Interaction of DNA with Cationic Lipid MixturesâInvestigation at

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Interaction of DNA with Cationic Lipid Mixtures – Investigation at Langmuir Lipid Monolayers Christopher Janich, André Hädicke, Udo Bakowsky, Gerald Brezesinski, and Christian Wölk Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b02014 • Publication Date (Web): 05 Sep 2017 Downloaded from http://pubs.acs.org on September 15, 2017

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Interaction of DNA with Cationic Lipid Mixtures – Investigation at Langmuir Lipid Monolayers Christopher Janich,a André Hädicke,b Udo Bakowsky,c Gerald Brezesinski,d Christian Wölk a, *

a Martin Luther University Halle-Wittenberg, Institute of Pharmacy, WolfgangLangenbeck-Strasse 4, 06120 Halle (Saale), Germany b CUO-Recherche, Hôpital du Saint-Sacrement, Centre de recherche du CHU de Québec and Département d'ophtalmologie, Faculté de médecine, and Regroupement stratégique PROTEO, Université Laval, Québec, Québec, Canada c Department of Pharmaceutics and Biopharmaceutics, University Marburg, Ketzerbach 63, 35037 Marburg, Germany d Max-Planck Institute of Colloids and Interfaces, Science Park Potsdam-Golm, Am Muehlenberg 1, 14476 Potsdam, Germany

*corresponding author Christian Wölk Institute of Pharmacy, Martin Luther University Halle-Wittenberg Wolfgang-Langenbeck-Strasse 4, 06120 Halle (Saale), Germany e-mail: [email protected]

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Abstract: Four different binary lipid mixtures composed of a cationic lipid and the zwitterionic co-lipids DOPE or DPPC, which show different DNA transfer activities in cell culture models, were investigated at the soft air/water interface to identify transfection efficiency determining characteristics. Langmuir films are useful models to investigate the interaction between DNA and lipid mixtures in a two-dimensional model system by using different surface sensitive techniques,

namely

epifluorescence

microscopy

and

infrared

reflection-absorption

spectroscopy. Especially, the effect of adsorbed DNA on the properties of the lipid mixtures has been examined. Distinct differences between the lipid composites were found which are caused by the different co-lipids of the mixtures. DOPE-containing lipid mixtures form fluid monolayers with a uniform distribution of the fluorescent probe in presence and absence of DNA at physiologically relevant surface pressures. Only at high non-physiological pressures the lipid monolayer collapses and phase separation was observed if DNA was present in the subphase. In contrast, DPPC-containing lipid mixtures show domains in the liquid condensed phase state in presence and absence of DNA in the subphase. The adsorption of DNA at the positively charged mixed lipid monolayer induces phase separation which is expressed in the morphology and the point of appearance of these domains.

Graphical abstract

Keywords cationic lipid, DNA, gene therapy, Langmuir monolayer, lipid mixing, malonic acid amide, phospholipids 2 ACS Paragon Plus Environment

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1. Introduction Lipid-based gene transfer (lipofection) vectors are the most commonly used class of non-viral gene delivery systems employed in clinical trials.1 Their ease of production, cost effectiveness and minimized immunological and carcinogenic risks, make lipofection vectors attractive as potential therapeutics.2,

3

Nevertheless, lipid-based transfection systems show lower

transfection efficiencies compared to viral vectors.4-6 Since Felgner et al. published their groundbreaking paper on lipofection in 1987,7 numerous cationic lipids have been synthesized for the purpose of gene delivery, and various theories about their cellular uptake and intracellular release of their nucleic acid payloads have been proposed.8-11 Also anionic liposomes in presence of divalent cations are promising nucleotide carriers.12, 13 Additionally, some universal transfection-influencing parameters have been determined, including physicalchemical characteristics such as the fluidity and length of lipid alkyl chains and the N/P-ratio (primary amino groups of the cationic lipid/phosphate groups of DNA) or charge density of head-groups.11,

14-16

Nevertheless, to date no universal optimal transfection-influencing

parameters have been identified, and different physical-chemical studies have produced contradictory results

17, 18

. Therefore, many unresolved questions regarding what constitutes

an optimal lipid formulation for gene transfer remained. It is our conjecture that optimal miscibility of the lipid components is a prerequisite for high transfection efficacies. Therefore, this study is designed to elucidate the correlation between miscibility and transfection efficiency. For this, we have selected four well-characterized binary mixtures (1/1 molar ratio) of cationic lipids with phospholipids as zwitterionic colipids. The cationic lipids are malonic acid diamides of the second generation (OH4 and TH4 in Figure 1). The phospholipids selected are DOPE and DPPC (Figure 1). In previous experiments, we performed a detailed screening of the four different cationic lipid/phospholipid mixtures at different molar mixing ratios and N/P ratios.19,

20

In this 3

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screening the equimolar ratio shows the best effectivity for each cationic lipid/phospholipid mixture, while in a direct comparison of different lipid composites OH4/DOPE mixtures exhibited the highest transfection efficiency, whereas TH4/DOPE has only moderate transfer rates, and OH4/DPPC and TH4/DPPC showed vanishing low transfection efficiencies.19,

20

Therefore, the focus is set on the equimolar mixtures for on-going research. The selfassembling of the binary lipid mixtures and the ternary lipid/DNA systems was investigated in bulk.21 In this study, these binary lipid mixtures were investigated using air/liquid interface monolayer techniques coupled with epifluorescence microscopy or infrared reflectionabsorption spectroscopy (IRRAS) in the presence and absence of DNA in the subphase in order to gain a better understanding of their mixing behavior.

Figure 1. Structures of the lipids used in this study: N-{6-amino-1-[N-(9Z)-octadec-9enylamino]-1-oxohexan-(2S)-2-yl}-N0-{2-[N,N-bis(2-aminoethyl)-amino]ethyl}-2hexadecylpropandiamide (OH4), N-[6-amino-1-oxo-1-(N-tetradecylamino)hexan-(2S)-2-yl]N0-{2-[N,N-bis(2-aminoethyl)amino]ethyl}-2-hexadecylpropandiamide (TH4), 1,2-dioleoylsn-glycero-3-phosphoethanolamine (DOPE), and 1,2-dipalmitoyl-sn-glycero-3phosphocholine (DPPC). Structural differences are highlighted.

2. Experimental 2.1 Materials


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Unless otherwise stated, all materials were purchased from Sigma-Aldrich. The 2-(Nmorpholino)ethanesulfonic acid (MES) buffer (10 mM) was prepared with Milli-Q Millipore water with a specific resistance of 18.2 MΩ cm and adjusted to pH 6.5 and filtered through a 0.2 mm cellulose acetate membrane before use. Lyophilized calf thymus DNA (ctDNA) was purchased from Sigma-Aldrich and dissolved in MES buffer to the appropriate concentration followed by 12 h stirring at 7°C before use. The synthesis of TH4 and OH4 was described previously.19, 22 DOPE and DPPC were purchased from Avanti Polar Lipids (Alabaster, AL, USA). The fluorescent dye BODIPY® 558/568 C12 (4,4-difluoro-5-(2-thienyl)-4-bora-3a,4adiaza-s-indacene-3-dodecanoic acid) (λexmax = 558 nm, λemmax = 568 nm) was purchased from ThermoFisher Scientific.

2.2 Methods Monolayer experiments For monolayer experiments, stock solutions of lipids were prepared in chloroform/methanol 9/1 (v/v). Lipid concentrations were 0.3 mM. The solutions were spread onto the subphase using an appropriate Hamilton syringe. In the absence of DNA, 10 min were given for the evaporation of the solvent. Experiments with DNA were started after 12 h given for adsorption of DNA to the monolayer compressed to 10 mN m-1 and allow the system to equilibrate. Therefore, we spread the lipid on the DNA containing subphase to ensure a homogeneous distribution of DNA, and compressed the monolayer to 10 mN m-1 before starting to collect the surface pressure data. The pressure/area isotherms were recorded on a computer-interfaced Langmuir trough (R&K, Potsdam, Germany) which is equipped with a Wilhelmy balance. The compression speed was 2 A2 molecule-1 min-1. The temperature was kept constant at 25 °C with an accuracy of 0.1 °C.

Epifluorescence microscopy 5 ACS Paragon Plus Environment

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Fluorescence microscopy imaging of monolayers at the air/water interface was performed using an Axio Scope A1 Vario epifluorescence microscope (Carl Zeiss MicroImaging, Jena, Germany). Underneath the microscope, a Langmuir Teflon trough with a maximal area of 264 cm2 and two moveable computer-controlled Teflon barriers (Riegler & Kirstein, Potsdam, Germany) was positioned on an x–y stage (Märzhäuser, Wetzlar, Germany) to be able to move the film surface with respect to the objective lens to any desired position. The x–y–z motion control was managed by a MAC5000 system (Ludl Electronic Products, Hawthorne, NY, USA). The trough was enclosed by a home-built Plexiglas hood to ensure a dust-free environment and to minimize evaporation of water. The temperature of 20.0 ± 0.1 °C was maintained with a circulating water bath, and the whole setup was placed on a vibrationdamped optical table (Newport, Darmstadt, Germany). The air/water interface was illuminated using the following set-up from Carl Zeiss MicroImaging (Jena, Germany): a 100 W mercury arc lamp (HXP 120 C), a long working distance objective (LD EC EpiplanNEOFLUAR 50x) and a filter/beam splitter combination (Filter Set 81HE), to select appropriate wavelengths for the excitation and detection of BODIPY® 558/568 C12. Images were recorded using an EMCCD camera (ImageEM C9100-13, Hamamatsu, Herrsching, Germany). Image analysis and data acquisition were done using the AxioVision software (Carl Zeiss MicroImaging, Jena, Germany). All presented images show areas of individually contrast-adjusted raw data. Lipid solutions with a concentration of 0.3 mM lipid in chloroform containing only 0.1 mol% fluorescently labelled BODIPY® 558/568 C12 were prepared separately. Lipid monolayer films were prepared by spreading this stock solution onto the water surface. The subphase may contain DNA. The monolayer was compressed using a compression speed of 2 Å2·molecule-1·min-1. Microscopy images were taken during the compression of the monolayer.

Infrared reflection-absorption spectroscopy (IRRAS) 6 ACS Paragon Plus Environment

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Infrared reflection–absorption spectra were recorded on a Vertex 70 FTIR spectrometer (Bruker, Ettlingen, Germany). The setup included a film balance (R&K, Potsdam, Germany) and an external air/water reflection unit, XA-511 (Bruker). A sample trough with two movable barriers for compressing the lipid monolayer, and a reference trough (bare subphase) allow the fast recording of sample and reference spectra by a shuttle technique. The setup with sample and reference troughs is placed in an air-tight plexiglas box to avoid fluctuations in the atmosphere. Sample and reference were filled to the same level (checked by the measured integrated intensity of the detector) to avoid effects of different filling levels. The infrared beam was focused on the liquid surface by a set of mirrors. The angle of incidence normal to the surface can be varied by means of moveable arms in the range between 30° and 72°. A KRS-5 wire grid polarizer was used to polarize the IR radiation, either in a parallel (p) or perpendicular (s) direction. After reflection from the surface, the beam was directed to a narrow-band mercury cadmium telluride detector (MCT) cooled with liquid nitrogen. The interference of the atmosphere and the aqueous subphase was removed by measuring the sample trough (subphase with lipid monolayer) and reference trough (bare subphase) in rapid succession (timescale of few minutes). Reflectance–absorbance spectra were obtained by using –log(R/R0), where R is the reflectance of the film-covered surface and R0 is the reflectance of the same subphase without the film. For each single-beam spectrum, 200 scans (s-polarized light) or 400 scans (p-polarized light) were added with a scanning velocity of 20 kHz and a resolution of 8 cm-1, apodized using the Blackman–Harris three-term function, and fast Fourier transformed after one level of zero filling.23 For data analysis, spectra obtained with s polarized light and an angle of incidence of 40° were used. All spectra were corrected for atmospheric interference (to correct weak fluctuations of the atmosphere inside the plexiglas box) using the OPUS software and baseline corrected using the spectral subtraction software. The spectra were not smoothed. To determine the exact position of the bands, a Lorentzian curve was fitted to the data points. 7 ACS Paragon Plus Environment

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3. Results and Discussion 3.1 Langmuir compression isotherms and epifluorescence microscopy of lipid mixtures First, the isotherms of DPPC mixed with a cationic lipid, OH4 or TH4, will be examined. To get a deeper understanding of the mixing behavior, binary mixtures of the cationic lipids with increasing amount of DPPC were investigated. The equimolar mixtures are of special biological relevance and will be considered in the later described experiments with DNA in the subphase.20 The effect of an increasing amount of either OH4 or TH4 in DPPC monolayers on the pressure-area isotherms at 25 °C is presented in Figure 2. The subphase was MES buffer (10 mM) adjusted to pH 6.5 referring to the environment of complex formation between the lipid mixtures and DNA used in previous studies.19-21 While compressing the DPPC monolayer a plateau region, indicating a first-order phase transition from the liquid-expanded (LE) to the liquid-condensed (LC) phase state, occurs at about 11 mN m-1. This transition was also proved by IRRAS experiments.19 Upon compression of monolayers composed of OH4 or TH4 no phase transition is observed and the lipids remain in the LE state (Figure 2A/C).19 Addition of cationic lipids to DPPC results in a shift of the isotherm to higher areas per molecule for both cationic lipids (compare Figure 2A for OH4 and 2C for TH4). The cationic lipids require a larger area per lipid due to the larger head-group (experimental results on MES 10 mM pH 6.5 Ao[OH4, 30 mN m-1] = 64.6 Å2, Ao[TH4, 30 mN m-1] = 63.1 Å2, Ao[DPPC, 30 mN m-1] = 47.6 Å2)

19

and the charge repulsion occurring between the cationic lipids at lower xDPPC

values (xlipid is defined as mole fraction of one lipid component, xlipid = nlipid ntotal-1). Furthermore, the plateau region becomes broader and less pronounced with increasing amount of cationic lipid (Figure 2A and C; xDPPC 0.8-0.67). For equimolar mixtures only a kink in the isotherms is visible (Figure 2A and 2C; xDPPC 0.5) in the case of OH4 as well as TH4. Additionally, the beginning of the phase transition shifts to higher surface pressures with 8 ACS Paragon Plus Environment

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increasing amount of cationic lipid (Fig. 2B [OH4] and 2D [TH4]). It should be emphasized that the average molecular areas of the binary mixtures are larger than the ones expected for ideal mixing behavior, as presented in Figure 2E/F for OH4/DPPC and TH4/DPPC at the physiological relevant pressure of 30 mN m-1. This positive deviation from the additivity rule (positive excess areas) indicates repulsive forces between the lipid components.24

Figure 2. Surface pressure – area isotherms of monolayers composed of OH4/DPPC (A) and TH4/DPPC (C) at varying molar ratios (n/n, the number given in brackets represents the mole fraction x of DPPC) on MES buffer pH 6.5 (10 mM). Additionally, the surface pressure of the beginning phase transition as function of xDPPC is given for OH4/DPPC (B) and TH4/DPPC (D) mixtures. The given values are taken from the surface pressure – area isotherms in combination with the compressibility curves (see SI Figure S1-S3). The average areas per molecule (A(30)) as function of xDPPC at 30 mN m-1 are given for OH4/DPPC (E) and TH4/DPPC (F). The dotted straight line demonstrates either ideal mixing behavior (additivity rule Aid12 = x1A1 + x2A2) or completely immiscibility. Complementing to the compression isotherm epi-fluorescence microscopy measurements were performed. This technique allows investigating the effect of the lipid composition on the domain formation in the LE/LC phase transition region. The results for OH4/DPPC mixtures are summarized in Figure 3. DPPC monolayer form condensed domains at the beginning of the LE/LC phase transition region at a surface pressure of 11.3 mN m-1 (Figure 3I). The 9 ACS Paragon Plus Environment

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condensed domains (LC) appear black, because the chain-labeled fluorescent lipid probe (BODIPY® 558/568 C12) is excluded from the more ordered regions. The dye is dissolved in the more expanded LE state. A clear coexistence of the LE and the LC states was observed until a surface pressure of around 20 mN m-1 (Figure 3 J-L). At higher surface pressures, the border between the two regions becomes indistinct (SI Fig. S4), a normal observation because the surface is covered with LC domains and the dye is enriched at the border of the condensed domains. Within the phase transition region of the isotherm, the domains grow with increasing surface pressure and branched protrusions occur at the domains. Typically, DPPC forms kidney-shaped domains, but fluorescence probes can induce shape instabilities 25. Also the used MES buffer cannot be excluded for promoting shape instabilities.

Figure 3. Fluorescence microscopy images of OH4/DPPC (n/n) or pure DPPC monolayers on MES buffer pH 6.5 (10 mM) at varying surface pressures (given red number in mN m-1). The scale bar indicating 20 µm is the same for all images. The fluorescent lipid probe was BODIPY® 558/568 C12 in a concentration of 0.1 mol%. Addition of the cationic lipid OH4 to DPPC changes the shapes of the condensed domains. For OH4/DPPC mixtures with a molar ratio of 1/2 (n/n) the size of the condensed domains decreases and the shape becomes star-like (Figure 3E-H). First domains appear with the 10 ACS Paragon Plus Environment

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beginning of the phase transition region at 15 mN m-1 and grow continuously with increasing surface pressure. OH4/DPPC mixtures with a molar ratio of 1/4 (n/n) show a comparable behavior with star-like domains appearing at the phase transition and growing with increasing surface pressure (data not shown). For equimolar OH4/DPPC mixtures, the relevant ratio for the comparison with the transfection efficiency, the condensed domains appear at higher surface pressure (33 mN m-1, Figure 3B) compared to the other mixtures with less cationic lipids. The occurrence of the condensed domains corresponds with the kink in the isotherm. The domains are quite small and the size decreases with increasing surface pressure. At a pressure above 40 mN m-1 (Figure 3D), the domains disappear or exhibit a size below the resolution of the microscope.

Figure 4. Fluorescence microscopy images of TH4/DPPC (n/n) monolayers on MES buffer pH 6.5 (10 mM) at varying surface pressures (given red number in mN m-1). The red scale bar indicating 20 µm is the same for all pictures. The fluorescent lipid probe was BODIPY® 558/568 C12 in a concentration of 0.1 mol%. The fluorescence behavior of TH4/DPPC mixtures is approximately comparable to OH4/DPPC mixtures (compare Figures 3 and 4). The appearance of the first condensed domains corresponds with the beginning of the phase transitions region in the Langmuir 11 ACS Paragon Plus Environment

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isotherms. The sizes of the domains increase with increasing surface pressure for the TH4/DPPC mixtures 1/4 (n/n) (Figure 4I-L). In case of the ratio 1/2, the domains firstly grow but get smaller at higher pressures (55 mN m-1) (Figures 4E-H). At the ratio 1/1, the domains also disappear at surface pressures above 40 mN m-1 (Figure 4A-D). At TH4/DPPC 1/4 and 1/2 (n/n), the domain shape is star-like (Figure 4E-L). At TH4/DPPC 1/1 (n/n), round domains occur at the beginning of the phase transition but disappear at high pressures. Whether the domains are really round or appear round due to the resolution limit of the microscope cannot be determined in our experiments. The above described results give insights into the mixing behavior of the two cationic lipids with DPPC. With the addition of the cationic lipid an increase of the surface pressure of the the LE/LC transition (Figures 2B and 2D) and a diversification of the domain shape is observed while the plateau of the transition becomes less pronounced. This indicates an incorporation of a certain amount of the cationic lipid in the condensed state of DPPC. A homogeneous mixing in the liquid-expanded phase is assumed. This is in line with DSC experiments (bilayer system) published earlier

19

. Nevertheless, the results described above

allow no differentiation between a partial and a complete mixing. A complete mixing in the LC phase can be excluded. The LC domains are enriched in DPPC, which has a phase transition in contrast to the cationic lipids. The LE phase is stabilized upon addition of the cationic lipid, which results in the shift of the phase transition to higher pressures with increasing amount of cationic lipid (Figures 2B and 2D). Although a certain amount of cationic lipid is in the LC domains as the line tension between the LE and LC domains is reduced which leads to the star-like shapes. If there would be a high line tension, a round shape is preferred (lowest boundary for a given area). Furthermore, the strange behavior of the equimolar mixtures (domains occur with beginning of the phase transition but become smaller and vanish with increasing surface pressure) has to be explained. If the domains really vanish, a homogeneous phase might be expected. This would mean the system is miscible at 12 ACS Paragon Plus Environment

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high surface pressure. If the domains are too small to be observed because of the resolution limit of the technique, there would be still a microphase separation at high surface pressure. Also the mentioned repulsive interactions between the lipids in the mixtures indicated by the positive excess areas demonstrate that the mixing behavior is not ideal, but give no final prove for lipid demixing. Therefore additional experiments with more molar ratios of the lipids including thermodynamic investigations are required (exceeds the scope of this article). We tend to say that there is at least a partial demixing between the condensed phase of DPPC and the cationic lipid. This is supported by observations made in bilayer experiments earlier.19, 21

Figure 5. Surface pressure – area isotherms of mixed monolayers and the pure components of the DPOE-mixtures (A) on MES buffer pH 6.5 (10 mM). Fluorescence microscopy images (B) of OH4/DOPE and TH4/DOPE 1/1 (n/n) monolayers on MES buffer pH 6.5 (10 mM) at varying surface pressures (given number in mN m-1). The scale bar indicating 20 µm is the same for all pictures. The fluorescent lipid probe was BODIPY® 558/568 C12 in a concentration of 0.1 mol%. The mixtures of the cationic lipids with DOPE show a quite different behavior compared to mixtures with DPPC. Figure 5A compares the surface pressure – area isotherms of the 13 ACS Paragon Plus Environment

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equimolar binary mixtures of the cationic lipids with either DOPE or DPPC. The isotherm of the OH4/DOPE 1/1 (n/n) mixture is shifted to higher areas compared to the OH4/DPPC 1/1 (n/n) mixture. No phase transition marked by a kink or plateau is observed until the film collapses at 42 mN m-1. In the fluorescence microscopy images of the OH4/DOPE monolayer no domain formation is observed, indicating that the monolayer is in the LE state (Figure 5B). The compression isotherm of the TH4/DOPE 1/1 (n/n) mixture overlays with those of TH4/DPPC 1/1 (n/n) till a surface pressure of approximately 20 mN m-1. At higher surface pressures the compressibility of the film remains at a low value and the isotherm is shifted to larger areas per molecule, because no phase transition from the LE to the LC state occurs as seen in mixtures of TH4/DPPC. The assumption of a stable LE state over the whole compression is also supported by fluorescence microscopy, showing a uniform distribution of the fluorescence probe (Figure 5B). Nevertheless, both mixtures with DOPE show positive excess areas Aex at 30 mN m-1 [OH4/DOPE 1/1 (n/n): Aex = +27.1 Å2; TH4/DOPE 1/1 (n/n): Aex = +12.1 Å2], indicating repulsive interactions between the components similar to the mixtures with DPPC. In summary, monolayers composed of binary mixtures of the cationic lipid with DOPE are in the liquid-expanded state until the film collapse. These binary mixtures with DOPE are less stable than the mixtures with DPPC because the collapse pressure is lower. Furthermore, there are no hints for demixing processes in the monolayer. Nevertheless, a demixing in two different liquid-expanded phases cannot be excluded, because the fluorescent probe used in epifluorescence microscopic studies has a high affinity to lipids in the LE state and a low affinity to the LC state. Different lipid associates in bulk system investigated by cryo transmission electron microscopy did not exclude phase separation. Also the repulsive forces determined by the excess areas of the lipid mixtures allow phase separation processes but give no final prove. Furthermore, grazing incidence X-ray diffraction experiments are not useful to investigate lipid demixing, because this technique needs a monolayer in the LC phase 26. 14 ACS Paragon Plus Environment

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3.2 Langmuir isotherms coupled with epifluorescence microscopy of mixed monolayers with DNA in the subphase Figure 6A shows the adsorption isotherms of monolayers composed of the 4 lipid mixtures spread on DNA containing subphase after compression to 10 mN m-1. We have chosen this experimental setup to avoid non-homogeneous distribution of the DNA (an effect which cannot be excluded for injection of DNA underneath a preformed monolayer) and multilayers of the lipid mixture at the air liquid interface (multilayers can be formed by spreading at high surface pressures). The adsorption isotherms show a fast decrease of the surface pressure by 1-2 mN m-1 during the first 30 min. Afterwards the surface pressure slowly increases by around 1 mN m-1 for OH4/DOPE and TH4/DPPC and 0.2 mN m-1 for TH4/DOPE. The surface pressure of OH4/DPPC further decreases by around 0.5 mN m-1. The effect of the adsorption of the DNA to cationic monolayers at 10 mN m-1 is not very pronounced, but it demonstrates that 12 h are needed to equilibrate the system. Furthermore, the weak changes in surface pressure indicate that the bound DNA adsorbs to the lipid head group region but did not penetrate into the monolayer. That DNA is adsorbed can clearly be seen in the IRRAS spectra (see section 3.3). As model DNA for this experiments ctDNA was chosen while plasmide DNA was used for the transfection experiments and the determination of the isoelectric points of lipoplexes.19,

20

For structural investigations without determining

lipid/DNA stoichiometric ratios this exchange is legitimized (own unpublished results and literature27). The presence of DNA in the subphase changes the compression behavior of the monolayer drastically compared to the behavior on pure buffer as subphase (SI Figure S5). The isotherms of the four investigated equimolar binary mixtures (OH4/DPPC, OH4/DOPE, TH4/DPPC, TH4/DOPE) in presence of DNA in the subphase differ only slightly (Figure 6B). Obviously, the adsorption of DNA to the lipid monolayers has an equalizing effect on the molecular areas. A strong condensing effect for the OH4/DOPE and OH4/DPPC monolayers is 15 ACS Paragon Plus Environment

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observed, decreasing the molecular areas of the lipids up to a surface pressure of 35 mN m-1 due to the adsorption of DNA (SI Figures S5A and S5C). The isotherms of TH4/DOPE monolayers on bare buffer or on DNA-containing buffer are comparable (SI Figure S5B). The slopes of the isotherms of TH4/DPPC monolayer in presence and absence of DNA in the subphase are comparable to each other till the phase transition of the TH4/DPPC monolayer without DNA in the subphase is reached. Above this pressure, a shift of the isotherm of TH4/DPPC to larger areas in the presence of DNA is monitored compared to the isotherm of the monolayer on DNA-free subphase. Interestingly, it is also described in literature that DNA is able to either fluidize or condense positively charged lipid monolayers 28-33. Upon addition of DNA into the subphase, the phase transition region in the compression isotherm observed for OH4/DPPC 1/1 (n/n) and TH4/DPPC 1/1 (n/n) between 25 and 30 mN m-1 disappears. DNA stabilizes the LE phase of the mixtures. The equalizing effect of DNA adsorbed to the cationic monolayers on the determined area per molecule is in concordance with IRRAS experiments published earlier, which demonstrate that all four lipid mixtures adsorb the same amount of DNA.20 In this report it was also shown that all four lipid composites have a charge density (ρ) of 10-17×10-3 e- Å-2

20

which is much

higher than the charge density of DNA which is estimated to ρDNA = 9×10-3 e- Å-2.34 Consequently, an electrostatically dominated attraction of the DNA to the lipid monolayer is induced by the cationic lipids. In the bound state, a comparable saturation of the cationic surface charge by DNA occurs for all tested lipid mixtures. The attracted DNA dominates the determined molecular area.


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Figure 6. A) Isotherms of surface pressure vs. time of monolayers composed of the four binary mixtures interacting with DNA in the subphase (10 mM MES buffer pH 6.5 with 0.1 mMnucleotides ctDNA) at an initial surface pressure of about 10 mN m-1. B) Surface pressure – area isotherms of monolayers composed of binary lipid mixtures at the molar ratio 1/1 (n/n) on MES buffer pH 6.5 (10 mM) containing ctDNA (0.1 mM nucleotides). The curves start at a surface pressure of about 10 mN m-1 because this was the surface pressure of the film for the DNA adsorption (see A). In contrast to the similar compression behavior of the monolayer composed of the four investigated lipid compositions with DNA present in the subphase, the fluorescence microscopy images of the lipid monolayers show different phase separation phenomena in dependence of the used phospholipid of the binary mixture. Both DOPE containing mixtures show an isotropic distribution of the fluorescent probe (Figure 7A und 7E) until probably multilayers are formed or material from the monolayer submerges into the subphase, what results in the pronounced plateau in the isotherm (Figure 6). The beginning of this plateau is connected with an anisotropic distribution of the fluorescent dye. Bright domains occur at the beginning of the plateau (Figure 7B: OH4/DOPE at 46.2 mM m-1 and 64 Å2 molecule-1; Figure 7F: TH4/DOPE at 49.4 mM m-1 and 42 Å2 molecule-1). Either there is a lipid phase which is preferred by the dye or this is just a clustering of the dye. These domains are a result of the interaction of DNA with the lipid monolayer because in absence of DNA comparable domains were not observed. The domains grow by compressing the monolayer (Figures 7B-


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D, 7F-H) while the measured pressure remains nearly constant. This behavior can be explained by two scenarios: i) Further compression results in the submergence of one part of the monolayer beneath another part. But in general this submergence should not result in a growth of the domains, because the pressure and lipid composition should be constant. This scenario is only relevant, if the further compression results in structural changes of the adsorbed DNA, for example the increase of ordered DNA domains like described by Dittrich et al. 35, and these structural changes would force an LC/LE coexistence. ii) The adsorbed DNA forms complexes with the lipids of the monolayer which submerge into the subphase. A comparable behavior was previously described by Zuhorn et al. for the interaction of DNA with SAINT-2/DOPE monolayers

36

(SAINT-2 = N-methyl-4-(dioleyl)methylpyridinium chloride). This scenario is more favored. If one of the two lipids has a higher affinity to the DNA, what is expected for the cationic lipid, this would be removed in a higher amount from the lipid monolayer and the composition changes and demixing processes with coexisting LE and LC domains occur. Charge interactions between the cationic lipid and the DNA are the driving force for complex formation. Most of the cationic lipids are bound to the DNA what changes the monolayer by the formation of cationic-lipid rich domains. The condensed areas of cationic lipid with bound DNA would be dark and the still fluid regions of DOPE would be bright, as observed. The decreasing area of the dark domains (ongoing compression in Figures 7CD and 7GH) supports this explanation. The submergence of cationic lipids complexed with DNA reduces the LC domains in the monolayer. In summary, in DOPE containing mixtures no condensed domains are observed till the beginning of the over-compression plateau and lipid mixing can be assumed. Phase separation 18 ACS Paragon Plus Environment

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processes can be only observed at non-physiological surface pressures where a plateau occurs due to over-compression processes.

Figure 7. Fluorescence microscopy images of monolayers composed of binary lipid mixtures at the molar ratio 1/1 (n/n) on MES buffer pH 6.5 (10 mM) containing ctDNA (0.1 mM nucleotides) at varying surface pressures (given number in mN m-1). The numbers in brackets represents the area per molecule in Å2 molecule-1. The scale bar indicating 20 µm is the same for all pictures. The fluorescent lipid probe was BODIPY® 558/568 C12 in a concentration of 0.1 mol%. In contrast, mixtures with DPPC show phase separation events into the LC and LE states from the beginning of the measured isotherm (approximately 10 mN m-1) when DNA was adsorbed to the lipid monolayer (dark domains in Figure 7I,M). The number of the condensed domains as well as the contrast increase with increasing surface pressure (Figures 7J-L and 7N-P). The domains have an irregular shape with a frayed border. A very low line tension is present. This behavior results in a fractal domain border. The size of the domains is quite big compared to the equimolar binary DPPC-containing mixtures without DNA in the subphase (Compare 19 ACS Paragon Plus Environment

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Figure 7I-P with Figures 3B/C and 4B/C). Only at very high surface pressures, smaller domains coexisting with the large domains occur (Figures 7L and 7P). Similar to crystal growth, there is a competition between growth of the existing domains and nucleation of smaller domains. The fact that the lateral pressure due to the external barriers still increases and the domains can’t grow further when they touch each other, small domains occur. Furthermore, an irregular distribution of the fluorescent probe within the LC domains can be observed. Some parts of these domains are brighter due to the incorporation of a certain amount of the fluorescent dye while some regions near the border to the LE phase appear darker because the amount of the incorporated fluorescent dye is lower (these are the regions of ongoing domain growth). This may be explained with different diffusion speeds and timescales in the experiment. The darker regions of the domains might be formed during the ongoing compression of the monolayer. The outer trigger of the barriers forces the lipid (DPPC) to the LC state and the diffusion of the dye is not fast enough to get out of the domains. Ongoing compression of the film results in a growing of the dark border region of the LC domains and an ongoing exclusion of the fluorescent dye from the domains what results in an increasing contrast (compare Figure 7J-L and 7N-P). What information can be got about the interaction between the DPPC-containing lipid mixtures and the DNA. The coupling of DNA to the lipid monolayer triggers a demixing process between LC and LE phase, which starts at a lower surface pressure compared to the equimolar DPPC-containing binary mixtures without coupled DNA. The DNA seems to have a condensing effect and stabilizes the LC domains. This phenomenon is the result of induced phase separation between the two lipids in the mixture. The coexisting region in mixtures containing DPPC between LC and LE occurs along the whole isotherm after adsorption of the DNA at 10 mN m-1. The permanent occurrence of LC domains in the DPPC containing systems is an argument against a first-order phase transition from LE to LC of the monolayer in presence of DNA, what is in line with the isotherms of the DPPC-containing mixtures after DNA 20 ACS Paragon Plus Environment

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adsorption (Figure 6). Summarizing, the coupling of DNA to the DPPC-containing lipid mixtures seems to induce phase separation processes.

3.3 Infrared Reflection-Absorption Measurements The monolayers composed of the equimolar binary mixtures, which are of special interest for the biological characterization, were investigated by infrared reflection-absorption spectroscopy. The results determined for the asymmetric and symmetric methylene stretching vibration (νasCH2 and νsCH2) at different surface pressures are presented in Figure 8. Because both methylene stretching frequencies have different sensitivities to chain conformational order and packing (the asymmetric one is less sensitive to packing changes),37 we monitored both. Both vibrational bands show the same dependence on the surface pressure. All investigated lipid mixtures show typical values for alkyl chains in the LE state.38 The wavenumbers never decrease below 2924 cm-1 (νasCH2) or 2854 cm-1 (νsCH2) for the mixtures OH4/DOPE, TH4/DOPE, and OH4/DPPC in presence and absence of DNA. Only the mixture TH4/DPPC exhibits slightly lower νasCH2 and νsCH2 values (Figure 8) which are still too high for lipids in the LC state (νasCH2 ≈ 2918 cm-1, νsCH2 ≈ 2849 cm-1). The presence of DNA in the subphase has no significant effect on the position of the νasCH2 and νsCH2 bands and consequently on fluidity of the alkyl chain (compare red circles with black squares in Figure 8). Dabkowska et al. also observed no effect of DNA on the averaged alkyl chain ordering using the IRRAS technique decrease

33

31

while also an increase

35

as well as a

of the average gauche conformers in the alkyl chains after interaction of lipid

monolayers with DNA is described in literature. The absence of a sudden shift of the νasCH2 band to lower values indicates the absence of a first-order phase transition from LE to LC what fits perfectly with the measured isotherms (compare Figure 5A and Figure 6 with Figure 8) in nearly all cases. The only exceptions are the isotherms of the binary DPPC-containing mixtures in absence of DNA, which exhibit a kink in the isotherm between 25 and 30 mN m-1. 21 ACS Paragon Plus Environment

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For the mixture OH4/DPPC, the kink is not connected with a clear jump of the νasCH2 band position to lower values, while for TH4/DPPC, the mixture with the more pronounced kink in the area – pressure isotherm, a pronounced shift of the νasCH2 band position to lower values was detected (Figure 8). Nevertheless, it has to be considered that the observed νasCH2 bands result from the average of all νCH2 vibrations and the resolution of the technique does not allow investigating separate νCH2 vibrations (symmetric as well as asymmetric ones). Following, also the demixing into LC and LE phase gives an average vibrational band which is in the case of our investigations in the range of alkyl chains in the LE phase. Nevertheless the dark domains observed by epifluorescence microscopy clearly indicate a certain amount of lipids in the LC state.

Figure 8. Wavenumber of the asymmetric (closed symbols) and symmetric (open symbols) methylene stretching vibration (νasCH2 and νsCH2) determined by IRRAS experiments at monolayers composed of the different lipid mixtures as a function of the surface pressure in presence (squares) and absence (circles) of ctDNA (0.1 mM nucleotides) in the subphase (10 mM MES buffer pH 6.5). For data analysis, spectra obtained with s-polarized light and an 22 ACS Paragon Plus Environment

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angle of incidence of 40° were used. The spectra obtained with p-polarized light show comparable results. The values are determined from the spectra (Figure 9A, Figure S6A, S7A, and S8A) by taking the maximum of the Lorentzian fit to the band. The IRRA spectra also give information about the interaction of the lipid monolayer with DNA. Recently, we described the IRRAS-based quantification of DNA adsorbed to Langmuir monolayers composed of the 4 different lipid composites, which are also topic of the present paper, and have demonstrated that all 4 lipid mixtures bind the same amount of DNA if a high excess of DNA is present in the subphase.20 A lot of typical DNA marker bands are present in the IRRA spectra which prove that DNA is adsorbed to the monolayer (Figure 9), although the pressure time curves show no pronounced effect on the surface pressure (Figure 6A). Consequently, the DNA attaches to the head group region what is confirmed by the increase of the OH stretching band at ca. 3600 cm-1 (sensitive for the film thickness) in presence of DNA in the subphase (Figure 9B),39 but did not penetrate inside the monolayer (this would increase the surface pressure). Also the increase of the symmetric and asymmetric phosphate diester valence vibrational bands showing distinct residual signals in the difference spectrum is an evidence for the adsorbed DNA (Figure 9D), although the orientation of the phosphor diester groups (changed transition dipole moment orientation) of the phospholipids have an effect on the signal intensities, too. The highest evidence for the attachment of DNA to the lipid monolayers is the appearance of the peak at 970 cm-1, which is only present in the spectra of monolayers with DNA in the subphase and, subsequently, in the difference spectra (Figure 9D). This signal results from the C-C stretching frequency of the DNA backbone and is typically for the B-form of DNA (B-DNA form is most common under the conditions found in cells).40 The IRRA spectra of the cationic lipids alone and of the mixtures in absence of DNA show a carbonyl valence vibration at 1740 cm-1. In presence of DNA attached to the monolayer this band shifts to lower wavenumbers and has a shoulder at higher wavenumbers which results from the lipid carbonyls. The new maximum is at 1719 cm-1, what fits perfectly with the C=O stretching vibrations of stacked guanine/thymidine base pairs of DNA and 23 ACS Paragon Plus Environment

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indicates that the bound DNA is still a double strand (single stranded DNA has the band at ca. 1692 cm-1).40, 41 Nevertheless, it should not be neglected that also a dehydration of the lipid head group and the interfacial area can result in a shift of the carbonyl valence vibration to lower wavenumbers. Additional information is given by the symmetric methylene bending mode δ(CH2) which appears as single band at 1463-1467 cm-1 for all spectra (Figure 9C, Figure S6C, S7C, and S8C) indicating a hexagonal or disordered chain packing, but disproves orthorhombic or triclinic phases for the alkyl chain lattice.38 We can also evaluate the amide I and amide II modes, but for interpretation of the signals of the spectra in presence of DNA we have to be careful due to multiple interferences with DNA derived signals (Figure 9C, Figure S6C, S7C, and S8C).40 Furthermore, the phospholipid containing monolayers have a pronounced H2O deformation band (broad positive peak at 1650 cm-1) which results for example from hydrated head groups and interferes with other bands. Nevertheless, the spectra of the pure cationic lipids and the mixtures without DNA in the subphase show shifts of the amide I mode to higher and the amide II mode to lower wavenumbers if phospholipids are mixed with the cationic lipids, what indicates the break of a certain amount of hydrogen bonds.42 That pure OH4 and TH4 form a hydrogen bond network could be demonstrated by X-ray and IRRAS studies.21 The addition of phospholipids breaks such hydrogen bond network. This will also explain the pronounced repulsive forces between lipids in the mixture (see 3.1). The partial loss of hydrogen bonds between cationic lipids leads to stronger repulsive forces between cationic charges in the head group region. Furthermore, the partial loss of hydrogen bonds due to the incorporation of phospholipids indicates also a certain degree of lipid mixing. Summarizing, the presented IRRAS data give information about the interaction between the positively charged monolayers and DNA and also about the interaction between the lipids, but


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could not indicate transfection determining differences between the mixtures (Figure 8, Figure 9, Figures S6, S7, and S8).

Figure 9: Sections of the IRRA spectra (40°, s-polarized) of OH4 in absence and OH4/DOPE 1/1 in presence and absence of DNA on MES buffer pH 6.5. A) methylene stretching frequency (region i in 9B) B) Relevant sections of all three IRRA spectra and the difference spectrum of OH4/DOPE 1/1 in presence and absence of DNA (ds). i = methylene stretching region, ii = amide/carbonyl region, iii = phosphate region. C) Section of IRRA spectra comparing amide, carbonyl stretching and the methylene bending frequencies (region ii in 9B). D) Section of IRRA spectra comparing the phosphate modes and the C-C stretching frequency of DNA (region iii in 9B).

3.4 Discussion The above described experiments show clear differences between the lipid mixtures with effective DNA transfer into cells (OH4/DOPE and TH4/DOPE) and the ineffective transfection mixtures (OH4/DPPC and TH4/DPPC). The binary lipid mixtures containing DOPE show a homogeneous distribution of the fluorescent dye at all surface pressures along the pressure – area isotherms till the collapse of 25 ACS Paragon Plus Environment

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the film (point of over-compression) in absence and presence of DNA in the subphase. Therefore, these films are in the LE state. The position of the methylene stretching vibrational bands supports this fact by indicating a high amount of gauche conformers in the alkyl chains. The adsorption of DNA to this monolayers has no effect on the chain fluidity demonstrated by epifluorescence microscopy and IRRAS. A mixing of lipids in the liquid crystalline phase of 3D systems

43, 44

and, hence, in the LE phase of 2D systems is very likely.45 Nevertheless,

liquid-liquid immiscibility in binary lipid mixtures is also described which depends either on the head group structure or the lipophilic part of the molecules.44,

46-49

Nevertheless, we

assume mixing of both components because the fluorescent probe is distributed homogeneously. Only at high surface pressures, the ternary systems OH4/DOPE/DNA and TH4/DOPE/DNA show clear phase separation in the collapse region of the isotherms (Figure 7). These demixing phenomena are induced by the electrostatic interaction of cationic lipids with DNA at high non-physiological surface pressures above 45 mN m-1. Values between 30 and 35 mN·m-1 are considered as the lateral pressure in bio-membranes.50 At such physiological relevant surface pressures no demixing is observed and consequently we can assume a homogeneous charge density and, hence, to some extend a homogeneous distribution of the coupled DNA layer (Figure 10). It has to be taken into account that DNA coupled to a lipid monolayer is not really homogeneously distributed but rather consists of unordered domains and domains with parallel aligned DNA strands.28,

35

But due to the

homogeneous charge density the potential binding quality of the DNA is equal all over the whole monolayer. This also explains the observation made in 3 different cell lines (A549, HeLa and LLC-PK1) that OH4/DOPE is a more effective lipofection system than TH4/DOPE.19,

20

OH4/DOPE monolayers have a charge density of ρ ≈ 12×10-3 e Å-2 and

TH4/DOPE monolayer of ρ ≈ 10×10-3 e Å-2.20 Consequently, only OH4/DOPE has a charge density in the optimal range for transfection with lamellar lipoplexes which is between 12×10-


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3

e Å-2 and 22×10-3 e Å-2 according to Ahmad et al.17 That both lipid composites build

lamellar lipoplexes was confirmed earlier.21

Figure 10. Schematic illustration of the mixing behaviour of the cationic lipids with DOPE or DPPC and the influence on the DNA coupling. The mixing of the cationic lipids and DOPE results in a homogeneous charge density (ρ) of the lipid film and, hence, a homogeneous DNA binding. A DNA-induced phase separation of cationic lipids and DPPC results in domains with either high or low charge density, and therewith in domains which bind higher amounts of compacted DNA and domains which only weakly can interact with DNA. The ineffective mixtures OH4/DPPC and TH4/DPPC show a quite different behavior compared to the DOPE containing mixtures. The ternary systems OH4/DPPC/DNA and TH4/DPPC/DNA show permanent coexistence of LC and LE domains. The domains are bigger (compare Figures 3B and 4B/C with 7I-P) and appear at quite lower surface pressures compared to the equimolar binary systems OH4/DPPC and TH4/DPPC without DNA in the subphase. This observation indicates a DNA-induced phase separation in the monolayers composed of the equimolar cationic lipid/DPPC mixtures. If the electrostatic interaction is the driving force for the DNA adsorption to the cationic lipid monolayer, these electrostatic interactions also induce the phase separation. In this context, the complex formation between the cationic lipid and the DNA seems to force the interaction of these lipoplexes at the air/water interphase between each other resulting in a squeezing out of the DPPC from this lipoplexes and a condensing effect on the DNA because of the increase of the local charge density in the cationic lipid enriched regions. A high amount of coupled and condensed DNA 27 ACS Paragon Plus Environment

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should be attached to the cationic lipid rich LE phase (cationic lipid rich phase appearing bright) which has a high charge density, and a low amount of coupled DNA is attached to the DPPC rich LC domains (dark domains) with low charge density (Figure 10). The observation of Symietz et al. 28 and Dittrich et al. 35 show that ordered DNA domains and unordered DNA strands can coexist even at single component cationic monolayers. The DPPC enriched domains have a very low charge density what means that only a small amount of unordered DNA or even no DNA is attached to these regions. It has to be mentioned that the IRRAS measurements could not prove the phase separation and the appearance of condensed DPPC because the technique only gives an average value. The use of DPPC with deuterated alkyl chains and non-deuterated cationic lipids allow investigating the alkyl chain rotational conformers of both components separately due to the different positions of the CD2 and CH2 stretching vibration bands.51, 52 Such experiments are planned for ongoing investigations. In literature, the direct correlation of monolayer properties of lipid composites with the transfection efficiency is poorly described. Dabkowska et al. investigated the influence of the mixing behavior of binary lipid mixtures composed of a cationic lipid (DODAB = dimethyldioctadecylammonium bromide) and either DOPE or cholesterol.32 The investigation demonstrate that the mixture DODAB/cholesterol 1/1 (n/n) is homogeneous, while the mixture DODAB/DOPE 1/1 (n/n) shows phase separation. The last mixture is the most effective lipid composition what seems to be contrary to our findings. Furthermore, the composition DODAB/DOPE 1/1 (n/n) has a charge density of 8.6×10-3 e Å-2 (outside the optimal charge density range proposed by Ahmad et al. 17), and DODAB/cholesterol 1/1 (n/n) has a charge density of 13.7×10-3 e Å-2 (in the optimal charge density range proposed by Ahmad et al.17).53 But these discrepancies can be explained. First, Zidovska et al. demonstrate that the behavior of cationic lipid composites using cholesterol as co-lipid is different compared to lipid compositions with phospholipids as co-lipids,54 and the Gaussian curve by 28 ACS Paragon Plus Environment

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Ahmad et al. is not applicable to cholesterol mixtures. Second, the demixing in the DODAB/DOPE system means that there are domains with a charge density lower than the calculated value of 8.6×10-3 e Å-2 but also domains with a higher charge density closer to the optimum proposed by Ahmad et al. (the discussion is permissible because Barreleiro et al. proved by small angle neutron scattering that DODAB/DOPE forms lamellar lipoplexe

55

).

The DNA attached to the DODAB/DOPE mixture can effectively be released in cells because of the optimal binding strength between DNA and the lipid mixture, even if Dabkowska et al. describe that less DNA (% per volume) is bound by this mixture compared to the DODAB/cholesterol composite.32 Furthermore we have to take into account that the DODAB mixtures act differently with DNA than the OH4 and TH4 mixtures. For example, the coupling of DNA to DODAB/DOPE 1/1 (n/n) monolayers has no influence on the size and shape of the domains while in the present report a drastic difference in size and shape of the domains for the phase separated systems TH4/DPPC 1/1 (n/n) and OH4/DPPC 1/1 (n/n) in presence and absence of DNA has been observed. Furthermore, DODAB is a permanently charged lipid which has a pH independent charge while the charge state of OH4 as well as TH4 depends on the pH value because of the primary amino functions (amino-functionalized lipids). Antipina et al. and Dittrich et al. could prove that DNA can promote the protonation of amino-functionalized lipids after interaction with the result that monolayers composed of such lipids can also couple DNA at high pH values where the single lipids are uncharged.29, 35 The same behavior can be expected for OH4 and TH4, but not for DODAB whose charge state cannot be influenced by the interaction with DNA. Consequently, DNA has more drastic effects on OH4 and TH4 containing films than on DODAB containing films. Paiva et al. used two binary mixtures composed of the cationic lipid DOTAP (1,2-dioleoyl-3trimethylammonium propane) and either cholesterol or a partially fluorinated cholesterol derivative.56 The mixture with cholesterol shows higher tendency for phase separation and 29 ACS Paragon Plus Environment

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binds more DNA compared to the mixture with the fluorinated cholesterol. The authors also discuss the influence of differences in charge density in the two systems and the influence on the DNA adsorption. Unfortunately, comparative transfection efficiency studies have not been presented. Zuhorn et al. investigated films composed of the cationic lipid SAINT-2 in equimolar mixtures with either DOPE or DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine).36 This article also describes a DNA-induced phase separation for the less effective SAINT2/DPPE mixture using epifluorescence microscopic studies. Strong support for our proposition that lipid demixing results in domains with ineffective charge density for efficient gene transfer, can be found in the work of Farago et al.57 They investigated cationic lipid/co-lipid mixtures by x-ray scattering and molecular simulations and identified the membrane cationic charge density as a key parameter for the degree of compaction of adsorbed DNA and correlated the compaction regimes with the transfection efficiency. One regime is characterized by lateral phase coexistence between cationic lipid rich domains in complex with DNA and neutral membrane domains. This regime perfectly fits to the model proposed for the cationic lipid/DPPC mixtures (Figure 10) investigated in this study. This regime is also connected with a low DNA transfer activity. Furthermore, they described a regime with efficient gene transfer and no phase coexistence. The charge density can be adjusted to the optimal ratio by varying the amount of the neutral co-lipid. This regime perfectly fits to the cationic lipid/DOPE mixtures (Figure 10) investigated in this study. Finally it has to be taken into account that the N/P ratio also affects the transfection efficiency.58,

59

The presented complex formation between monolayer and DNA were

performed with huge excess of DNA in the subphase. This allows the saturation of the positively charged monolayer with DNA but the direct N/P ratio of the complexes at the interface can’t be determined. To demonstrate the effect of the N/P ratio on the transfection efficiency we refer to previous works.19, 20 30 ACS Paragon Plus Environment

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Future research should focus on the investigation of the arrangement of DNA attached to the cationic monolayers by atomic force microscopy and X-ray diffraction experiments. The results of such studies would complete the model of the lipid/DNA complexes at the soft air/water interface which is proposed in Figure 10 for all 4 lipid mixtures.

4. Conclusion This study demonstrates that DNA has drastic effects on the lipid mixing using the twodimensional Langmuir monolayer system at the soft air/liquid interface, a useful model to the theoretical study of processes of relevance for bilayer systems such as pore formation, phase separations, miscibility, and domain formation.60 The model can be considered as half of a bilayer, and characteristics at physiological relevant surface pressures (around 30 mN m-1) give information which can be transferred to bulk systems (for example liposomes). Our findings show that phase separation processes can drastically affect the charge density distribution and, hence, the quality of DNA binding with consequences for the gene transfer efficiency. We could demonstrate that the equimolar mixtures of two different cationic lipids (OH4 and TH4) with DOPE result in fluid monolayers with no indication for phase separation phenomena at physiological relevant surface pressures of about 30 mN m-1. The adsorption of DNA to these systems does also not induce phase separation. The charge distribution along the lipid surface is homogeneous and the efficiency of the system depends on the overall charge density. In contrast, the equimolar mixtures of two different cationic lipids (OH4 and TH4) with DPPC result in monolayers which show the coexistence of liquid-expanded and condensed phases at physiological relevant surface pressures. The adsorption of DNA induces a lipid phase separation which has drastic effects on the transfection efficiency referring to previous biological studies.19,

20

Cationic lipid enriched regions with high charge density

coexists beside DPPC rich domains with low charge density. This phase separation is connected with inefficient DNA transfer. 31 ACS Paragon Plus Environment

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All in all, this study demonstrates that in the search for cationic lipid composites for lipofection it is not sufficient to modify the charge density of a cationic lipid formulation by adding the appropriate amount of a neutral co-lipid to reach the optimal ratio. Furthermore, it is important to create mixtures which do not phase separate, what results in domains with charge densities above and below the optimal value. It is also important to ensure that the interaction with DNA does not induce such phase separation processes in the lipid mixtures.

Acknowledgement We thank Prof. Dariush Hinderberger and Prof. Alfred Blume for providing us excess to the epifluorescence microscope.

Supplemental Information Supplemental compressibility data, fluorescence microscopy images, IRRA spectra and Langmuir isotherms are available.

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Figure 1. Structures of the lipids used in this study: N-{6-amino-1-[N-(9Z)-octadec-9-enylamino]-1oxohexan-(2S)-2-yl}-N0-{2-[N,N-bis(2-aminoethyl)-amino]ethyl}-2-hexadecylpropandiamide (OH4), N-[6amino-1-oxo-1-(N-tetradecylamino)hexan-(2S)-2-yl]-N0-{2-[N,N-bis(2-aminoethyl)amino]ethyl}-2hexadecylpropandiamide (TH4), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). Structural differences are highlighted. 286x124mm (150 x 150 DPI)


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Figure 2. Surface pressure – area isotherms of monolayers composed of OH4/DPPC (A) and TH4/DPPC (C) at varying molar ratios (n/n, the number given in brackets represents the mole fraction x of DPPC) on MES buffer pH 6.5 (10 mM). Additionally, the surface pressure of the beginning phase transition as function of xDPPC is given for OH4/DPPC (B) and TH4/DPPC (D) mixtures. The given values are taken from the surface pressure – area isotherms in combination with the compressibility curves (see SI Figure S1-S3). The average areas per molecule (A(30)) as function of xDPPC at 30 mN m-1 are given for OH4/DPPC (E) and TH4/DPPC (F). The dotted straight line demonstrates either ideal mixing behavior (additivity rule Aid12 = x1A1 + x2A2) or completely immiscibility. 500x283mm (150 x 150 DPI)


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Figure 3. Fluorescence microscopy images of OH4/DPPC (n/n) or pure DPPC monolayers on MES buffer pH 6.5 (10 mM) at varying surface pressures (given red number in mN m-1). The scale bar indicating 20 µm is the same for all images. The fluorescent lipid probe was BODIPY® 558/568 C12 in a concentration of 0.1 mol%. 388x196mm (150 x 150 DPI)


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Figure 4. Fluorescence microscopy images of TH4/DPPC (n/n) monolayers on MES buffer pH 6.5 (10 mM) at varying surface pressures (given red number in mN m-1). The red scale bar indicating 20 µm is the same for all pictures. The fluorescent lipid probe was BODIPY® 558/568 C12 in a concentration of 0.1 mol%. 387x196mm (150 x 150 DPI)


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Figure 5. Surface pressure – area isotherms of mixed monolayers and the pure components of the DPOEmixtures (A) on MES buffer pH 6.5 (10 mM). Fluorescence microscopy images (B) of OH4/DOPE and TH4/DOPE 1/1 (n/n) monolayers on MES buffer pH 6.5 (10 mM) at varying surface pressures (given number in mN m-1). The scale bar indicating 20 µm is the same for all pictures. The fluorescent lipid probe was BODIPY® 558/568 C12 in a concentration of 0.1 mol%. 229x297mm (150 x 150 DPI)


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Figure 6. A) Isotherms of surface pressure vs. time of monolayers composed of the four binary mixtures interacting with DNA in the subphase (10 mM MES buffer pH 6.5 with 0.1 mMnucleotides ctDNA) at an initial surface pressure of about 10 mN m-1. B) Surface pressure – area isotherms of monolayers composed of binary lipid mixtures at the molar ratio 1/1 (n/n) on MES buffer pH 6.5 (10 mM) containing ctDNA (0.1 mM nucleotides). The curves start at a surface pressure of about 10 mN m-1 because this was the surface pressure of the film for the DNA adsorption (see A). 424x161mm (150 x 150 DPI)


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Figure 7. Fluorescence microscopy images of monolayers composed of binary lipid mixtures at the molar ratio 1/1 (n/n) on MES buffer pH 6.5 (10 mM) containing ctDNA (0.1 mM nucleotides) at varying surface pressures (given number in mN m-1). The numbers in brackets represents the area per molecule in Å2 molecule-1. The scale bar indicating 20 µm is the same for all pictures. The fluorescent lipid probe was BODIPY® 558/568 C12 in a concentration of 0.1 mol%. 365x260mm (150 x 150 DPI)


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Figure 8. Wavenumber of the asymmetric (closed symbols) and symmetric (open symbols) methylene stretching vibration (νasCH2 and νsCH2) determined by IRRAS experiments at monolayers composed of the different lipid mixtures as a function of the surface pressure in presence (squares) and absence (circles) of ctDNA (0.1 mM nucleotides) in the subphase (10 mM MES buffer pH 6.5). For data analysis, spectra obtained with s-polarized light and an angle of incidence of 40° were used. The spectra obtained with ppolarized light show comparable results. The values are determined from the spectra (Figure 9A, Figure S6A, S7A, and S8A) by taking the maximum of the Lorentzian fit to the band. 440x310mm (150 x 150 DPI)


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Figure 9: Sections of the IRRA spectra (40°, s-polarized) of OH4 in absence and OH4/DOPE 1/1 in presence and absence of DNA on MES buffer pH 6.5. A) methylene stretching frequency (region i in 9B) B) Relevant sections of all three IRRA spectra and the difference spectrum of OH4/DOPE 1/1 in presence and absence of DNA (ds). i = methylene stretching region, ii = amide/carbonyl region, iii = phosphate region. C) Section of IRRA spectra comparing amide, carbonyl stretching and the methylene bending frequencies (region ii in 9B). D) Section of IRRA spectra comparing the phosphate modes and the C-C stretching frequency of DNA (region iii in 9B). 303x216mm (150 x 150 DPI)


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Figure 10. Schematic illustration of the mixing behaviour of the cationic lipids with DOPE or DPPC and the influence on the DNA coupling. The mixing of the cationic lipids and DOPE results in a homogeneous charge density (ρ) of the lipid film and, hence, a homogeneous DNA binding. A DNA-induced phase separation of cationic lipids and DPPC results in domains with either high or low charge density, and therewith in domains which bind higher amounts of compacted DNA and domains which only weakly can interact with DNA. 339x117mm (150 x 150 DPI)


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