS:G4 89:10:1 mole ratio in a droplet of the 9:1 lipid vesicle solution and 98:1:1 in the 99:1 solution. This corresponds to 1.6x1014 and 1.9x1013 negatively charged head-groups per droplet, respectively, while the injection of dendrimer introduces 1.4x1015 positive charges. Thus, electrostatic attractions in the bulk solution could promote the formation of DPhPC:DPhPS/G4 aggregates, but still not cause a significant reduction in the concentration of free dendrimer.


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Figure 11. Microscopy image of aggregates visible in a droplet containing DPhPC:DPhPS 9:1 vesicles after injection of G4. Combining these electrophysiological observations with the data from the surface sensitive techniques confirm our analysis of the NR experiment that G4 dendrimers, at neutral pH, permeabilize and cross lipid bilayers upon contact with negatively charged lipids present in the bilayers. Consequently, at pH 10.6, when the dendrimer are close to neutral, no effect on the membrane permeability is observed (Figure S5). The results indicate that the substrate does not play a major role promoting the translocation of the dendrimers across the bilayers. Discussion Dendrimer translocation across solid supported lipid bilayers Surface sensitive techniques and electrophysiology measurements showed that PAMAM G4 translocates through model biomembranes containing PS lipids both at low and high ionic strength (samples prepared in solutions of 10 or 100 mM of 1:1 electrolyte). The adsorbed amount after the addition of dendrimers increased and was similar for bilayers with different charge density. However, the amount was larger and showed stronger dependence on the dendrimer concentration at low ionic strength compared to the data at high ionic strength. The adsorption of the cationic dendrimer is reduced when the ionic strength increases, which can be attributed to screening of the electrostatic interactions. We note that the adsorbed amount


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determined by ellipsometry is typically significantly lower than the corresponding QCM-D data, except for the lipid bilayer before dendrimer adsorption (Table 4). This is expected as QCM-D mass also includes the coupled solvent, which is high after the dendrimer injection. A key question is the location of G4 in relation to the model membranes, which could be revealed by NR measurements. In fact, the experimental reflectivity profiles show very good agreement with a model where the dendrimers have translocated across the bilayer and are located between the membrane and the surface. In addition some dendrimers are adsorbed on top of the lipid bilayer. This shows that PAMAM dendrimers can translocate across a negatively charged bilayer, which is consistent with findings from previous work on the ability of PAMAM dendrimers to translocate across model membranes of zwitterionic lipids only.15 The surface excess of G4 was found to be slightly lower for the lower charge density bilayer (POPC:POPS 9:1). The bilayer coverage was also reduced to a greater extent (46 % by volume) compared to the POPC:POPS 8:2 bilayers (79 % by volume), when the cell was flushed with salt solution after adsorption of the dendrimers. From previous work on POPC bilayers, we know that the coverage after rinsing was reduced to 47 % while maintaining the integrity of the bilayer.15 Thus, the results show that the bilayer coverage increases as the amount of anionic lipid in the bilayer increases. The dendrimer layer underneath the bilayer is highly hydrated as a result of the balance between the electrostatic attraction to the oppositely charged substrate/bilayer and the intermolecular repulsion. This explains the large amount of coupled water measured by QCM-D (Table 4). Effect of dendrimer/lipid aggregates in bulk solution on the interfacial layer Additional information on the layer structure can be extracted from the QCM-D results. For all the systems studied, rinsing with dendrimer-free salt solution gave a large decrease in frequency


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and increase in dissipation. Reviakine et al. pointed out that dissipation of energy in QCM-D data can occur for both laterally homogeneous as well as laterally heterogeneous films. For the laterally homogeneous layers, the dissipation increase occurs due to internal motion of molecules in the layer, while for the laterally heterogeneous it results from the motion of particles and occurs at the liquid-particle boundary.58 Here, the observed increase in mass (wet mass) from QCM-D measurements could be attributed to swelling of the layers. In fact, the ellipsometry data for the subsequent rinses with a salt solution showed an increase in the films thickness, although the surface excesses (dry mass) decreased. Possibly, this finding could be taken to indicate a restructuring of the adsorbed film in such a way that more solvent is coupled to the layer. From the modelling of the NR data it was also found that the total adsorbed amount (G4 and lipids) after flushing with the salt solution is reduced. In fact, as observed from the ellipsometry results, it is even lower than the surface excess of the bilayer before dendrimer addition. Here we must note that the surface excess from ellipsometry was calculated based on the refractive index increment of the individual components and does not allow us to resolve the composition of the layers. DLS measurements showed that dendrimer/lipid aggregates are formed in bulk solution upon rinsing the QCM-D flow cells with G4 solutions. Previous work on the adsorption from mixtures of cationic polyelectrolytes and anionic surfactants on silica (negatively charged surface) showed that the adsorption at high surfactant concentrations (above charge neutrality) of polyelectrolyte/surfactant complexes could in fact increase upon dilution when exchanging the polymer-surfactant solution with water. This was attributed to precipitation of the complexes on the surface as the system enters the two-phase region (phase separation) upon dilution during the rinsing process. In this case, the dendrimer can be considered as a dispersing agent that solubilizes the lipids removed from the bilayer and, thus, the final rinsing step could potentially


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promote the precipitation of a few aggregates as the system is diluted. In fact, the ellipsometry data indicates an increase in layer thickness, compared to the layers before rinsing with salt, but the surface excess is low suggesting that there are only a few particles adsorbed to the surface. QCM-D is, compared to ellipsometry and NR, very sensitive for discrete particles adsorbed on the surface, as the amount of coupled solvent can be significant even if the coverage is very low.59 Åkesson, Cardenas et al., have investigated the interactions between PAMAM G6 and lipid bilayers formed by mixtures of POPC and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1´-racglycerol) (POPG) (negatively charged) both in bulk solution and on solid supports. It is important to note that their experiments were performed at different salt conditions (phosphatebuffered saline (PBS) solution containing 10 mM sodium phosphate and 100 mM NaCl at pH 7.4), with higher generation of dendrimers and a different type of anionic phospholipid. They showed the formation of bulk aggregates from POPC:POPG 9:1 vesicles and PAMAM G6, with apparent hydrodynamic diameters from 214 to 248 nm.60 These aggregates are smaller than the ones found in this study formed by PAMAM G4 and POPC:POPS bilayers However, the differences in the size of the aggregates can be attributed to the dissimilarities of the two systems as mentioned above. They also investigated the addition of PAMAM G6 to bilayers on silica formed from vesicles with POPC:POPG at 3:1 mole ratio 8 and the adsorption from the mixtures of PAMAM G6 and POPC:POPG vesicles with supported lipid bilayers of different negative charge densities.61 Under the conditions employed, and contrary to the present study, their NR data showed that upon the addition of 8 µM G6 (1.2x1021 positive charges per mL) to POPC:POPG 3:1 solid supported bilayers, the dendrimer adsorbed on top of the membrane and did not translocate through it. Our previous data on zwitterionic lipid bilayer also show that the


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dendrimers of larger generation had a detrimental effect on the bilayer integrity and also formed a layer of dendrimers on top of the bilayer.15 Åkesson et al. also observed partial removal of lipids and an increase in layer thickness, consistent with a model with patches of lipid stacked with dendrimer molecules sandwiched between the lipid bilayers.8 Although G6 was not able to translocate across the POPC:POPG bilayers, the ‘dendrimer/lipid stacks’ are likely closely related to the adsorption of dendrimer-lipid aggregates obtained in the present work. Furthermore, they recently showed that the adsorption from PAMAM G6 dendrimer/ POPC:POPG mixtures produces layers with a similar interfacial structure as observed in this study if the substrate is located below the sample.61 On the other hand, they showed the adsorption of aggregates with a lamellar order if the substrate was placed above the solution phase. The differences were attributed to a transport mechanism mediated by gravity and electrostatic force. It cannot be ruled out that such phenomena can potentially influence our results. However, the extent of aggregation is expected to be significantly lower as in our system they only appear during the rinsing stage.

Leakage of the model biomembrane induced by dendrimer/bilayer interaction The electrophysiological response to the injection of dendrimer in the presence of PS was significant and increased with the PS concentration. However, in the absence of the anionic lipid and if the data was recorded at high pH, when the charge of the dendrimers is close to neutrality, no effect was observed on the membrane structure. This indicates that the electrostatic interactions play a major role in the interactions between the dendrimers and the lipid bilayers. Dendrimer addition disrupts and even destroys DIBs as the fraction of PS in the bilayers increases. One might suspect that the dendrimer/lipid aggregates may also contribute to the


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observed effects since G4 aggregates with the free DPhPC:DPhPS vesicles in the droplets. Furthermore, changes in the membrane permeability are only observed when the bilayer contains negatively charged lipids and it is likely that it is indeed the free dendrimers that interact with and transverse the membrane. Some of the electrophysiology results can be taken to be in contradiction to the findings from previous work where G4 translocated through supported lipid bilayers of POPC only. However, there are some important differences between the setups used (supported membranes vs. DIBs) that could explain the different results. One major distinction between the methods is that, during the electrophysiology measurements, the bulk solution in the droplets contains a high number of free liposomes when the dendrimer is added, which is not the case for the bulk solution on measurements with solid supported membranes. The measurements showed the formation of large aggregates between G4 and vesicles containing PS but the interactions with PC only liposomes are much weaker. Thus, even if the dendrimers cross the membranes to a low extent, the bilayer might regenerate fast due to the free DPhPC lipid vesicles present in the aqueous phase and thus no current changes are measured while the DPhPC:DPhPS/G4 aggregates formed could hinder the regeneration of the bilayer. This study has shown that PAMAM dendrimers can penetrate also an anionic lipid bilayer, while retaining the bilayer structure. However, depending on the bilayer, charge the penetration can induce leakage of this simple model biomembrane. Thus, PAMAM G4 dendrimers can induce the formation of holes in the lipid membranes that allow the dendrimers to transport other molecules to their interior, but also implies that they can potentially be hemolytic and thus toxic. It is clear that more realistic membrane models could help to tune the balance between toxicity and the capability of dendrimers as penetration enhancers. One possible way to manipulate the


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interaction between dendrimer-based vehicles and cell membranes could be to conjugate the dendrimer with other molecules, which modulate the extent of biomembrane surface interaction.

Conclusions One of the challenges for the potential application of PAMAM dendrimers as drug or gene delivery vehicles is to control dendrimer uptake into the cell. An understanding of the mechanism is important to support pharmacokinetic and toxicity data that will be vital for acceptance and development of dendrimers as a pharmaceutical agent. Most cell membranes have a net negative charge and electrostatic interactions with the cationic dendrimers play an important role. We have shown that PAMAM G4 dendrimers can translocate through model membranes containing PS lipids. This was observed for solid-supported bilayers and for DIBs formed by vesicles with different PC:PS mole ratios. The electrostatic attraction between the dendrimer and the lipid bilayers is screened to a larger extent at high salt concentrations and at high pH (>10). Based on the observed effects of the bilayer charge density and the solution conditions, we can conclude that the electrostatic attraction between the cationic dendrimer and the anionic bilayer promote the translocation process. As a result, the lipid density in the bilayer decreases at least locally and the membrane becomes more permeable. Additionally, G4 forms aggregates in bulk solution with the oppositely charged lipids and those aggregates can deposit at the interface of the membrane or they could hinder the regeneration of the DIBs. The ability of PAMAM G4 dendrimers to disrupt model biomembranes of net negative charge could explain how dendrimers used as drug delivery vehicles cross the cell membrane but it might also have implications on their cytotoxicity.


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ASSOCIATED CONTENT Supporting Information. Parameters for the Voigt model and the description of β, QCM-D frequency, dissipation and surface excess versus time plots, ellipsometry angles as function of time plots, electrophysiology data of the effect of pH on the interactions of G4 with DPhPC:DPhPS DIBs and correlation functions from the DLS measurements on vesicles and dendrimer/lipid aggregates. This material is available free of charge via the Internet at http://pubs.acs.org. ACKNOWLEDGMENT This work was supported by the Swedish Research Council (VR) through the Linnaeus grant Organizing Molecular Matter (OMM) center of excellence (239-2009-6794). The Knut and Alice Wallenberg foundation funded the acquisition of the QCM-D instrument. We thank the ILL for beam time allocations on D17 and Max Planck Society at the Heinz Maier-Leibnitz Zentrum (MLZ) for beam time allocation on NREX. We also thank Costanza Montis for assistance during the neutron reflectometry (NREX) experiment at Heinz Maier-Leibnitz Zentrum, FRM II, in Munich. References 1. Kesharwani, P.; Jain, K.; Jain, N. K. Dendrimer as nanocarrier for drug delivery. Prog. Polym. Sci. 2014, 39 (2), 268-307. 2. Tomalia, D. A. Birth of a new macromolecular architecture: dendrimers as quantized building blocks for nanoscale synthetic polymer chemistry. Prog. Polym. Sci. 2005, 30 (3–4), 294-324. 3. Eichman, J. D.; Bielinska, A. U.; Kukowska-Latallo, J. F.; Baker Jr, J. R. The use of PAMAM dendrimers in the efficient transfer of genetic material into cells. Pharm. Sci. Technol. Today 2000, 3 (7), 232-245. 4. Dufès, C.; Uchegbu, I. F.; Schätzlein, A. G. Dendrimers in gene delivery. Adv. Drug Delivery Rev. 2005, 57 (15), 2177-2202. 5. Esfand, R.; Tomalia, D. A. Poly(amidoamine) (PAMAM) dendrimers: from biomimicry to drug delivery and biomedical applications. Drug Discovery Today 2001, 6 (8), 427-436.


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Figure 1. (a) Circuit diagram describing how the electrodes in the two droplets separated by a bilayer are connected. A potential, Vm, is applied between the electrodes and controlled by an arbitrary function generator. The changes in current and voltage as a function of time are related to the capacitance, Cm, of the membrane given in pF. The resistance of the membrane, Rm, can be calculated according to Ohm’s law. (b) Schematic of the two DIBs containing the same concentration of K+ (red dots) and Cl- (blue dots) ions. G4 (green circles) is injected in one of the drops. The injection of dendrimer in the droplet with mobile electrode always causes a dendrimer concentration gradient in one direction from the mobile to the fixed electrode droplet, while the direction of the potential gradient depends on the signed of the applied potential. 70x36mm (300 x 300 DPI)



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Figure 2. (a) Frequency (∆f, closed blue symbols) and dissipation (∆D, open red symbols) change as a function of time for the addition of G4 dendrimers to a pre-adsorbed POPC:POPS 9:1 bilayer on silica using QCM-D. The overtones displayed are 5 (circles), 7 (squares) and 9 (diamonds) and the corresponding fit of the Voigt model (lines between the points) to the experimental data is shown. (b) Adsorbed amount including coupled solvent (∆m) obtained from data modeling using the Voigt model. Time zero corresponds to the injection of the lipid vesicle solution to the cuvette. The dashed lines correspond to injections of: (i) NaCl 100 mM, (ii) NaCl 10 mM, (iii) G4 0.06 mg mL-1, (iv) G4 0.15 mg mL-1, (v) G4 0.30 mg mL-1, and (vi) NaCl 10 mM. 135x70mm (150 x 150 DPI)


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Figure 3. (a) Frequency (∆f, closed symbols) and dissipation (∆D, open red symbols) change as a function of time for the addition of G4 dendrimers to a pre-adsorbed POPC:POPS 9:1 bilayer on silica using QCM-D at high salt concentration. The overtones displayed are 5 (circles), 7 (squares) and 9 (diamonds), and the corresponding fit of the Voigt model (lines between the points) to the experimental data is shown. (b) Adsorbed amount including coupled solvent (∆m) obtained from data modeling using the Voigt model. Time zero corresponds to the injection of the lipid vesicle solution to the cuvette. The dashed lines correspond to injections of: (i) NaCl 100 mM, (ii) G4 0.06 mg mL-1, (iii) G4 0.15 mg mL-1, (iv) G4 0.30 mg mL-1, (v) NaCl 100 mM. 135x70mm (150 x 150 DPI)



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Figure 4. Ellipsometry data for the time evolution of the thickness (d, closed black circles) and surface excess (Γ, open red squares) for the interactions of G4 dendrimers with a POPC:POPS (9:1) bilayer supported on silica in (a) 10 mM NaCl and (b) 100 mM NaCl. Time zero corresponds to the addition of the vesicle solution to the cuvette. The dashed lines correspond to replacement of solvent (“rinsing”) (a) with: (i) NaCl 100 mM, (ii) NaCl 10 mM, (iii) G4 0.06 mg mL-1, (iv) G4 0.15 mg mL-1, (v) G4 0.30 mg mL-1, and (vi) NaCl 10 mM; and in (b) with: (i) NaCl 100 mM, (ii) G4 0.06 mg mL-1, (iii) G4 0.15 mg mL-1, (iv) G4 0.30 mg mL-1, (v) NaCl 100 mM. 142x70mm (150 x 150 DPI)


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Figure 5. Neutron reflectivity (plotted as log10R) as a function of momentum transfer (Q), recorded using D17, for surface-deposited POPC:POPS (9:1) bilayers in the (a) absence and (b) presence of 0.30 mg mL-1 G4 dendrimers in 10 mM NaCl. Data correspond to the steady state interfacial parameters observed using ellipsometry and QCM-D in Figure 2 and 4a. Three contrasts were used to characterize the layers, D2O (red circles), H2O (green squares) and cmSi (blue triangles). Lines correspond to the neutron reflectivity profiles calculated from multilayer model fits to the experimental data. The insets correspond to the volume fraction (v) profiles as a function of the distance (d) to the silica-water interface of G4 (blue - · -), lipid headgroups (red ―) and lipid tails (black - -) obtained from the modelling of the reflectivity profiles. 140x69mm (150 x 150 DPI)



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Figure 6. Neutron reflectivity (plotted as log10R) as a function of momentum transfer (Q), recorded using NREX, for a surface-deposited d31-POPC:d31-POPS (8:2) bilayer in the (a) absence and (b) presence of 0.30 mg mL-1 G4 dendrimers in 10 mM NaCl. Data corresponds to the steady state interfacial parameters observed using QCM-D in Figure S1. The layer has been characterized in two contrasts, D2O (red circles) and H2O (green squares). The solid lines correspond to the neutron reflectivity profiles calculated from multilayer model fits to the experimental data. The insets correspond to the volume fraction (v) profiles as a function of the distance (d) to the silica-water interface of G4 (blue - · -), lipid headgroups (red ―) and lipid tails (black - -) obtained from the modelling of the reflectivity profiles. In (b), the dashed lines correspond to the calculated multilayer models to the D2O contrast were the dendrimer is (− ∙ ∙ −) only on top of or (- ) spanning the membrane. 141x69mm (150 x 150 DPI)


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Figure 7. DLS intensity-averaged size distribution for (a) a vesicle solution of POPC:POPS 9:1 with a concentration of 0.5 mg mL-1, (b) the samples collected when flushing through 0.06 mg mL-1 G4 over the POPC:POPS 9:1 supported solid bilayers and (c) the final salt solution rinse. The measurements were done in 100 mM NaCl. 246x185mm (72 x 72 DPI)



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Figure 8. Current flow (I) through DPhPC:DPhPS membranes of different mole ratios while applying a constant potential of +10 mV, before (closed symbols) and after injection of G4 to a final concentration of 1 mg mL-1 (open symbols). The mole ratios were: 1:0 (red circles), 99:1 (blue triangles) and 9:1 (green squares). 66x69mm (150 x 150 DPI)


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Figure 9. Current flow (I) through a DPhPC:DPhPS (99:1) membrane after injection of G4 to a final concentration of 1 mg mL-1 while applying a constant potential of + 10 mV (black filled circles) or - 10 mV (red open squares). 64x70mm (150 x 150 DPI)



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Figure 10. Current flow (I) through a DPhPC:DPhPS (9:1) membrane after injection of G4 to a final concentration of 1 mg mL-1 under a tension of + 1 mV (light blue inverted triangles), - 1 mV (dark blue triangles), - 2 mV (green squares), - 3 mV (black diamond) and - 10 mV (red circles). 67x70mm (150 x 150 DPI)


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Figure 11. Microscopy image of aggregates visible in a droplet containing DPhPC:DPhPS 9:1 vesicles after injection of G4. 43x43mm (150 x 150 DPI)



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Interactions of PAMAM Dendrimers with Negatively Charged Model

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