Structural modifications of DPPC bilayers upon inclusion of an

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Structural modifications of DPPC bilayers upon inclusion of an antibacterial cationic bolaamphiphile Marianna Mamusa, Annalisa Salvatore, and Debora Berti Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b01689 • Publication Date (Web): 05 Jul 2018 Downloaded from http://pubs.acs.org on July 7, 2018

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Structural modifications of DPPC bilayers upon inclusion of an antibacterial cationic bolaamphiphile

M. Mamusa*, A. Salvatore, D. Berti CSGI and Department of Chemistry “Ugo Schiff”, University of Florence, Italy

* corresponding author: [email protected], Phone: +39 055 4573025

ABSTRACT The emergence of antibiotic-resistant bacterial strains has fostered fundamental research to develop alternative antimicrobial strategies. Among the several systems proposed so far, the association complexes (nanoplexes) formed by transcription factor decoys (TFD), i.e. short oligonucleotides targeting a crucial bacterial transcription factor, and a bolaform cationic amphiphile, 12-bis-THA, have demonstrated their potential in vitro and in vivo. The application of these nanoplexes is hampered by a scarce colloidal stability, that can be addressed by including the bolaamphiphile in a liposomal carrier, which is then associated to the TFD. The present study reports an investigation on the effects of 12-bis-THA on the structure of synthetic lipid bilayers, to assess the morphology of the mixed assemblies, gain insight into the location of the host within the bilayer, and determine the loading capacity of the carrier. Our results demonstrate that 12-bis-THA promptly inserts within DPPC bilayers, bending its C-12 spacer chain to adopt a cone-like shape, and shifting the gel-liquid crystalline transition of the chains to lower temperatures. The host liposomal structure is retained for a bolaamphiphile concentration up to 3.2 %mol to DPPC, while higher concentrations lead to the destabilization by means of a detergency-like mechanism, with the simultaneous existence of different lamellar-based structures, such as liposomes, bicelles, rafts, in which DPPC and 12-bis1 ACS Paragon Plus Environment

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THA could be present in different molar ratios. Overall, these results shed light on the interaction of the bolaamphiphile with a lipid bilayer, and provide valuable insight to better formulate the antimicrobial amphiphile in liposomal carriers to circumvent the colloidal instability of nanoplexes.

KEYWORDS Antibacterials, Nanoplexes, Bolamphiphiles, Lipid Bilayers, Drug Delivery, Small Angle X-Ray Scattering

SUPPORTING INFORMATION Differential scanning calorimetry, Dynamic light scattering, Small-angle X-ray scattering supplementary data.

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Introduction The emergence and spread of antimicrobial resistance in the last few decades1 has determined a real healthcare crisis,2 which is motivating researchers to find new strategies against infections by multidrug resistant microorganisms. Among the several alternative approaches proposed,3–6 the use of an oligonucleotide-based therapeutic,7 obtained by the association of transcription factor decoys (TFD)8 and a bolaform amphiphile (12-bis-THA, Scheme 1),9 has recently been reported.10 The cationic bolaamphiphile 12-bis-THA has a fundamental role in forming nanosized complexes (termed nanoplexes) with excess positive charge, where the oligonucleotide is reversibly condensed into a psi-form. The low colloidal stability of nanoplexes in saline media of biological relevance was circumvented by reformulating them in liposomal carriers. The liposomal formulation proved able to cross the bacterial membrane in vitro,11,12 but the details of interaction between 12-bis-THA and the bilayer at the molecular scale are yet to be elucidated. The bolamphiphile 12-bis-THA has a strong affinity for lipid bilayers composed of phosphatidylcholine, with or without phosphatidylethanolamine, and even in the presence of another cationic species, ethylphosphocholine.12 The interaction has been observed regardless of the sequence of addition of the bolaamphiphile, i.e. directly in a dry lipid film formed by organic solvent evaporation, or by surface decoration of pre-formed aqueous liposomal dispersions.11 However, the exact localization of the bolaform molecule in the lipid membrane is not known. The maximum length of the stretched molecule is not sufficient to span a phospholipid bilayer (see Scheme 1); therefore, a bent conformation can possibly be adopted, to accommodate the hydrophobic spacer chain and the two polar headgroups in the bilayer regions with similar polarity, respectively. Previous studies on the self-assembly behavior of 12-bis-THA in water have suggested that such bent conformation is however unfavored.10 Detailed information at the nanometric scale on the structural features of lipid bilayers hosting the bolaamphiphile would enable to tailor the liposomal structures and the formulation composition. 3 ACS Paragon Plus Environment

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For example, whether 12-bis-THA exposes one or both headgroups towards the continuous medium is related to the amount of therapeutic oligonucleotide that can be encapsulated per surface area and the stability of the formed complexes. The toxicity of the formulation itself is also directly connected with the concentration of bolamphiphile. While the TFD encapsulation needs to be maximized, the toxicity should be minimized, and a compromise could be designed by having a more detailed picture of the local structure. Moreover, the possible existence of a free-bound equilibrium for 12-bis-THA between the phospholipid membrane and the dispersion medium could tell us more about the origin of 12-bis-THA toxicity towards the cells. While the existence of free (i.e., not incorporated in liposomes) 12-bis-THA/TFD complexes has been ruled out in previous studies,11,12 we do not know whether the toxic effects are due entirely to the bilayer-bound 12-bisTHA or whether there is enough free bolaamphiphile to play a tangible role. Finally, it is not clear how much 12-bis-THA can be loaded in a liposome before the carrier structure is damaged. The molecular structure of this bolaamphiphile strongly evokes the possibility of amphiphilic properties that may lead to the solubilization of lipid molecules and the consequent destruction of the bilayers,13 which has been reported to be even more significant for bolaamphiphiles than for the corresponding single-headed surfactants.14 In this work, we present a physical-chemical investigation to assess the effect of 12-bis-THA on the structure of a lipid bilayer model system formed by the zwitterionic 1,2-dipalmitoyl-sn-glycero3-phosphocholine (DPPC, Scheme 1). The choice of DPPC resides in the enormous amount of studies available in the literature on the structure and properties of bilayers formed by this lipid. DPPC has often been used as a model system to study the interactions of drugs with eukaryotic membranes.15 We should mention that the gel-to-fluid transition of DPPC bilayers at full hydration takes place at 42 °C, 5 °C above the body temperature of a healthy human being. Even if this feature could appear to suggest the unsuitability of this lipid for studies with a biologic target, DPPC is commonly used for structural studies since the effect of the inclusion of other molecular species in the bilayers is easily monitored by means of calorimetric methods. In this work, 4 ACS Paragon Plus Environment

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differential scanning calorimetry is used along with small-angle X-ray scattering, light scattering, ζpotential measurements, and electron transmission microscopy to probe the structure of DPPC dispersions containing added 12-bis-THA at different concentrations.

Scheme 1. Structural formulae of (a) 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and (c) 12,12’(dodecane-1,12-diyl)bis(9-amino-5,6,7,8-tetrahydroacridinium) chloride (12-bis-THA). (b) and (d): their respective 3D space-filling molecular structures (hydrogens are not explicit).


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Materials and methods.

DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, >99% purity) was purchased from Avanti PolarLipids (Alabaster, AL). 12-bis-THA (1,1'-(dodecane-1,12-diyl)-bis-(9-amino-1,2,3,4-tetrahydroacridinium) chloride was synthesized with 95% purity by Shanghai Chempartners & co. LTD., and kindly provided to us by Procarta Biosystems (Norwich, UK). Milli-Q water was used as dispersion medium.

PREPARATION OF SAMPLES DPPC and 12-bis-THA were dissolved and thoroughly mixed in a chloroform/methanol mixture. The solvents were evaporated using a gentle N2 flow, and the lipid films were further dried by vacuum pumping for at least 8 hours. The films were hydrated with ultrapure water and the mixture was vortexed until homogenization.

DIFFERENTIAL SCANNING CALORIMETRY (DSC) DSC analysis was performed with a Q2000 DSC, TA Instruments (New Castle, USA). Roughly 20 mg of each sample were placed in aluminum hermetic pans, and they were analyzed by scanning the temperature between 0 °C and 50 °C at 5 ºC/min. Data modelling was carried out with IGOR Pro, using the Multi-peak fitting macro package. The melting transition peaks were isolated, and a cubic polynomial baseline was applied for each thermogram, while transition peaks were fitted using the exponentially-modified Gaussian model.16,17


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DYNAMIC LIGHT SCATTERING (DLS) DLS measurements were carried out on a Brookhaven Instruments apparatus 90Plus. The signal was detected by an APD detector. The light source was a 35 mW diode laser, λ = 659 nm, C30902SH-DTC. The normalized electrical field time autocorrelation functions of the normalized intensity time autocorrelation of the scattered light were measured at 90° and analyzed according to the Siegert relationship (Eq. (1)), which connects the first order or field normalized autocorrelation function g1(q, s) to the measured normalized time autocorrelation function g2(q, τ):

, = 1 + ,

Eq. 1

with b being the spatial coherence factor, which depends on the geometry of the detection system. The functions were also normalized to vary between 0 and 1 for display purposes. The field autocorrelation functions were analyzed through a cumulant analysis stopped to the second order. Samples were diluted 10 times before measurements.

ZETA-POTENTIAL MEASUREMENTS ζ-potential measurements were taken using a ζ-potential analyzer (Zeta Plus, Brookhaven Instruments Corporation, Holtsville, NY). Zeta-potentials were obtained from the electrophoretic mobility µ, according to Helmholtz–Smoluchowski equation:

=

∙

Eq. 2

with η being the viscosity of the medium and ε the dielectric permittivity of the dispersing medium. The ζ-potential values are reported as averages from 10 measurements on each sample.


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SMALL ANGLE X-RAY SCATTERING (SAXS) SAXS analysis of DPPC vesicles was conducted at the Austrian SAXS beamline (Elettra Synchrotron, Trieste, Italy). The samples were placed in quartz Mark capillaries of 1 mm thickness, enclosed in a steel cell; the same capillary was used for the blank to subtract to each sample. Scattering patterns were recorded at room temperature, on a Mar300-image-plate detector (MarResearch, Norderstedt, Germany), by irradiating the samples with an X-ray beam at an 8 keV energy. Irradiation times were in the order of 30 seconds, and for each sample 3 spectra were acquired and averaged. The sample-to-detector distance of 1308 mm allowed to access a 0.00670.46 Å-1 Q-range. Data reduction and background subtraction were performed with the software IGOR Pro (Wavemetrics, Inc.).18 Data modelling was carried out with IGOR Pro and with the software GAP,19 kindly provided by Prof. Georg Pabst (Graz University, Austria) free of charge. In a SAXS experiment,20 a sample is illuminated with an X-ray beam of wavelength λ, which interacts with the different regions of the matter according to their electron density, and this registers as fluctuations of scattered intensity I(Q) as a function of the scattering vector (Q = 4π sin θ/λ, where θ is the angle between the incident beam and the scattered beam). I(Q) is proportional to two functions: the form factor P(Q), which contains information on the shape and size of the scatterers, and the structure factor S(Q), which describes the interactions between scatterers. The form factor of the lateral cross-section of a lipid bilayer is given by P(Q) = 〈|| 〉, with:

=

Eq. 3

which is the Fourier transform of the electron density profile ρ of the bilayer along its z axis.21 In the present work, the electron density of the lipid bilayers is modelled using a three-Gaussian profile:22


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= !"#$ $ % /2(% ) + !"#$ + % /2(% ) $ * !"$ /2(+

Eq. 4

Here, two Gaussians represent the headgroups, centered at zH and of width σH, while one Gaussian is used to represent the terminal −CH3 group at the bilayer’s center. ρr is the ratio between the electron density amplitude of the terminal methyl group to that of the headgroup: + $ , / % $ , , with the electron density of the solvent (water) ρs arbitrarily set to zero. The Fourier transform of Eq. 4 is:23

= -2. ⁄ 0#2(% !"$(% /2 123 $ (+ * !"$(+ /2)

Eq. 5

A typical bilayer electron density profile (ρ(z) as a function of the distance z from the bilayer center), along with the salient structural features of a model bilayer, is depicted in Scheme 2. Using Eq. 4 allows to calculate the bilayer thickness dB (i.e. the headgroup-to-headgroup thickness of the lipid double-layer) as:

4

= 2% + 2(%

Eq. 6

Scheme 2. Pictorial representation of a model lipid bilayer and the corresponding electron density profile, where: dB (Å) = bilayer thickness; dH (Å) = headgroup thickness; dC (Å) = half thickness of hydrophobic region; zH (Å) and σH (Å) = center and width, respectively, of the Gaussians representing the headgroups. 9 ACS Paragon Plus Environment

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TRANSMISSION ELECTRON MICROSCOPY (TEM) DPPC/12-bis-THA dispersions were first diluted 50 times in milli-Q water (final lipid concentration of 0.08 mg/mL) in order to be suitable for TEM imaging. Briefly, we prepared a 2 %wt aqueous solution of phosphotungstic acid and we mixed equal quantities of sample and stain (10-20 µL). Then we placed a drop of this mixture onto a formvar grid held by tweezers for ~20 s and we removed almost all the solution with filter paper. The grids were left to air dry for 24 h before measurements. Transmission electron microscopy analyses were performed on a TEM CM12 PHILIPS, equipped with an OLYMPUS Megaview G2 camera.


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Results and discussion.

The affinity of the bolaform cationic surfactant 12-bis-THA for zwitterionic lipid bilayers, and its effect on the double layer integrity, were assessed by mixing the bolaamphiphile with DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) at different ratios, ranging between 1:60 and 1:4. These correspond to bolaamphiphile mole percentages ranging from 1.6% to 20.8%. The DPPC concentration was kept at 20 mg/mL, to yield concentrated dispersions of multilamellar vesicles (MLV) in water by vortex mixing, Figure 1 shows the DSC thermograms for the DPPC samples containing increasing concentrations of 12-bis-THA. The thermogram of neat DPPC bilayers presents the expected endothermic peak with Tm, centered at 41.8 ºC, due to the main thermotropic phase transition associated to the gel-to-liquid crystalline melting. This is preceded a lower intensity signal, between 36 and 39 ºC, attributed to the a Lβ - Pβ’ pre-transition due to a transient ripple

Heat Flow (mW/mg)

phase.24

0.05

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20.8%

11.6%

6.2%

3.2% 1.6% 0%

12-bis-THA increase

41.8 °C

30

35

40

45

50 T (°C)


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Figure 1. DSC thermograms (heating mode, exo up) obtained for DPPC samples containing growing amounts of 12-bis-THA ([DPPC] = 20 mg/mL), indicated for each curve in the figure as mol%. The curves have been offset along the heat flow axis for clarity of presentation.

The presence of a small amount of bolaamphiphile (1.6 %mol, or 12-bis-THA:DPPC = 1:60 mole ratio) leads to the disappearance of the pre-transition, and to the shift of the main transition to a lower temperature. By increasing the surfactant concentration, the temperature of the gel-to-liquid transition decreases further (Figure 2a). Both the disappearance of the pre-transition and the decrease of the melting temperature are well-known effects that take place in the presence of detergent molecules,25,26 consistent with the intercalation of species with short alkyl chains (< 10-C) into the lipid bilayer.27,28 Peak areas and widths at half maximum (FWHM) were obtained by fitting the peaks with an exponentially modified Gaussian (EMG) model (see SI, Figure S1). For 12-bis-THA concentrations from 0 to 6.2%, fitting could be achieved using a single EMG, while the convolution of two such curves was necessary to correctly model the peaks corresponding to [12-bis-THA] = 11.6% and 20.8%. Peak areas were used to obtain the transition enthalpies (∆H), which undergo an initial marked reduction, followed by a momentary increase (up to 6.2% 12-bis-THA) and finally a plateau (Figure 2b). An increase of ∆H has been linked to the existence of interactions between the polar heads of the lipid and the detergent.29 The FWHM values, plotted in Figure 2c, increase until [12bis-THA] = 3.2% and then decrease again. This may indicate a transitory disruption of the gel-tofluid transition cooperativity.27 However, the growing peak asymmetry for high 12-bis-THA concentrations, and the fact that these peaks were modelled as the convolution of two EMG, make it difficult to estimate the real trend of ∆H and FWHM values. Indeed, complex peak shapes and growing asymmetry may suggest that part of the lipid is present in a different phase, which also gives an endothermic transition, but with a different associated enthalpy.29,30 Unfortunately, in the 12 ACS Paragon Plus Environment

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absence of a linear regime for these data, we are unable to determine and quantify a free-bound equilibrium for 12-bis-THA.27

a

42

40 39

b

2.0

∆ H (J/g)

41

1.6 1.2 0.8

38

2.4

c

2.2 FWHM (°C)

43

Tm (°C)

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2.0 1.8 1.6 1.4

37

1.2 0

5 10 15 20 [12-bis-THA] %mol

0

5 10 15 20 [12-bis-THA] %mol

0

5 10 15 20 [12-bis-THA] %mol

Figure 2. Plots of (a) the main transition temperature, Tm, (b) the transition enthalpy ∆H, and (c) the width at half-maximum, FWHM, as a function of 12-bis-THA concentration (%mol).

In this picture, we can infer that 12-bis-THA inserts into the DPPC bilayer and destabilizes the gel phase by disrupting the packing of the acyl chains. The length of the extended bolaamphiphile molecule is too low to span the entire DPPC double layer, however these DSC results confirm the interaction of 12-bis-THA with the lipid hydrophobic chains and with the polar headgroups: we can speculate that 12-bis-THA could bend its 12-C spacer chain, assuming a cone-like shape, so that its alkyl moiety can intercalate among the phospholipid molecules, while the two cationic headgroups can rest at the water-facing interface (Scheme 3).


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Scheme 3. Pictorial representation of the intercalation of 12-bis-THA in a DPPC bilayer, according to interpretation of the DSC results.

The naked-eye observation of the samples could provide some information on the colloidal dispersion. Indeed, the sample containing pure DPPC appeared immediately as a MLV suspension, turbid and colloidally unstable (precipitation of the lipid component occurred in a matter of hours). On the other hand, the presence of even a small amount of bolaamphiphile (1.6%, or 1:60 mole ratio) induced the formation of a slightly opalescent dispersion, stable over time, suggesting the presence of unilamellar vesicles (ULV) with sizes in the 100-500 nm range. This hypothesis was verified through dynamic light scattering (DLS) analysis. For these measurements, the samples were diluted in order to eliminate the possible effects of multiple scattering and interactions between particles; the highest possible dilution factor was 20×, yielding an average scattering intensity about ten times higher than that of pure water. Dilution factors between 5× and 20× yielded superimposable DLS autocorrelation functions for each sample (data not shown), demonstrating the absence of interparticle interactions that could have led to artefacts in data treatment. The autocorrelation functions of the scattered intensity, reported in Figure 3a, could not be analyzed through the common cumulant fitting, due to the presence of two exponential decays; the data were thus modelled through a Laplace inversion using the CONTIN algorithm.31 As reported in SI, Figure S2, two main populations of scatterers can be identified, the first one peaked at around 500 nm and the second one at around 5 µm hydrodynamic diameter. Interestingly, the integral of the distribution highlights that increasing the amount of bolaamphiphile leads to an increase of the percentage contribution to the total scattering of the smallest population, while the largest population percentage decreases, in agreement with the formation of ULV and reorganization of the phospholipids in different structures. Nevertheless, a third even smaller population starts to appear in the sample with 6.2% bolaamphiphile (12-bis-THA:DPPC ratio of 1:15), which becomes more evident in the sample with 20.8% 12-bis-THA. Since the scattered 14 ACS Paragon Plus Environment

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intensity depends on the squared volume of the scattering particles, we can speculate that the relative amount of the smallest population, is highly predominant by number in the sample. ζpotential analysis of the samples showed that the average electrostatic (positive) surface charge of the object in suspensions increased, as expected, by increasing the concentration of bolaamphiphile (Figure 3b).

Figure 3. a) Representative DLS autocorrelation functions acquired for DPPC samples containing increasing amounts of 12-bis-THA. The arrow over the curves indicates the increase in concentration. The legend indicates the 12-bis-THA concentrations in %mol. b) ζ-potential profile in comparison with hydrodynamic diameter obtained from multimodal size distribution (MSD) at different concentrations of 12-bis-THA. Measurements were performed on dilute samples (total lipid concentration = 0.4 mg/mL). The black arrow indicates the ζ-potential values (black full circles), while the white arrow indicates the MSD number (open circles).

The disruption of lipid vesicles due to detergent action is a well-known but complex and poorly understood process.32 Several mechanisms have been proposed to describe this process, such as the “three-stage model” which suggests the initial partitioning of the detergent in the bilayer, followed by the co-existence of mixed vesicles and mixed micelles, and then mixed micelles only, as the


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surfactant concentration in the system is increased.33 Experimental evidence, however, has shown that the most diverse structures can form in the presence of lipid and surfactant mixtures. The preferential structures will depend on the thermodynamics of the system and in particular on the packing parameter of the molecules involved.34 Common structures are rippled and/or perforated vesicles; in many cases, mixtures of phospholipids and surfactants having different packing parameters can give rise to bicelles,35 bilayered disks in which the faces are composed of the molecular species with the packing parameter closest to 1, while the curved rim is constituted preferentially by the other molecules.36 Rafts can also be observed, which are lipid clusters of submicron size deriving from the fragmentation of larger lipid membranes, stabilized by detergents or proteins among many possible molecular species.37 In the presence of high detergent concentrations, lipid vesicles can finally transition to thread-like,38 bicontinuous, or even octopuslike micelles.39 In the present case, if we suppose the bending of 12-bis-THA into a cone-like shape as suggested by the DSC results discussed previously, the bolaamphiphile could likely participate to the stabilization of DPPC bilayer fragments by forming the rims of bicelles, as observed with other surfactants.40 This hypothesis would be consistent with the absence of macroscopic phase separation in the samples containing both DPPC and 12-bis-THA, since bicelles can be considered as defects of a lamellar phase and thereby coexist with vesicles and other bilayered structures of similar composition and density.41 The co-existence of lamellar-based nanoaggregates with different shapes or structures is further reinforced by the DSC analysis discussed earlier, which suggested that the peaks of the systems with 11.6% and 20.8% bolaamphiphile could be the convolution of two endothermic transitions occurring at roughly the same temperature but with different enthalpic content. However, other than demonstrating the co-existence of several types of scattering objects of different sizes, DLS analysis cannot yield more precise information on the shape of each of these aggregates. A deeper investigation at the nanometric scale using small-angle X-ray scattering


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(SAXS) could provide further insight on the local structure of the aggregates and shed light on the modification of the lipid bilayers induced by the presence of the cationic bolaamphiphile.

I(Q) (arb. units)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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4 2

100

8 6 4 2

10

8 6 4 2

1

8 6

12-bis-THA %mol 0 1.6 3.2 6.2 11.6 20.8 2

3

4

5 6 7 8 9

0.01

0.1

2

3

4 -1

Q (Å )

Figure 4. SAXS patterns obtained for DPPC (4 mg/mL) samples containing increasing amounts of 12-bis-THA. The legend indicates the 12-bis-THA concentrations in %mol. The curves have been offset along the intensity axis for clarity of presentation.

Figure 4 reports the SAXS patterns for the six samples with varying 12-bis-THA mole percentages. The pattern for the sample without 12-bis-THA presents a series of peaks originating from the diffraction of X-rays by adjacent bilayers in MLV,42 the first one occurring at Q0 = 0.098 Å-1 and the next following at regular intervals, according to the relationship Q1/Q0 = 2, = Q2/Q0 = 3. This series of Bragg peaks identifies the structure factor of a lamellar phase43 with a characteristic spacing d = 2π/Q0 = 63.8 Å, a typical value for hydrated DPPC bilayers in the Lβ phase.44 With the addition of small amounts of bolaamphiphile, namely 1.6 %mol and 3.2 %mol, the SAXS profiles change dramatically: no diffraction peaks are present, and the patterns consist of the pure form factor of positionally uncorrelated bilayers, i.e. the diffuse scattering from unilamellar 17 ACS Paragon Plus Environment

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vesicles. This is in agreement with earlier macroscopic visual observation, which suggested the sole presence of unilamellar liposomes in these samples. The spontaneous formation of ULV upon addition of a charged species to a lipid bilayer is a known phenomenon:15 the onion-like structure of MLV, observed in the neat DPPC sample, is now unfavored due to electrostatic repulsion between bilayers. The electrostatic charge also affects the bending rigidity of the bilayers by inducing a progressively more bent conformation (and therefore smaller vesicles, in agreement with DLS results), as observed, among others, in systems of coexisting non-ionic and ionic surfactants.45 Fitting of these two experimental curves (Figures 5a,b) was carried out by modelling the electron density profiles according to Eq. 5. The misfit in correspondence of the first node (Q ~ 0.05 Å-1) is likely due to bilayer asymmetry:46 indeed, since the wedged 12-bis-THA molecules (see Scheme 2) present a bulky headgroup, they should be more concentrated in the outer leaflet, where the curvature helps to minimize the headgroup-to-headgroup electrostatic repulsion, and this would generate some asymmetry in the electron density profile. Using Eq. 6, we calculated the bilayer thickness dB = 55.7 Å for the 1.6% sample and 56.3 Å for the 3.2% sample, respectively. These values are comparable with the dB of pure DPPC in the gel phase, 52.4 Å,47 with an increase justifiable by the presence of 12-bis-THA. However, the apparent bilayer thickness increase by 0.5 Å with higher 12-bis-THA is within the fitting procedure error bar, and it is therefore not significant. Moreover, it is very difficult to define a clear-cut separation between headgroup and hydrophobic regions in the electron density profiles in the presence of a mixture of lipids,23 therefore we cannot extract any further detailed structural information from these SAXS curves.


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Figure 5. SAXS patterns obtained for (a) 1.6% and (b) 3.2% 12-bis-THA:DPPC. Experimental patterns are represented by markers, while red lines are curve fits. Insets: electron density (ρ) profiles as a function of the distance (z) from the bilayer center.

The formation of ULV here can be considered the result of a discontinuous unbinding transition.48 Indeed, for lipid membranes swollen in excess water, the intermembrane distance is determined by the interactions occurring between adjacent bilayers.49 In the present case, the DPPC membrane goes from a state characterized by low surface roughness (low-amplitude thermal undulations), favoring lamellar stacks or onion-like vesicles,50 to an infinite bilayer separation induced by the positive charge of 12-bis-THA (electrostatic repulsion dominates the intermembrane potential).51 This hypothesis is further corroborated by the fact that preparing the 3.2% sample in saline solution (NaCl), where the ionic strength serves to screen the electrostatic repulsion, leads to the recovery of the MLV structure as demonstrated by the presence of Bragg peaks in the SAXS patterns (see SI, Figure S3). The samples with neat DPPC and with 3.2% 12-bis-THA, respectively, were heated up to 65 °C and SAXS patterns were recorded at 5 °C intervals (Figure 6). As expected,52,53 in the pure DPPC MLV the lamellar spacing d decreases from 63.8 Å at 25 °C to smaller values during the pretransition to the ripple phase (35 °C) and just before the main transition (occurring at 42 °C): here, 19 ACS Paragon Plus Environment

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the long-range order is lost as evidenced by the enlarged first-order diffraction peak and the disappearance of the higher-order Bragg peaks. After the main transition, the order of the events is reversed as the Bragg peaks appear narrow (only the first two are observed here) and the lamellar spacing increases. Above 60 °C, the peaks are enlarged again as the thermal disorder prevails.

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The 3.2% sample presents a discontinuity in the scattering curves around 40 °C, in agreement with the endothermic transition recorded by DSC. Above the transition, the position of the maximum is unchanged (Q = 0.1 Å-1, corresponding to a real-space length of 62.8 Å, still close to the DPPC lamellar spacing). However, we observe the disappearance of the minimum at Q = 0.05 Å-1, which could be due to extreme polydispersity in bilayer thickness induced by thermal disorder. Returning to the SAXS patterns in Figure 4, as the concentration of bolaamphiphile is increased further (6.2%, 11.6%, and 20.8%) the shape of the curves is still partly reminiscent of the liposomal form factor; however, the mathematical model used so far proved no longer adequate to fit these 20 ACS Paragon Plus Environment

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data, and fitting with other common form factors, such as core-shell disks (bicelles) or polydisperse spheres, also failed. Considering the size polydispersity of the objects observed in the DLS analysis, we can infer that the presence of 12-bis-THA at %mol > 3.2 determines the destabilization of the ULV bilayers and partial solubilization of the lipid molecules, with the consequent rearrangement of the systems in different types of structures.40 The SAXS curves therefore reflect the diversity of aggregate shapes simultaneously present in solution, which could be a mixture of vesicles, bicelles, and micelles, each with their own bolaamphiphile/lipid compositions, so that model fitting becomes a very difficult (if not impossible) task. Nevertheless, the coexistence of spherical vesicles and discoidal aggregates (bicelles) seems to be confirmed by TEM micrographs; by increasing the amount of bolaamphiphile, we notice the reshaping of the objects from being spherical to discoidal, respectively at 1.6% (Figure 7a) and 20.8% bolaamphiphile (Figure 7b), in agreement with DLS and SAXS measurements.

Figure 7. TEM micrographs of DPPC samples in the presence of 12-bis-THA: a) 1.6% (1:60 mole ratio); b) 20.8% (1:4 mole ratio).


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Even though accurate SAXS fitting has proven extremely complicated for these samples, we can still extrapolate the trend of the average bilayer thickness by considering dB = 2π/Qmax (where Qmax is the “top of the bump” for each SAXS curve) and plotting it against 12-bis-THA concentration. These values are obviously affected by greater error than a thorough fitting of the electron density profiles, however they can serve to our purpose. Figure 8 compares this trend to that of the melting temperature determined by DSC: the correlation between the two functions is evident and demonstrates the link between the variations of microscopic and macroscopic properties due to the intercalation of the cationic detergent in the DPPC bilayer and the progressive restructuration of the latter.

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38.5 0

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Figure 8. Plot of the average bilayer thickness (inferred from SAXS data) and the gel-to-liquid crystal melting temperature (determined by DSC) as a function of 12bis-THA concentration.


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Conclusions.

We have investigated the effect of including the cationic bolaform amphiphile 12-bis-THA in model membranes constituted of dipalmitoylphosphocholine (DPPC). The aim of such study was to assess the stability of liposomal carriers, proposed for possible in vivo applications, where this bolaamphiphile is used to encapsulate oligonucleotide therapeutics and target bacterial cells.11,12 12-bis-THA has shown high affinity for lipid bilayers as demonstrated by the progressive lowering of the gel-to-liquid crystal transition temperature of DPPC, as expected for the intercalation of a short amphiphile. SAXS analysis has revealed that the addition of the bolaamphiphile to DPPC multilamellar vesicles also determined the spontaneous formation of unilamellar vesicles due to a discontinuous unbinding transition. Upon growing 12-bis-THA concentration, we observed a detergency effect: DLS, SAXS, and DSC data strongly hint at the simultaneous existence of different lamellar-based structures, such as liposomes, bicelles, stabilized bilayer fragments (lipid rafts), in which DPPC and 12-bis-THA could be present in different molar ratios. The steep increase of the ζ-potential with added bolaamphiphile confirms that 12-bis-THA is retained in the formed aggregates. In particular, the presence of bicellar aggregates is supported by TEM imaging, which revealed discoidal objects. From the formulative point of view, these results show that the liposomal structure is retained for a bolaamphiphile concentration up to 3.2 %mol to DPPC, while higher concentrations lead to the destabilization by means of detergency. This value sets the experimental limit for the loading capacity of the cationic amphiphile in the liposomal carriers, and confirms the liposomal stability and bilayer integrity at the concentrations used in previous work.11,12 The DSC results have allowed us to obtain a more detailed idea of the way in which 12-bis-THA interacts with a lipid bilayer: the bolaamphiphile most likely bends its 12-C alkyl chain spacer in a cone-like shape so that it can quite literally wedge into the DPPC layer. In order for the surfactant to span the entire bilayer, the length of the spacer chain should match the thickness of the lipid bilayer obtained by SAXS 23 ACS Paragon Plus Environment

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analysis: this may offer a way to stabilize the liposomes against bilayer solubilization at higher bolaamphiphile concentrations. However, in the present system each 12-bis-THA molecule displays both its polar heads facing the continuous medium, providing an excellent “forceps” mechanism to anchor the complexed TFD at the liposomal surface. This suggests that a longer-chained bolaform surfactant, presenting its polar heads on each side of the lipid bilayer, may not ensure a similarly efficient DNA-complexing performance. In this picture, it appears that 12-bis-THA indeed has the appropriate molecular structure to bind the TFDs to liposomal structures. The use of low 12-bisTHA concentration is nonetheless warranted by the cytotoxicity due to the cationic charge. Ultimately, this study supports the use of 12-bis-THA as an oligonucleotide transfection vector in liposomal carriers in a maximum surfactant-to-lipid molecular ratio of 3%.


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Acknowledgements. This work was funded under the 7-People Framework – Marie Curie Industry and Academia Partnerships & Pathways scheme (grant agreement nr. 612338). We gratefully acknowledge technical assistance by Dr. Heinz Amenitsch (synchrotron SAXS; Elettra, Trieste, Italy). The authors thank Prof. Georg Pabst (University of Graz, Austria) for providing the GAP software free of charge; Dr. Francesca Ridi and Prof. Sandra Ristori (CSGI and Department of Chemistry, University of Florence, Italy) for fruitful discussion and precious suggestions.


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Abstract Graphic


Structural modifications of DPPC bilayers upon inclusion of an

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