Fluorescent and Highly Stable Unimodal DMPC Based Unilamellar

May 17, 2012 - Design, Synthesis, and Optical Properties of Na-DSA ..... UNIK research initiative of the Danish Ministry of Science, Technology, and I...
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Fluorescent and Highly Stable Unimodal DMPC Based Unilamellar Vesicles Formed by Spontaneous Curvature Dong Shi,† George Sfintes,† Bo W. Laursen,*,† and Jens B. Simonsen*,‡ †

Nano-Science Center & Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen Ø, Denmark ‡ Department of Basic Science and Environment, Faculty of Life Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark S Supporting Information *

ABSTRACT: The formation of uniform and highly stable unilamellar vesicles (ULVs) and the theory behind it are ongoing tasks within the vesicle community. Herein, we report the formation of highly stable, fluorescent, and unimodal 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) based ULVs with an average size of ∼100 nm, as determined by cryogenic transmission electron microscopy (cryo-TEM) and dynamic light scattering (DLS). The ULVs are formed by mixing a two-component powder mixture or mixed lipid film of DMPC and 5 mol % of a novel amphiphilic carbenium salt, sodium 2-didecylamino-6,10-bis(N-methyltaruino)-4,8,12-trioxatriangulenium (Na-DSA) in aqueous solution when subjected to shaking. We propose that the high stability and the unimodal size distribution of the 5% DSA ULVs confirmed by DLS studies are a product of spontaneous curvature. UV−vis absorption/emission studies reveal that the structure of DSA promotes a strong interaction between the DMPC and the DSA to take place due to the complementary charge distribution of the DSA and DMPC head groups. The strong interaction may introduce an asymmetric amphiphile composition in the inner and outer leaflet of the bilayer which drives the spontaneous curvature.

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Jung et al.10 and others,11,12 thermodynamic equilibrium state ULVs can result from two scenarios: (i) spontaneous curvature that promotes an energetically favorable radii or (ii) thermal fluctuations, such as Helfrich undulations of the bilayer,13 that makes the formation of an extra bilayer too costly entropically. In addition to this, a broad size distribution as well as a large number of ULVs (translational entropy) can also contribute to the entropy driven mechanism.14 Introduction of surface charges that result in strong electrostatic repulsions between bilayers is also an efficient way to produce stable ULVs and is often used to form kinetically stable ULVs. Surface charges may also result in thermodynamically stable ULVs, in which case the effect of surface charges is accounted for within the theory of (i) and (ii), through the changes in the bending modules and the saddle-splay modulus.15,16 Keeping this in mind, we are motivated to propose a chemical approach toward spontaneous formation of stable phospholipid based ULVs in a proposed equilibrium state. ULVs formed by spontaneous curvature were described for the first time more than two decades ago and were based on cationic/anionic surfactant mixtures.17 Later on, mixtures of, for example, long-chain and short-chain lipids have

INTRODUCTION The formation of unilamellar vesicles (ULVs) is an important area of research from fundamental biological, biophysical, and applied perspectives. ULVs can serve both as a model of biomembrane systems and as a drug delivery system for the transportation of either hydrophobic or hydrophilic drugs. Several reviews on the manifold applications of vesicles in general have appeared recently. Here are listed just a few of them dealing with issues such as delivery of potent cancer medicine,1,2 peptide and protein drug delivery,3 siRNA delivery,4 food and cosmetics applications,5 and novel analytical applications.6 ULVs are often generated by mechanical methods such as sonication7,8 and extrusion.9 However, both methods possess intrinsic limitations. Sonication produces small unilamellar vesicles that exhibit low encapsulation efficiency in an irreproducible way due to uncontrollable process conditions. It also causes degradation of the lipids and potential damage to the encapsulated material. On the other hand, extrusion is timeconsuming and leads to loss of material to the filter. Overall the mechanical approaches tend to form ULVs trapped in a kinetically meta-state, which confers an inherent instability to the system. In order to obtain spontaneous formation of stable ULVs, these structures need to be in the thermodynamic equilibrium state as opposed to multilayer sheets. According to © 2012 American Chemical Society

Received: April 9, 2012 Revised: May 16, 2012 Published: May 17, 2012 8608

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shown the spontaneous formation of ULVs. There seems to be a mutual agreement in the literature, at least from a theoretical point of view, that a nonsingle amphiphile composition is needed to form ULVs based on the spontaneous curvature, since an asymmetric distribution of the amphiphiles in the inner and outer leaflet drives the spontaneous curvature.18,19 DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) is one of many lipids within the phosphatidylcholine lipid family that is highly abundant in biological membranes and has therefore drawn much attention. Nevertheless, to our knowledge, only a few examples of spontaneous formed ULVs based on these phosphocholine lipids exist. Nieh and co-workers12,20 show that the mixture of DMPC/DHPC (1,2-dihexanoyl-sn-glycero-3phosphocholine) forms stable ULVs if the DMPC/DHPC composition is mixed with either negatively charged DMPG (1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol)) or Ca2+ ions. However, these ULVs exhibit a polydispersity, measured by neutron scattering, of 0.1−0.2. Claessens et al. have shown that both pure DOPC (1,2-dioleoyl-sn-glycero-3phosphocholine)21 and DOPG (1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol))22 and the DOPC/DOPG mixtures11 form stable ULVs after multiple freeze−thaw cycles. Claessens et al. suggest that these ULVs are stabilized by entropic translational and undulation contributions. It should be pointed out that pure DOPC forms only stable ULVs in a very limited range of phospholipid concentrations and ionic strengths and does not form stable ULVs when using biologically relevant NaCl(aq) electrolytes.21 Lecithin (POPC) (1-palmitoyl-2oleoyl-sn-glycero-3-phosphocholine) and DMPC based ULVs have been prepared by dilution23 and temperature-jump24 methods, respectively. In this study, we show that extremely stable, fluorescent, ∼100 nm ULVs with very low size polydispersity can be obtained by adding 5 mol % of a novel synthetic rhodamine-like amphiphile, Na-DSA (sodium 2-didecylamino-6,10-bis(9Nmethyltaruino)-4,8,12-trioxatriangulenium) to a suspension of DMPC (Figure 1). The monodisperse ULVs are obtained spontaneously from a broad range of concentrations and ionic strenghts, simply by vortexing the powders in aqueous solutions. Our formulation combines ease of assembly and reproducibility. The ULVs assembled by this method reach a proposed equilibrium state most likely by the spontaneous curvature mechanism. We do address the hypothesis about the equilibrium state by various approaches, since any statement regarding equilibrium state ULVs should be treated with great circumspection.

Figure 1. Molecular structure of Na-DSA and DMPC.

cationic, the novel DSA derivative has been given an overall negative charge by appending sulfonic acid groups on two of the amine substituents. The molecular design of DSA serves to enhance the amphiphilic character of the dye via strongly hydrated sulfonic acid groups and at the same time obtains an electrostatic profile complementary to the zwitterionic DMPC phospholipid. Thus, docking of DSA into a DMPC membrane should be facilitated by reverse-oriented dipoles (see Figure 1). We hypothesize that the formation of a DSA-DMPC complex in aqueous solution and additional nonideal mixing may lead to the formation of an asymmetric distribution of the amphiphiles in the inner and outer leaflets of a bilayer and thereby form highly stable and monodisperse ULVs. The relation between complex formation/nonideal mixing and the formation of an asymmetric bilayer has been pointed out by Claessens et al.,11 while Lasic et al.19 propose that the asymmetric bilayer drives the spontaneous curvature. In our case, strong cationic−anionic interactions take place between the zwitterionic DMPC and the charged DSA. These electrostatic interactions are similar to the interactions taking place in the cationic(cetyltrimethylammonium bromide)/anionic (sodium perfluorooctanoate) system of Jung et al.10 The work of Jung et al. is one of only a few ULV systems proposed to be stabilized by a spontaneous curvature. The Na-DSA salt was synthesized by analogy to other ATOTA+ salts via selective aromatic nucleophilic substitution of the para methoxy group in tris(2,4,6-trimethoxyphenyl)carbenium tetrafluoroborate, TMP-BF4 (Scheme 1).26,34 The key step in the Na-DSA synthesis is the one-pot preparation of the intermediate compound 1 in 41% yield by substitution, first with didecylamine, then followed by addition of excess of Nmethyltaurine and raising the temperature. LiI catalyzed ring closure of 1 gave Na-DSA in 50% yield after column purification and recrystallization. Diluted solutions of Na-DSA in methanol display spectral properties typical of the rhodamine like ATOTA+ chromophore

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RESULTS AND DISCUSSION Design, Synthesis, and Optical Properties of Na-DSA. The amphiphilic Na-DSA salt belongs to the family of triangulenium ions, which are characterized by a rigid and planar triphenylmethylium chromophore. The bridging of all ortho positions by heteroatoms leads to extreme cation stability.25−29 When the triangulenium core is equipped with amino substituents in the resonant para positions, like in DSA, the optical properties of the system becomes those of an extended threefold symmetric rhodamine with very intense absorption and fluorescence in the visible region.29−31 The amino groups in these tris(dialkylamino)trioxatriangulenium ions (ATOTA+) also favor further delocalization of the positive charge, resulting in a pronounced tendency to form columnar aggregates in the solid state, Langmuir and Langmuir−Blodgett films.26,32,33 While previous ATOTA+ salts all have been 8609

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Scheme 1. Synthetic Route for the Preparation of Na-DSA Surfactanta

a (i) Didecylamine (1.2 equiv), N,N-diisopropyl-N-ethylamine (catalytic amount), DMSO, room temperature, 67 h. (ii) N-Methyltaurine sodium (10 equiv), DMSO, 70 °C, 15 days. (iii) LiI (20 equiv), N,N-diisopropyl-N-ethylamine (catalytic amount), NMP, reflux, 3 h.

(spectra given in the Supporting Information).30,31 However, the introduction of negatively charged sulfonic acid groups results in drastic change in the solubility, compared to the cationic ATOTA+ analogues. Thus, solutions of Na-DSA in chloroform were found to be in a clearly aggregated state, as witnessed by the broad and blue-shifted absorption λmax = 460 nm (see Figure 2) and a significantly red-shifted fluorescence at

DSA and DMPC. Such cosolutions of 5 mol % Na-DSA and 95 mol % DMPC are used as starting material for preparation of ULVs. Preparation of ULVs. When a lipid film prepared from an organic mixture of 95 mol % DMPC and 5 mol % Na-DSA (labeled “5% DSA” throughout the text) or a mixture of the corresponding amount of powders were hydrated in Milli-Q water by vortexing at 40 °C, the sample changed from an initial opaque, turbid suspension to a clear orange solution. It was on the other hand not possible to prepare transparent solutions from lipid films of either 0% DSA (100% DMPC) or 1% DSA by following the same straightforward procedure, even after 24 h of continuous vortexing. The vortexed solutions with different relative amounts of DSA are shown in Figure 3. All the DSADMPC solutions were prepared in pure Milli-Q water if nothing else is specified.

Figure 2. (A) Normalized absorption and emission spectra (solid and dotted lines, respectively) of Na-DSA in CHCl3 in the absence (blue lines) and presence of DMPC (Na-DSA/DMPC = 5/95 by molar) (red lines). (B) Na-DSA dissolved in CHCl3. (C) Na-DSA/DMPC (5/95 by molar) dissolved CHCl3.

Figure 3. Image of the vortex-processed suspensions of DMPC containing 0, 1, 5, and 10 mol % DSA (vortexing time from left to right: 24, 24, 4, and 4 h). The total component concentration is 1 mM for all four suspensions.

λmax = 580 nm. These spectroscopic features are a clear fingerprint of the formation of face-to-face columnar Haggregates of the dyes in CHCl3.30,32,33 The minor emission peak at ∼500 nm is assigned to a small fraction of nonaggregated dyes, as confirmed by the excitation spectra (Figure S2). When DMPC was added to the aggregated CHCl3 solution of Na-DSA, the aggregates were broken up, and the sharp spectra and intense fluorescence of the well-solvated ATOTA+ type dyes is observed (Figure 2). We interpret this significant effect as a clear indication of the attractive interactions between

Structural Studies by Cryo-TEM. Cryogenic transmission electron microscopy (cryo-TEM) was applied to obtain information about the geometrical structure of the DSADMPC aggregates. The cryo-TEM study of the 5% DSA solution shows that the hydrated DSA-DMPC aggregates form well-defined ULVs. The peripheral gray contrast that defines the interface between the inner and outer part of the aggregate corresponds to a wall thickness of ∼6 nm which is comparable with the DMPC bilayer thickness of ULVs determined by 8610

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SANS and SAXS studies of ULVs (∼4 nm).35 The discrepancy is assigned to the cryo-TEM contrast that is a result of a convolution over a curved surface and this gives rise to a broadening of the apparent shell thickness seen in the cryoTEM images. The lower homogeneous intensity inside the gray ring indicates that the aggregates are unilamellar bilayer vesicles rather than mutilamellar vesicles. The ULVs exhibit a radius of ∼50 nm. The low number of observed ULVs rules out a statistically robust estimate of the size distribution. Thus, we employed dynamic light scattering to get more detailed information about the bulk size distribution of the formed ULVs.

the average size of the ULVs obtained from cryo-TEM (radius ∼50 nm) and the DLS results (Rh = 68 nm) could be due to following two reasons: (i) The average size determined from the second order cumulant analysis is based on the intensity and not concentrations/numbers. The slight shift toward smaller size when going from the intensity to numbers is illustrated in the CONTIN based size distributions in Figure 5. (ii) The ionic concentration can affect the particle diffusion speed40 by changing the thickness of the electric double layer called the Debye length. Therefore, a low conductivity medium will produce an extended double layer of ions around the particle, reducing the diffusion speed and resulting in a larger, apparent hydrodynamic diameter. Conversely, higher conductivity media will suppress the electrical double layer and the measured hydrodynamic diameter. The latter effect (ii) was pronounced at NaCl(aq) concentrations below 20 mM as shown in Figure 6A, while Figure 6B shows that the unimodal size distribution of the ULVs is preserved even at physiological salt concentrations 100−200 mM of NaCl(aq). The hydrodynamic size of the ULVs at [NaCl] above 20 mM (Rh ≈ 58 nm) corroborated the size of the ULVs determined by cryoTEM (R ≈ 50 nm). The Proposed Mechanism of the Formation of Stable Unimodal ULV. The stability of the ULVs formed in 5% DSA solutions was probed by DLS. The 5% DSA ULVs show both high thermal stability and good long-time stability. A 5% DSA solution (1 mM) exposed to 70 °C for 30 min (Rh = 70.0 nm and PI = 0.08) and a 5% DSA solution stored at rt for 7 months (Rh = 70.4 nm and PI = 0.07) show only a negligible deviation in size and polydispersity from the fresh 5% DSA solution (Rh = 68.9 nm and PI = 0.07) (Table 1). Although it is difficult to prove that the 5% DSA ULVs are truly equilibrated structures, a number of properties suggest this: (1) the high reproducibility of the size of the formed ULVs (the size of the 5% DSA ULVs vary less than 0.5 nm as shown in table 1), (2) the low polydispersity, (3) the long-term stability, (4) the stability at elevated temperatures, and finally (5) the observation that different methods of preparation leads to the same final structure. Altogether these observations provide compelling evidence that an equilibrium state has been reached, contrary to a trapped metastable state. Similar arguments were used by Jung et al. in there paper entitled The Origins of the Stability of Spontaneous Vesicles.10 Additionally, we list a few features that support that the spontaneous curvature is the most likely origin of the stability and equilibration of the DSA-DMPC ULVs. The rather low size polydispersity of the DSA-DMPC ULVs indicate according to Jung et al.10 that the origin of the stability of the ULVs is caused by a favorable spontaneous curvature introduced by the DSA. The fact that the ULV size is found to be independent of the lipid concentration in the full range from 0.1 to 5 mM further supports a nonentropic based stabilization of the formed ULVs, according to the work by Claessens et al.21 Additionally, the strong DSA-DMPC interactions enable an asymmetric distribution of the amphiphiles in the inner and outer leaflets of the ULV bilayer, which is essential for driving the spontaneous curvature. We assume that the DSA has a preference for the outer leaflet based on the concept of the packing parameter of amphiphiles. We have previously shown that the molecular area taking up by the ATOTA core without the additional sulfonic units at the air− water interface (∼60 Å2) is larger than the hydrophobic footprint at the water−air interface of the didecylamino chain (∼40 Å2).32 We would like to point out that only two examples

Figure 4. Cryo-TEM images of the 5% DSA sample. The dark solid features that have a size 5−10-fold smaller than the ULVs correspond to water crystals. These crystals are abundant due to use of pure water as solvent.

Structural Studies by DLS. We only studied the 5% and 10% DSA solutions by DLS, since the 0% and 1% solutions were opaque. The opacity is caused by the scattering from the large multilamellar vesicles (MLV) and/or multimodal ULVs. The autocorrelation function measured by dynamic light scattering from the 5% DSA solution was fitted by CONTIN36,37 revealing a unimodal size distribution as shown in Figure 5. The fact that the size distribution was unimodal enabled us to use the coefficients of the of the second order cumulant analysis38,39 to determine the average hydrodynamic radius and the polydispersity of the unimodal size distribution. The second order cumulant fit is shown in Figure 5, while the fitted second order coefficients are listed in Table 1. We did not investigate the 10% DSA solution further, because the CONTIN analysis of the corresponding autocorrelation function gave rise to more than one distinct size distribution. The DLS results and the opaqueness of the 0 and 1% DSA solutions show clearly that fine-tuning the relative amount of Na-DSA to DMPC is of high importance when we are aiming for ULVs that exhibit a unimodal size distribution. A 5% DSA solution turned out to form highly monodisperse ULVs with a hydrodynamic radius (Rh) of 68 nm. The discrepancy between 8611

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Figure 5. (A) Autocorrelation function measured by dynamic light scattering from the 5% DSA solution (black dots) and the corresponding fit based on CONTIN (red dashed line). (B) Autocorrelation function (black dots) (the same to the one used in (A)) and the corresponding second order cumulant fit (red dashed line). Resulting Rh distributions by intensity (C) or number weight (D) based on the CONTIN fit shown in (A).

Table 1. DLS Data Based on the Second Order Cumulant Analysis of the 5% DSA Solutiona method mixed mixed mixed mixed mixed mixed

lipid film lipid film lipid film lipid filme lipid filmf powders

C (mM)

Rh (nm)b

SE of Rh (nm)c

PId

0.1 1 5.0 1 1 1

68.0 68.9 71.2 70.4 70.0 68.1

0.3 0.3 0.4 0.3 0.3 0.3

0.05 0.07 0.09 0.07 0.08 0.08

a

The DLS results were averaged from measurements of two independently prepared samples for each case. Each sample was equilibrated for ∼30 min at 25 °C before collecting data, and the duration time for each single measurement was 3−5 min. b Hydrodynamic radius (Rh) is based on the first order cumulant coefficient of the second order fit. cSE denotes the standard error from the mean value. dPI denotes the polydispersity index which is based on the second order cumulant coefficient. e7 months old sample. f Exposed to 70 °C for 30 min. Figure 6. (A) Rh values of the 5% DSA ULVs obtained from the second order cumulant analysis as a function of the NaCl concentration. (B) Corresponding polydispersity index (PI) values for each sample in (A). All the data were averaged from two DLS measurements, and the time duration was set to be 3 min for each measurement. The total component concentration is 1 mM for all the suspensions.

in which the average size of the vesicles is independent of the lipid concentration were found in the literature in 2004 according to Nieh et al.12 Finally, we would like to emphasize that we assume that the shaking is needed to speed up the process and suggest that that the chemical approach triggering the spontaneous curvature is the overall driven force toward the formation of unimodal 5% DSA ULVs. The extended mechanical shaking without the DSA does not enable the formation of a unimodal distribution of pure DMPC ULVs. This observation supports the idea that the shaking is only enhancing the speed of the 5% DSA ULV formation rather the being the primary cause of the unimodal ULV formation. Nevertheless, we were not able to prepare a clear 5% DSA

solution without the vortexing step. A 5% DSA dispersion in Milli-Q water was not clarified after 3 weeks of stirring at 40 °C, which suggests that the formation of the DSA-DMPC ULVs is a very slow process. Optical Properties of DSA-DMPC ULVs. The structure and properties of the DSA-DMPC ULVs are clearly indicative 8612

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the protocol and the opportunity to track the ULVs by either absorption or emission assays due to the presence of DSA are all properties that are highly desirable within the vesicle community.

of an uneven distribution of the DSA amphiphiles in the DMPC membrane. Another indication of the complexity of this bicomponent nanostructure is found in the optical properties of the DSA-DMPC ULVs. Thus, when the ULVs are formed, the absorption spectrum of the DSA dye becomes strongly broadened and blue-shifted as is expected for an H-aggregate (Figure 7). However, the fluorescence is similar to that of the

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EXPERIMENTAL SECTION

Synthesis. Compounds 1 and Na-DSA were synthesized and identified by 1H NMR and 13C NMR (shown in the Supporting Information), FAB-MS, and elemental analysis. Tris(2,4,6trimethoxyphenyl)carbenium tetrafluoroborate (TMP-BF4) was synthesized according to the reported procedure.26 Sodium 4-Didecylamino-2,6-dimethyoxyphenyl-bis(4-N-methyltaurino-2,6-dimethyoxy-phenyl)carbenium (1). In the presence of N,N-diisopropyl-N-ethylamine (2.5 mL), the mixture of TMP-BF4 (0.80 g, 1.33 mmol) and didecylmaine (0.48 g, 1.61 mmol) in DMSO (40 mL) was stirred for 67 h at room temperature. N-Methyltaurine sodium (2.15 g, 13.3 mmol) was added, and the reaction mixture was heated to 70 °C and stirred for 15 days. After removal of the solvent and drying under vacuum for 24 h at room temperature, the dark crude material was purified through column chromatography (slica gel, CH3OH/CH2Cl2 = 1/5 by volume ratio). The TLC pure fraction was collected and concentrated. Finally, reprecipitation from CH2Cl2/nhexane (1/10) yielded the pure product 1 (0.55 g, 41%). 1H NMR (300 MHz, DMSO) δ 5.87 (s, 4H), 5.76 (s, 2H), 3.68 (t, J = 7.5 Hz, 4H), 3.40 (s, 22H), 3.06 (s, 6H), 2.71 (t, J = 7.5 Hz, 4H), 1.56 (t, J = 7.5 Hz, 4H), 1.28−1.21 (m, 28H), 0.82 (t, J = 6.7 Hz, 6H). 13C NMR (75 MHz, DMSO) δ 163.72, 163.20, 155.69, 154.81, 115.94, 115.56, 89.91, 56.54, 51.05, 49.68, 48.59, 31.96, 29, 28.04, 26.94, 22.76, 14.62. MALDI-TOF (dichloromethane, NaBF4 added) m/z = 1036.0, Calcd for C51H80N3Na2O12S2+: [M•2Na]+ = 1036.4973. High resolution ESP+ (NaBF4 added): found, m/z = 1036.7916. Sodium 2-Didecylamino-6,10-bis(N-methyltaruino)-4,8,12-trioxatriangulenium (Na-DSA). In the presence of N,N-diisopropyl-Nethylamine (2.0 mL), the mixture of 1 (0.50 g, 0.49 mmol) and LiI (1.34 g, 10.01 mmol) in NMP (40 mL) was refluxed and stirred for 3 h under N2. After being cooled down to room temperature and removal of the solvents by reduced pressure distillation, the dark brown crude product was purified through silica gel column (CH3OH/CH2Cl2 = 1/ 3 by volume ratio). The TLC pure fractions were collected and concentrated. The concentrated TLC pure product was washed with CH3CN (3 × 50 mL). Then the product was dissolved in CHCl3 (50 mL) and washed with brine (5 × 50 mL) and distilled water (3 × 50 mL). Finally, recrystallization from ethyl acetate (50 mL) afforded the target product Na-DSA (0.22 g, 50%). 1H NMR (300 MHz, DMSO) δ 6.52 (s, 2H), 6.47 (s, 2H), 6.42 (s, 2H), 3.71 (t, J = 6.7 Hz, 4H), 3.42 (t, J = 6.7 Hz, 2H), 3.11 (s, 6H), 2.78 (t, J = 6.7 Hz, 4H), 1.56 (t, J = 6.4 Hz, 4H), 1.30−1.24 (m, 28H), 0.82 (t, J = 6.0 Hz, 6H). 13C NMR (126 MHz, methanol) δ 158.24, 157.93, 155.19, 154.99, 95.97, 95.73, 95.66, 95.48, 40.26, 33.11, 30.86, 30.76, 30.66, 30.54, 28.37, 28.02, 23.78, 14.49. MALDI-TOF (dichloromethane, NaBF4 added) m/z = 898.0 ([M·2Na]+), Calcd for C45H62N3Na2O9S2+: m/z = 898.372. High resolution ESP+ (acid added): calcd for C45H63N3NaO9S2+, m/z = 876.3898; found, m/z = 876.9765 ([M·H·Na]+). Elemental analysis: Calcd for C45H62N3NaO9S2: C, 61.69; H, 7.13; N, 4.80. Found: C, 59.99; H, 7.14; N, 4.69. UV−Vis, Emission, and Excitation Measurements. The electronic absorption spectra were recorded with a lambda 1050 spectrometer. The emission and excitation spectra were recorded with the PerkinElmer LS50 spectrometer. Sample Preparation. The samples were prepared through a vortex-processed procedure. The two components of DMPC and NaDSA were mixed according to the desired molar ratios in the form of mixed powders or mixed lipid films and then suspended into Milli-Q water or NaCl(aq) with specified concentrations (2, 5, 10, 20, 50, 100, and 200 mM). The suspension was vortexed for hours (24 h for 0 and 1 mol % DSA solutions, 8 h for 5 mol % DSA solution with overall concentration of 5 mM, 14 h for the mixed powders containing 5 mol % DSA with overall concentration of 1 mM, and 4 h for the rest). The shaking procedure started at room temperature (24 C°). Then the

Figure 7. Normalized electronic absorption and emission (excited at 450 nm) spectra of a 5% DSA solution.

monomer in organic solution. These observations suggest that most of the DSA in the ULVs is present as nonfluorescent columnar aggregates, while a minor fraction of monomers is responsible for the fluorescence from the ULVs. The most obvious use of the stable and well-defined DSA ULVs would relate to applications where the visible absorption and/or fluorescence would help track the position or interactions of the ULVs. For in vivo applications, the biocompatibility of the DSA dye must of course first be investigated.

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CONCLUSIONS We have synthesized a new amphiphilic carbenium salt, NaDSA, featuring two sulfonic units and a didecyl amino unit attached to the corners of a planar, fluorescent triangulenium core. The molecular asymmetry introduced by the polar and apolar moieties gives rise to the strongly amphiphilic nature of DSA. The addition of 5 mol % DSA to DMPC triggers the formation of unimodal ULVs with an average size of ∼100 nm by shaking the two components in aqueous solution. The ULV structure was confirmed mainly by cryo-TEM images and supported by DLS data. The DMPC based ULVs exhibit following properties: (1) long-term and (2) thermal stability, (3) they are highly reproducible, and (4) the final product turns out to be independent of the mixing procedure we apply. These four observations are consistent with a state of equilibrium. Three additional observations suggest that DSA promotes spontaneous curvature and thus equilibrium: (5) UV−vis absorption/emission studies show that the DSA structure enables a strong interaction between the DMPC and the DSA. The strong interaction drives the asymmetric amphiphile composition in the inner and outer leaflet of the bilayer; asymmetry is essential for spontaneous curvature to take place. (6) The rather narrow size distribution (PI < 0.1) and (7) that the size of the ULVs is independent of the total amphiphile concentration. It should be emphasized that vesicles stabilized by spontaneous curvature are rarely observed. We would also like to point out that the interesting features listed above (1), (2), (3), (6), and (7) together with the easy of 8613

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sample was heated up to 40 °C automatically within 1 h due to the thermo effect converted from the mechanical driven force of the laboratory vortexer, and this temperature was maintained. The mixed lipid films were prepared by mixing the two components in CHCl3. Then solvent was removed under N2 flow at 40 °C. The lipid films were further dried under vacuum (∼5.0 × 10−2 Torr) for 24 h at room temperature. Cryo-TEM Measurements. Cryogenic transmission electron microscope measurements were performed using a Philips CM120 instrument equipped with a GATAN CCD camera (1024 × 1024 pixels). Samples were prepared by applying 5 μL of the vesicle suspension (1 mM in Millipore water) on a carbon grid and the excess liquid was removed by tissue. The grid was immediately frozen to −180 °C by the plunging method and kept at this temperature during imaging. DLS Measurements. The ULV sizes were determined by dynamic light scattering (DLS) using the BI-200SM light scattering system from Brookhaven Instruments Corporation.

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(9) Olson, F.; Hunt, C. A.; Szoka, F. C.; Vail, W. J.; Papahadjopoulos, D. Preparation of Liposomes of Defined Size Distribution by Extrusion Through Polycarbonate Membranes. Biochim. Biophys. Acta 1979, 557, 9−23. (10) Jung, H. T.; Coldren, B.; Zasadzinski, J. A.; Iampietro, D. J.; Kaler, E. W. The Origins of Stability of Spontaneous Vesicles. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 1353−1357. (11) Claessens, M. A. E.; van Oort, B. F.; Leermakers, F. A. M.; Hoekstra, F. A.; Stuart, M. A. C. Bending Rigidity of Mixed Phospholipid Bilayers and The Equilibrium Radius of Corresponding Vesicles. Phys. Rev. E. 2007, 76, 011903. (12) Nieh, M. P.; Harroun, T. A.; Raghunathan, V. A.; Glinka, C. J.; Katsaras, J. Spontaneously Formed Monodisperse Biomimetic Unilamellar Vesicles: The Effect of Charge, Dilution, and Time. Biophys. J. 2004, 86, 2615−2629. (13) Helfrich, W. Steric Interaction of Fluid Membranes in Multilayer Systems. Z. Naturforsch. 1978, 33A, 305−315. (14) Simons, B. D.; Cates, M. E. Vesicles and Onion Phases in Dilute Surfactant Solutions. J. Phys. II (Paris) 1992, 2, 1439−1451. (15) Claessens, M. M.; van Oort, B. F.; Leermakers, F. A.; Hoekstra, F. A.; Cohen Stuart, M. A. Charged Lipid Vesicles: Effects of Salts on Bending Rigidity, Stability, and Size. Biophys. J. 2004, 87, 3882−3893. (16) Pincus, P.; Joanny, J. F.; Andelman, D. Electrostatic Interactions, Curvature, Elasticity, and Steric Repulsion in Multimembrane Systems. Europhys. Lett. 1990, 11, 763−768. (17) Kaler, E. W.; Murthy, A. K.; Rodriguez, B. E.; Zasadzinski, J. A. N. Spontaneous Vesicle Formation in Aqueous Mixtures of Singletailed Surfactants. Science 1989, 245, 1371−1374. (18) Safran, S. A.; Pincus, P. A.; Andelman, D.; MacKintosh, F. C. Stability and Phase Behavior of Mixed Surfactant Vesicles. Phys. Rev. A 1991, 43, 1071−1078. (19) Lasic, D. D. Sterically Stabilized Vesicles. Angew. Chem., Int. Ed. 1994, 33, 1685−1698. (20) Nieh, M. P.; Kucerka, N.; Katsaras, J. Spontaneously Formed Unilamellar Vesicles. Methods Enzymol. 2009, 465, 3−20. (21) Claessens, M. M. A. E.; Leermakers, F. A. M.; Hoekstra, F. A.; Stuart, M. A. C. Entropic Stabilization and Equilibrium Size of Lipid Vesicles. Langmuir 2007, 23, 6315−6320. (22) Claessens, M. M.; van Oort, B. F.; Leermakers, F. A.; Hoekstra, F. A.; Cohen Stuart, M. A. Charged Lipid Vesicles: Effects of Salts on Bending Rigidity, Stability, and Size. Biophys. J. 2004, 87, 3882−3893. (23) Egelhaaf, S. U.; Schurtenberger, P. Micelle−to−Vesicle Transition: A Time−Resolved Structural Study. Phys. Rev. Lett. 1999, 82, 2804−2807. (24) Lesieur, P.; Kiselev, M. A.; Barsukov, L. I.; Lombardo, D. Temperature−induced Micelle to Vesicle Transition: Kinetic Effects in The DMPC/NaC System. J. Appl. Crystallogr. 2000, 33, 623−627. (25) Sabacky, M. J.; Johnson, C. S.; Smith, R. G.; Gutowsky, H. S.; Martin, J. C. Triarylmethyl Radicals. Synthesis and Electron Spin Resonance Studies of Sesquixanthydryl Dimer and Related Compounds. J. Am. Chem. Soc. 1967, 89, 2054−2058. (26) Laursen, B. W.; Krebs, F. C.; Nielsen, M. F.; Bechgaard, K.; Christensen, J. B.; Harrit, N. 2,6,10-Tris(dialkylamino)trioxatriangulenium Ions. Synthesis, Structure, and Properties of Exceptionally Stable Carbenium Ions. J. Am. Chem. Soc. 1998, 120, 12255−12263. (27) Laursen, B. W.; Krebs, F. C. Synthesis of a Triazatriangulenium Salt. Angew. Chem., Int. Ed. 2000, 39, 3432−3434. (28) Laursen, B. W.; Krebs, F. C. Synthesis, Structure, and Properties of Azatriangulenium Salts. Chem.Eur. J. 2001, 7, 1773−1783. (29) Søresen, T. J.; Laursen, B. W. Synthesis and Optical Properties of Trioxatriangulenium Dyes with One and Two Peripheral Amino Substituents. J. Org. Chem. 2010, 75, 6182−6190. (30) Laursen, B. W.; Reynisson, J.; Mikkelsen, K. V.; Bechgaard, K.; Harrit, N. 2,6,10 Tris(dialkylamino)trioxatriangulenium Salts: a New Promising Fluorophore. Ion-pair Formation and Aggregation in Nonpolar Solvents. Photochem. Photobiol. Sci. 2005, 4, 568−576. (31) Westerlund, F.; Elm, J.; Lykkebo, J.; Carlsson, N.; Thyrhaug, E.; Akerman, B.; Sørensen, T. J.; Mikkelsen, K. V.; Laursen, B. W. Direct

ASSOCIATED CONTENT

S Supporting Information *

Additional optical spectra and DLS data including the NMR spectra of the Na-DSA and the precursors. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(J.B.S.) Telephone: +45 3533 2338. E-mail: [email protected]. (B.W.L.) Telephone: +45 3532 1881. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Gunnel Karlsson, Lund University for her assistance with the cryo-TEM measurements. We gratefully acknowledge financial support from the “Center for Synthetic Biology” at Copenhagen University funded by the UNIK research initiative of the Danish Ministry of Science, Technology, and Innovation, and from the Danish National Research Foundation under the Danish-Chinese Centre for Self-Assembled Molecular Electronic Nanosystems. J.B.S. would also like to acknowledge the VKR Foundation and the “IMK Almene” Foundation for funding. Finally, deep thoughts go to friends and family of George Sfintes who passed away in April 2012. He will be missed.

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