10032
J. Phys. Chem. B 2007, 111, 10032-10039
Properties of Mixed DOTAP-DPPC Bilayer Membranes as Reported by Differential Scanning Calorimetry and Dynamic Light Scattering Measurements S. Cinelli, G. Onori, and S. Zuzzi Dipartimento di Fisica, UniVersita´ di Perugia and CEMIN (Centro Eccellenza Materiali InnoVatiVi Nanostrutturati) Via A. Pascoli, I-06123, Perugia, Italy and INFM-CRS SOFT, Unita´ di Perugia
F. Bordi, C. Cametti,* and S. Sennato Dipartimento di Fisica, UniVersita´ di Roma “La Sapienza” Piazzale A. Moro 5, I-00185- Roma, Italy and INFM-CRS SOFT, Unita´ di Roma 1
M. Diociaiuti Istituto Superiore di Sanita´ Viale Regina Elena 205, I-00185- Roma, Italy ReceiVed: March 2, 2007; In Final Form: June 12, 2007
We have investigated the effect of a cationic lipid [DOTAP] on both the thermotropic phase behavior and the structural organization of aqueous dispersions of dipalmitoyl-phosphatidylcholine [DPPC] by means of highsensitivity differential scanning calorimetry and dynamic light scattering measurements. We find that the incorporation of increasing quantities of DOTAP progressively reduces the temperature and the enthalpy of the gel-to-liquid crystalline transition. We are further showing that, in mixed DOTAP-DPPC systems, the reduction of the phase transition temperature is accompanied by a reduction of the average size of the structures present in the aqueous mixtures, whatever the DOTAP concentration is. These results, which extend a previous investigation by Campbell et al. (Campbell, R. B.; Balasubramanian, S. V.; Straubinger, R. M.; Biochim. Biosphys. Acta 2001, 27, 1512.) limited to a DOTAP concentration below 20 mol %, confirm that the insertion of cationic head groups in zwitterionic phosphatidylcholine bilayers facilitates the formation of stable, relatively small, unilamellar vesicles. This self-assembling restructuring from an aqueous multilamellar structure toward a liposomal phase is favored by decreasing the phospholipid phase transition temperature and by increasing the temperature of the system. This reduction of the average size and the appearance of a stable liposomal phase is also promoted by a heating and cooling thermal treatment.
1. Introduction Because of their entrapping ability, liposomes, formed when phospholipids are dispersed in an aqueous medium, represent potent drug carriers to specific tissues and cells.1-8 However, despite the current effort in improving lipidic vectors with minimal toxicity, the molecular mechanisms of liposome-cell interactions are not completely elucidated yet, and moreover little is known about the parameters influencing the transfection efficiency. Among these parameters are the composition of the liposomes, their surface charge density, and their size, in addition to the transfection protocol. In particular, size and charge should play a key role in drug delivery9,10 and have a strong influence on dosage, targeting, and rate of clearance from the body. A great number and a wide variety of synthetic vectors have been prepared and transfection efficiency has been evaluated both in Vitro studies and in clinical trials. Although some positive and interesting results have been obtained to date, the crucial problem remains a more efficient formulation of the carriers.11 Mixed liposomes composed of cationic and zwitterionic lipids have been extensively studied by means of a variety of * To whom correspondence cesare.cametti@ roma1.infn.it
should
be
addressed.
E-mail:
experimental techniques, because of their promising therapeutic interest owing to their certain drug entrapping and delivering properties.12 In this work, we present a study of the effects of a cationic lipid [DOTAP] on the thermotropic phase behavior and on the structural organization of DPPC bilayers, on the basis of a combined use of differential scanning calorimetry [DSC] and dynamic light scattering [DLS] techniques. DSC is a sensitive and nonperturbing technique extensively used in the investigation of thermotropic phase transitions, both in model lipid bilayers and in biological membranes.13 DLS furnishes the correlation function of the intensity-intensity scattered light from the lipidic structure as a whole and, consequently, is able to probe the hydrodynamic properties of these structures (average size and size distribution).14 These two techniques allow us to evaluate the size and the membrane domain structure of both multilamellar DOTAP-DPPC mixed structures and spontaneously (or thermally induced) DOTAP-DPPC mixed liposomes. Among cationic lipids, DOTAP, which is a two-chained amphiphile whose acyl chains are linked to the propyl ammonium group, is one of the most popular lipids available for transfection purposes. We selected zwitterionic DPPC and cationic DOTAP lipids since their mixture allows the modulation of the surface charge density of the resulting structure and,
10.1021/jp071722g CCC: $37.00 © 2007 American Chemical Society Published on Web 07/31/2007
Properties of Mixed DOTAP-DPPC Bilayer Membranes
Figure 1. The chemical structure of the two lipids employed: (A) DOTAP; (B) DPPC.
moreover, PC-containing liposomes are known to be rather stable and suitable for drug delivery.15 The self-assembling is a hierarchical process where aggregates can form at different levels of complexity, giving rise to various structures that involve large-scale conformational complexity with an intrinsic structural heterogeneity. Moreover, these structures undergo a structural reorganization from low-level to high-level of a self-assembly hierarchy. Because of the complexity of the self-assembling process, it is reasonable to expect heterogeneous aggregates that manifest different structural features on a macroscopic scale, which determine the final efficiency in gene therapy approaches. One objective of this investigation was to provide information about the interactions between DOTAP and DPPC to evaluate how the presence of a cationic lipid might affect the stability and the structural organization of zwitterionic lipids in the bilayers. This study extends a previous investigation by Campbell et al.16 to a wider range of DOTAP concentrations, up to 80 mol %. Our results show that DOTAP exerts an efficient function in enhancing the colloidal stability of the complexes and may markedly contribute to the formation of stable, small size aggregates that resemble unilamellar vesicular structures. Moreover, DOTAP induces a marked broadening of the main phase transition, from a gel-like to a liquid-like structure, increasing the fluidity of the membrane and favoring processes relevant to medical applications of liposomes. 2. Materials and Methods Materials. The zwitterionic lipid, dipalmitoylphosphatidylcholine (DPPC, C40H80NO8P), was purchased from Sigma Chemical Co. (St. Louis, Mo). The cationic lipid dioleoyltrimethylammonium propane (DOTAP, C42H80NO4Cl) was obtained from Avanti Polar Lipids Inc. (Alabaster, AL). All lipids, whose purities were >98%, were used without further purification. The chemical structures of the two lipids employed are shown in Figure 1. Samples were prepared in clean glass tubes by mixing appropriate weights of DOTAP and DPPC lipids to obtain the required lipid molar ratio XDOTAP ) NDOTAP/ (NDOTAP + NDPPC) with NDOTAP and NDPPC as molar concentration of DOTAP and DPPC, respectively. We investigated DOTAP-DPPC mixed systems as the DOTAP concentration is progressively increased from 0 (pure DPPC) up to 80 mol %. Liposome Preparation. DPPC and DOTAP-DPPC aqueous mixtures were prepared by lipid-film hydration. Lipid mixtures were dissolved in methanol-chloroform (1:1 vol/vol) and, after 3 h vacuum evaporation of the organic solvents, the resulting lipid film was rehydrated with Milli-Q quality water (electrical conductivity less than σ ) 10-6 Ω-1 cm-1, at room temperature), to the final concentration of 5 mg/mL. The rehydration
J. Phys. Chem. B, Vol. 111, No. 33, 2007 10033 process was carried out for 1 h at a temperature of 47 °C, above the main phase transition temperature of pure DPPC (Tm = 42 °C) and of DOTAP/DPPC mixtures. Differential Scanning Calorimetry [DSC]. The differential scanning calorimetry [DSC] experiments were performed on a Micro-DSC II Setaram calorimeter. This instrument, calibrated with indium standard, has a resolution, in both heating and cooling cycles, higher than 2 µW. The thermograms were recorded in the range from -10 to 50 °C, using a heating rate, dT/dt, of 0.05 °C/min. DSC technique17 measures the heat flow, dQ/dt, from which -1 -1 the excess heat capacity is obtained (Cex p ) dQ/dt(dT/dt) n , with n ) number of moles). A mass of 0.830 g of the sample and of the reference (water) was inserted in the sample and in the reference cells, respectively. The weights of the sample and of the reference cells were always matched. The excess power versus temperature scan for the lipid mixtures was obtained by subtracting the power input of the thermal scan of solvent versus solvent (water versus water) from the power input scan of the solution versus solvent. For each sample, the peak height, its width, and the enthalpy were measured. The transition temperature refers to the maximum of the excess heat capacity peak. Enthalpies, ∆H, were determined from the area under the transition thermal profile18-20
∆H )
∫TT C exp dT f
(1)
o
The results were largely independent of DSC scan rate. The reproducibility of the DSC data was checked by measuring each sample more than three times. At some selected DOTAP concentrations, DOTAP-DPPC mixtures were investigated by means of DSC measurements during iterated heating and cooling cycles. The resulting thermograms do not differ appreciably. Dynamic Light Scattering [DLS]. We employed dynamic light scattering [DLS] measurements to characterize the size and size distribution of pure DPPC, DOTAP and mixed DOTAP-DPPC aqueous structures. Each sample was heated from 10 to 50 °C at intervals of 1 °C, after 10 min of thermalization. An optical fiber probe (Brookhaven FOQELS) has been employed for all DLS measurements, in conjunction with a Brookhaven 9000 AT logarithmic correlator. In this fiberoptic probe, the Gaussian laser beam transmitted by a monomode optical fiber illuminates the scattering volume and a second fiber, positioned at a fixed angle of 137.5°, collects the scattered light. The main advantage of this apparatus, when compared to more traditional ones, consists in its inherent larger insensitiveness to multiple scattering effects.21 DLS technique measures the normalized (second-order) time autocorrelation function g(2)(τ) of the intensity of the scattered light which is related to the normalized (first-order) autocorrelation function g(1)(τ) by the Siegert relationship22
g(2)(τ) ) 1 + β|g(1)(τ)|2
(2)
where β is a spatial coherence factor dependent on the geometry of the detection system. In a dilute suspension of monodisperse particles, g(1)(τ) decays exponentially with a decay rate Γ ) q2D, where q is the magnitude of the scattering wavevector and D the translational diffusion coefficient which is related to the hydrodynamic radius RH through the Stokes-Einstein relationship23
RH )
KBT 6πηD
(3)
10034 J. Phys. Chem. B, Vol. 111, No. 33, 2007
Cinelli et al.
where KBT is the thermal energy and η the viscosity of the aqueous phase. In a polydisperse system, g(1)(τ) can be expressed as
g(1)(τ) )
∫0∞ G(Γ) exp(-Γτ) dΓ
(4)
where G(Γ) is the distribution function for decay rate. However, for moderately polydisperse systems the method of cumulants24 can be applied. The autocorrelation function g(1)(τ) can be expanded in series as
1 g(1)(τ) ) exp -〈Γ〉τ + µ2τ2 + ... 2
(
)
(5)
where 〈Γ〉 ) Dq2 is the mean of Γ and µ2 is the variance of the distribution. The index of polydispersity is given by Q ) xµ/Γ. Transmission Electron Microscopy [TEM]. Transmission electron microscopy [TEM] measurements were carried out by means of a ZEISS 902 microscope operating at 80 kV, equipped with an electron energy loss filter (EF-TEM). A droplet of the suspension of the lipid mixture was deposited onto 300-mesh copper grids (for electron microscopy) and covered with a very thin amorphous carbon film (about 20 nm). The liquid excess was removed by placing the grid on a filter paper. The staining solution, filtered by polycarbonate 0.2 µm filters, consists of 2% (w/w) of phosphotungstic acid (PTA) in a buffered aqueous solution at pH ) 7.3 (NaOH). The image acquisition was performed by a digital charge-coupled device camera model PROSCAN HSC2 (1 k × 1 k pixels), thermostated by a Peltier cooler. Image analysis was carried out by a digital analyzer SIS 3.0, which allows the contrast and sharpness of the acquired images to be enhanced and morphological quantification and statistics to be performed.25 The overall attainable resolution can be evaluated on the order of 2 nm. 3. Results and Discussion Differential Scanning Calorimetry. Differential scanning calorimetry [DSC] measurements have been carried out to investigate the thermotropic phase behavior of pure DPPC and mixed DOTAP-DPPC spontaneously forming aqueous mixtures. The overall baseline-corrected DSC scans of DOTAPDPPC complexes, at varying DOTAP concentrations up to 80 mol %, are shown in Figure 2. The data are stacked as a function of the increasing mole fraction of DOTAP. Each measured thermogram is normalized against the total mole number of the two lipids in the samples. In the absence of DOTAP, DPPC heating scan shows two sharp endothermic peaks, centered at 33.5 and 41.2 °C, which correspond to the pretransition (lamellar gel phase to rippled gel phase, Lβ′ f Pβ′) and to the main transition (rippled gel phase to lamellar liquid-crystalline phase, Pβ′ f LR), respectively. These values are in good agreement with literature data.26 By increasing the DOTAP mole fraction, the main transition temperature shifts gradually to lower values, and, at the same time, a broadening of DPPC peak is observed (bottom panel of Figure 2). The DSC scans of mixed DOTAP-DPPC systems exhibit a small enthalpic pretransition at low DOTAP concentrations up to about 16.1 mol % and this pretransition is progressively incorporated in the main transition peak at DOTAP concentrations larger than 20 mol %. The pretransition peaks are shown in the bottom panel of Figure 2. The effect of DOTAP on the main phase transition of DPPC deserves a further comment. The calorimetric data clearly show
Figure 2. Upper panel: Calorimetric heating scans of DOTAP-DPPC mixed aqueous suspensions, at the concentration of 5 mg/mL. The thermograms shown are acquired at the DOTAP molar concentration XDOTAP ) (NDOTAP/NDOTAP + NDPPC) indicated. Curves have been shifted along the y-axis to avoid overlap and to facilitate visibility. The thermograms for DOTAP-DPPC mixtures at concentrations from 16.1 to 80 mol % were plotted on the y-axis twofold expanded relative to all others. Bottom panel: enlarged region of the calorimetric scans, showing the evolution of the pretransition temperature at low DOTAP mole fractions, up to 16.1 mol %.
that, at moderate DOTAP concentrations up to about 20 mol %, DPPC bilayers exhibit asymmetric thermograms, indicating that at least two different thermal events overlap. With the DOTAP increase, the DSC endotherm consists of a sharp component that is progressively reduced in temperature and area (A2) under the transition thermal profile and a broad component that decreases in temperature but increases in area (A1). With the increase of DOTAP concentration, the sharp component reduces as the broad component grows. The deconvolution analysis of the DSC thermograms (a typical example is shown in Figure 3) shows that, of these two components, one is considerably sharper and its peak temperature decreases slightly than the other. Data presented in Figure 3 are an example of the curve-fitting analysis of the DSC thermograms in the low DOTAP concentration range. For all the other DOTAP concentrations investigated, the system behaves similarly. These results indicate that the thermograms can be simulated by the summation of two components. This complex behavior, resolved in sharp and broad components, can be attributed to the differential melting of DOTAPpoor and DOTAP-rich lipid domains, respectively. The sharp component results from the melting of the DOTAP-poor and the broad component from the melting of the DOTAP-rich membrane domain. The presence of multicomponent DSC endotherms without the need of assuming a two-state transition
Properties of Mixed DOTAP-DPPC Bilayer Membranes
Figure 3. Representative decomposed endotherm of the main phase transition of DOTAP-DPPC 6.1 mol %. The lower melting curve represents the broad component (with area A1) and the higher melting curve the sharp component (with area A2). Decompositions of the other systems at different DOTAP concentrations behave similarly (data not shown). The inset shows the effect of the increasing amount of DOTAP on the sharp component area A2 to the total area (A1 + A2) ratio.
J. Phys. Chem. B, Vol. 111, No. 33, 2007 10035 DOTAP have been summarized in the plot of Figure 4 where the main thermodynamic parameters (the main transition temperature Tm, the transition temperature of the sharp and broad component in the low DOTAP concentration range, and the enthalpy associated with the whole peak) are shown as a function of DOTAP concentration. The temperature-composition diagram can be described in two segments, with DOTAP content less than or greater than about 20 mol %. For DOTAP concentrations of 0-20 mol % in DOTAP-DPPC bilayers, with increasing temperature, we cross the pretransition (Lβ′ f Pβ′), which persists up to 18-20 mol % DOTAP. For DOTAP concentration from 20 to 80 mol %, the endothermic peak of the main phase transition is composed by only one progressively broader unresolved component. The ratio of the area of the sharp component (A2) to the total peak area (A1 + A2) approaches to zero (inset of Figure 3). Although the mixed system shows miscibility in both phases, its behavior deviated from ideality, as shown in Figure 4. The ideal phase diagram can be obtained from the relationships30
X(l) ) (1 - R)/(β - R) X(s) ) βX(l)
(6)
where X is the DOTAP mole fraction and the superscripts l and s refer to the liquid and solid curves, respectively. Here, R and β are defined as
R ) exp
{ {
β ) exp
Figure 4. Phase diagram of DOTAP-DPPC mixtures derived from differential scanning calorimetry measurements. Liposomes contained varying concentrations of DOTAP from 0 to 80 mol %. The phase transition temperature Tm (9) of the main transition of DPPC and DOTAP-DPPC mixture are shown as a function of DOTAP concentration. The upper and lower full lines represent the ideal phase diagram calculated according to eq 6. The pretransition temperature (b) is clearly evidenced up to a DOTAP concentration of about 20 mol %. Upper inset shows in detail the Tm values obtained from the sharp (O) and broad (0) components of the main phase transition and the pretransition temperature (b). Bottom inset shows the overall main transition chain melting enthalpy, ∆H, as a function of the DOTAP mole fraction.
nor of applying curve-fitting routines can be also gained from a thermogram-subtraction technique, as suggested by McMullen et al.27 A similar thermal deconvolution has been previously observed by McMullen et al.27 in the interaction of cholesterol with DPPC and more recently by Mannock et al.28 in the interaction of lanosterol and cholesterol with DPPC. For higher DOTAP content mixtures, from 20 to 80 mol %, the progressive shift of the main transition temperature to lower values prevents clear observation of the pretransition temperature. The gradual incorporation of the pretransition in DSC heating scans as a function of DOTAP concentration is shown in Figure 4. It is worth noting that, for mixtures with a DOTAP concentration up to about 16.1 mol %, the pretransition temperature remains constant to approximately the value of 33.5 °C. The formation of DOTAP-rich domain in the DPPC matrix has been previously evidenced by surface pressure measurements (Langmuir isotherms) that clearly demonstrate deviation from ideal mixing.29 The overall calorimetric data of aqueous multilamellar dispersions of DPPC containing up to 80 mol %
( (
∆HDOTAP 1 1 R T TmDOTAP ∆HDPPC 1 1 R T TmDPPC
)}
)}
(7)
(8)
where ∆HDOTAP and ∆HDPPC are the transition enthalpies of the pure DOTAP and pure DPPC lipids and TmDOTAP and TmDPPC their absolute temperature transitions. The ideal behavior has been calculated assuming ∆HDPPC ) 35 kJ/mol, TmDPPC ) 41.9 °C, ∆HDOTAP ) 18 kJ/mol and TmDOTAP ) -8 °C31 Deviation of the phase diagram of DOTAP-DPPC mixtures from an ideal phase diagram could be, at least partially, justified by the dissimilarity between tails of the two lipids. The DPPC is a saturated lipid with a 16 carbon chain, while the DOTAP is an unsaturated lipid with an 18 carbon chain. The different chain lengths affects the transition temperature and the peak width. Mabrey et al.32 have analyzed different lipids and have observed that the phase diagram of a lipid mixture, with the increase of the difference in the carbon tail length, progressively deviates from an ideal phase diagram. Dynamic Light Scattering. Dynamic light scattering [DLS] measurements (Figures 5 and 6) were used to investigate the effect of temperature and DOTAP concentration on the size and size distribution of pure DPPC and mixed DOTAP-DPPC spontaneously forming aqueous mixtures. For all the lipid mixtures investigated, the increase of the temperature reduced the mean average size of DPPC multilamellar aggregates, the greatest effect being observed in the low DOTAP concentration, both above and below the main phase transition temperature. Moreover, the most relevant aggregate size reduction occurs at temperatures close to the mean phase transition temperatures. Figure 5 shows, in the case of DOTAP-DPPC mixture 4.2 mol %, the almost complete correspondence of the temperatures at which the size reduction occurs and at which the main phase transition manifests. The thermotropic behavior and the structural organization of the DOTAP-DPPC mixture was in part previously investigated
10036 J. Phys. Chem. B, Vol. 111, No. 33, 2007
Figure 5. Influence of temperature on the average size and on the thermotropic phase behavior of DOTAP-DPPC aqueous mixture 4.2 mol %. Here, we show the correspondence between the temperature at which the reduction of the average size occurs and the temperature of the main phase transition.
Figure 6. Effect of temperature on the average size of multilamellar DOTAP-DPPC aqueous mixture prepared at varying DOTAP to DPPC molar ratio: (9) 4.2 mol %; (4) 8.1 mol %; ( left-pointing solid triangle), 16.1 mol %; (O) 28.2 mol %; (b) 41.2 mol %; (left-pointing open triangle) 80 mol %. The temperature was varied from 10 to 50 °C, steps of 1 °C after 10 min of thermalization. The inset shows the effect of DOTAP concentration on the average size of the aggregates at different temperatures: (9) T ) 10 °C; (b) T ) 20 °C; (2) T ) 30 °C; (1) T ) 40 °C.
by Campbell et al.,16 who took into consideration only the low DOTAP concentration range (up to 20 mol %). We have extended this concentration range up to 80 mol % and, as far as the average sizes are concerned, a typical scenario is summarized in Figure 6. We show the average size of the mixed DOTAP-DPPC liposomal structures at varying DOTAP concentrations during the heating run starting from about 10 up to 50 °C. The average size has been derived from the first cumulant of the series expansion of the intensity-intensity correlation function C(τ). In the low DOTAP concentration range, up to 20 mol %, the mixed DOTAP-DPPC structures, starting from average size values relatively high (of the order of 2.5 µm), progressively reduce in size to values of the order of 800 nm as the temperature is increased from 10 to 50 °C. Moreover, this size reduction is particularly marked, as already abovementioned, by crossing the main transition temperature, giving support to the role played by the membrane fluidity in the reduction of the vesicle size. As pointed out by Campbell et al.,16 alterations induced by DOTAP at the membrane interface could facilitate the decrease of the bilayer radius of curvature as a result of phospholipid-cationic head group interactions. A qualitatively different behavior is observed in the high DOTAP concentration range (from 28.1 to 80 mol %), where the structure, starting from relatively lower values (of the order of 800 nm) reduces its average size to values of the order of 100-200 nm, without any marked effect, from crossing the main transition temperature.
Cinelli et al.
Figure 7. Effect of heating and cooling cycles on the sample DOTAPDPPC 8.1 mol %, during different thermal cycles: (9) first heating run (10-50 °C); (4) first cooling run (50-10 °C); (b) second heating run; (left-pointing triangle) second cooling run; ([) third heating run; (0) third cooling run. The sample was heated (cooled) from 10 to 50 °C (from 50 to 10 °C) at 1 °C steps, after 10 min of thermalization. The arrows indicate the heating and cooling cycles.
This overall behavior reflects the whole thermotropic behavior where the progressive shift of the main transition temperature is accompanied by a progressive reduction of the average size of the aggregates and moreover, to the broadening of the thermal peak, it corresponds a more or less pronounced size reduction. However, the effect of the temperature is more complex than the data shown in Figure 6 suggest, and this is clearly evidenced when, for example, the same sample undergoes a thermal treatment consisting of sequential heating and cooling runs. In this case, substantial differences between the different thermal cycles were observed. As an example, in Figure 7, we show the behavior of DOTAP-DPPC mixed aggregates (8.1 mol %) during a series of subsequent heating and cooling cycles (in the range from 10 to 50 °C and viceversa). As can be seen, starting from an average diameter of the order of 2.5 µm, the mixture undergoes a structural rearrangement at the end of the third thermal cycle resulting in a vesicular structure with hydrodynamic diameter of about 100-150 nm. These structures are stable in time and, from this point on, further thermal cycles do not induce further changes in their average size. It is worth nothing that this thermal process is characterized by an intermediate state when structures of the order of 1 µm are formed, during the cooling treatment, after the first heating run (Figure 7). These intermediate structures further evolve toward the small-sized, stable, structures during the third heating run. All the DOTAP-DPPC mixtures investigated, with DOTAP concentration up to 16.1 mol %, behave similarly. Moreover, the influence of the gel-to-liquid-phase transition of the lipid chains on the average size of the aggregates must be parted-out. At the beginning of the process, during the first cycle, crossing of the main transition temperature results in a marked decrease of the average size (from 1.7 µm to 700 nm). During the second heating cycle, this decrease is still detectable, the average size decreasing from 1 µm to 600 nm. At the beginning of the third heating run, aggregates have reached their minimum size that remains constant over the whole temperature range investigated, and crossing of the main transition temperature does not induce any further size reduction. The temperature-induced decrease of the average size is also evidenced by the shift of the correlation function of the scattered light intensity toward smaller correlation times, as shown in Figure 8. Here, we compare the correlograms of DOTAPDPPC mixtures (8.1 mol %) at the beginning (10 °C) and at the end (50 °C) of the first and the third heating cycle, respectively. As can be seen, the correlation functions maintain their usual shape, but the average relaxation time is considerably
Properties of Mixed DOTAP-DPPC Bilayer Membranes
J. Phys. Chem. B, Vol. 111, No. 33, 2007 10037
Figure 8. Correlograms of the DLS measurements of DOTAP-DPPC aqueous mixtures (8.1 mol %) at the beginning and at the end of the first and third cycle, respectively: (continuous lines) correlation function at the beginning (T ) 10 °C) of the first and third heating run; (dotted lines) correlation function at the end (T ) 50 °C) of the first and third heating run.
Figure 9. Effect of heating and cooling thermal cycles on pure DOTAP aqueous mixtures: (9) first heating run (10-50 °C); (b) first cooling run (50-10 °C); (4) second heating run (10-50 °C). The data concerning the further cooling and heating runs are not shown for clarity of presentation. However, they overlap the values of the second heating run. The sample has been heated (cooled) from 10 to 50 °C (from 50 to 10 °C) at 1 °C steps, after 10 min of thermalization. Insets show the size distribution of the resulting aggregates during the thermal cycles obtained from a statistical analysis of the TEM images: (A) first heating run, at the temperature of 25 °C; (B) after the third heating run, at the temperature of 25 °C.
reduced after the first heating cycle and reaches its minimum value at the end of the third heating cycle. Finally, we investigated the effect of thermal (heating and cooling cycles) treatment on the average size of pure DOTAP and pure DPPC temperature-induced structures, starting from an aqueous multilamellar mixture. In Figure 9, we show the progressive size reduction of pure DOTAP aqueous mixture from a value of about 650 nm at 10 °C to the value of about 100 nm at the end of the first heating run. This value is maintained constant during the subsequent thermal cycles, suggesting that stable structures are formed. Their average size recalls that of unilamellar liposomes that are obtained from the aqueous mixture (at a constant temperature) after sonication or pressure extrusion procedure. Moreover, once reaching their minimal size, the structures remain stable over a long period of time (weeks or months). Further evidence for the above stated phenomenology is gained from electron microscopy measurements [TEM] that furnish a direct visual inspection of the size of the aggregates. These images, collected for a pure DOTAP aqueous mixture (at a concentration of 5 mg/mL) before and after the thermal treatment confirm the results obtained from DLS measurements
Figure 10. TEM images of DOTAP aqueous mixture (lipid concentration 0.1 mg/mL) before the thermal cycles (upper panel) and after the thermal cycles (bottom panel). The insets show a typical large aggregate and smaller aggregates, respectively, as a consequence of the thermal treatment. The bar is 100 nm.
Figure 11. Effect of heating and cooling thermal cycles on pure DPPC aqueous mixture. (9) heating runs (10-50 °C); (O) cooling runs (5010 °C). The cycles have been made continuously, the sample being heated (cooled) from 10 to 50 °C (from 50 to 10 °C) at 1 °C steps, after 10 min of thermalization. For sake of clarity, only the first, third, and fourth cycles are shown.
(see, for example Figure 10. A typical example is shown in the inset of Figure 9, where we illustrate the size distribution of the DOTAP mixtures both before and after a thermal cycle obtained from a statistical analysis of more than one hundred aggregates collected in different TEM images. The agreement is quite satisfactory, taking into account that the size distribution
10038 J. Phys. Chem. B, Vol. 111, No. 33, 2007
Cinelli et al. region of the phase diagram. This more fluid phase should favor the formation of small aggregates, resembling in size single liposomes. The chemical structure of the two lipids allows a spontaneous (or temperature induced) vesicular self-assembly. For a mixed lipid structure, the packing factor P, related to the geometrical configuration assumed by the self-assembling aggregates, can be written as34
Figure 12. The scattered light intensity correlation functions of pure DPPC aggregates (at the concentration of 1 mg/mL) as a function of the correlation time τ, after the thermal treatment described in Figure 7. Measurements have been taken at the temperature of 25 °C. As the time goes on (up to 50 h from the beginning), the correlation function shifts toward higher correlation time, indicating an increase of the average size of the aggregates. The inset shows the increase of the average size of the aggregates as a function of time, calculated from the first cumulant of the correlation function.
depends on the method used, since the distributions are weighted according to the measurement technique employed.33 In fact, the dynamic light scattering method yields a distribution shifted toward larger-sized objects and is generally smoother. However, in the present case, both the two techniques yield the same qualitative results, with the presence of a smaller-sized structure, with a narrower-sized distribution, after thermal cycles. A slightly different behavior is observed in the case of pure DPPC aqueous mixtures, subjected to the same thermal heating and cooling treatment. The behavior of pure DPPC is similar to the one of mixtures of pure DOTAP. In this case, starting from objects 2-3 µm in size, after the fourth heating cycle, aggregates reach a relatively small size (of the order of 80100 nm), as can be seen in Figure 11. However, these structures are intrinsically unstable, being subject to a further evolution toward larger average size as time goes on. Figure 12 enforces this statement, showing the evolution of the scattered light intensity correlation functions of pure DPPC aggregates over time, at the temperature of 25 °C, after the iterated thermal cycles. As can be seen, the shift toward higher correlation times and the curve-shaped broadening are characteristic of a continuous increase of the typical average size of the aggregates, indicating a further restructuring of the DPPC aqueous mixture, starting from the relatively small structures induced by the thermal treatment. It is worth noting that the transition between these larger morphologies is relatively slow (taking place within several days) and can be hindered by external stress, for example thermal treatment. This phenomenology does not occur in cationic lipids (DOTAP), where the surface charge once vesicular structures are formed prevents further aggregation nor in mixed cationic-zwitterionic lipid mixtures (DOTAP-DPPC mixtures). The sizes at the end of the heating run are those typical of unilamellar vesicles obtained from aqueous lipidic mixtures by means of pressure extrusion or sonication. However, we have also observed that the thermotropic behavior of DOTAP-DPPC mixtures after sonication markedly differs from that of untreated samples (data not shown). In this case, thermograms display a series of peaks strongly dependent on the thermal heating and cooling cycles. This finding gives evidence to a structural reorganization of the multilamellar mixture toward a vesicular structure of smaller size. In this case, at these relatively high DOTAP concentrations, the DPPC bilayer undergoes a phase transition temperature shifted toward lower temperatures, approaching a homogeneous liquid-crystalline phase in a wide
VDPPCXDPPC + VDOTAPXDOTAP P)2 (a0DPPCXDPPC + a0DOTAPXDOTAP)lc
(9)
where Xj is the molar fraction of the lipid j in the assembly, Vj is the volume occupied by an alkyl chain whose maximum length is lc (assumed the same for the two lipids), and a0 the area of the lipid polar head. The value of P for the DOTAPDPPC mixtures, calculated assuming a0DPPC ) 52.3 Å2, a0DOTAP ) 65 Å2, V ) (27.4 + 26.9nc) Å3, lc ) (1.54 + 1.26nc) Å with nc ) 16 for DPPC and nc ) 18 for DOTAP,35 results between 0.40 and 0.33, in agreement with the value predicted for a vesicular geometry. Thus the mixture of these two lipids is expected to spontaneously form vesicles. The effect of thermal treatment on size of the DOTAP-DPPC mixtures could be related with the spontaneous formation of stable unilamellar vesicles, even if this mechanism is not yet completely understood.36 Lipid mixtures spontaneously evolve by self-assembly to form relatively small vesicles thanks to an entropic gain. Therefore, the temperature could simply favor and speed up the process that would have taken place in time without any input of energy, be it mechanical, chemical, or electrochemical. It is well-known37 that the behavior of singlecomponent liposomes is different and the spontaneous formation of small unilamellar vesicles is not favored, the driving force for the self-assembly being the reduction of the unfavorable hydrocarbon-water interface between lipid tails and aqueous solvent. Because of geometrical packing constraints, this free energy contribution favors the formation of larger aggregates. This is only partially true in the case of pure DOTAP, where the thermal cycles induce the formation of small and stable unilamellar vesicles evidencing the important role of the surface charge for the stability of the vesicles.38,39 Thermal cycles, also in this case, induce a structural rearrangement, the stability of which depends on the chemical properties of the lipids involved. 4. Conclusions The combined use of differential scanning calorimetry and dynamic light scattering in the investigation of lipid mixtures results in a powerful tool to analyze the thermotropic phase behavior and the structural organization of aggregates at different levels of complexity. We have observed the role of cationic lipid DOTAP on the reduction of average size of DPPC aggregates. The average size decrease occurs both with the increase of DOTAP concentration in the mixed DOTAP-DPPC systems and with a thermal treatment consisting of subsequent heating and cooling cycles. During the temperature cycles, this size reduction is particularly marked by crossing the main transition temperature, when lipids pass through the gel to the liquid-crystalline phase. The mixed DOTAP-DPPC aggregates, obtained after the thermal treatment, have qualitatively both the same average size and the stability in time of the structures obtained after a sonication procedure. The thermotropic phase behavior of these systems was previously investigated up to 20 mol % DOTAP by Campbell
Properties of Mixed DOTAP-DPPC Bilayer Membranes et al.16 We have extended the DOTAP concentration up to 80 mol % and have defined in detail the phase diagram on the basis of an accurate deconvolution of the DSC theromgrams as composed of two sharp and broad components ascribed to the different melting of DOTAP-poor and DOTAP-rich lipid domains. We find that inclusion of DOTAP up to 80 mol % decreases the average size of spontaneously forming aggregates, decreases the particle heterogeneity, and moreover increases the time stability. In particular in the low DOTAP concentration range, the thermotropic behavior reflects, in a very close way, the structural characteristics of the aggregates. In fact the sharp component is progressively reduced, and simultaneously the average size decreases. As the sharp component vanishes (at concentrations larger that 20 mol %) the aggregate size tends toward a constant value in the whole temperature range investigated. DOTAP-containing liposomes have been studied most extensively for DNA delivery. The possibility of reducing the average size or increasing of the membrane fluidity through the main transition peak broadening (as observed by DSC measurements) might be an important parameter in drug delivery applications. References and Notes (1) Lasic, D. D. Liposomes in Gene DeliVery; Boca Raton, FL, 1997. (2) Lasic, D. D.; Templeton, N. S. New directions in liposome gene delivery. Mol. Biotechnol. 1999, 11, 175. (3) Forssen, E.; Willis, M. Ligand-targeted liposomes. AdV. Drug. DeliV. 1998, 29, 249. (4) Chonn, A.; Cullis, P. R. Recent advances in liposome technologies and their applications for systemic gene delivery. AdV. Drug. DeliV. ReV. 1998, 30, 73. (5) Maurer, N.; Mori, A.; Palmer, L.; Monck, M. A.; Mok, K. W. C.; Mui, B.; Akhong, Q. F.; Cullis, P. R. Lipid-based systems for the intracellular delivery of genetic drugs. Mol. Membr. Biol. 1999, 16, 129. (6) Langner, M.; Kra, T. E. Liposome-based drug delivery systems. Pol. J. Pharmacol. 1995, 51, 211. (7) Langner, M. Effect of liposome molecular composition on its ability to carry drugs. Pol. J. Pharmacol. 2000, 22, 3. (8) Ulrich, A. S. Biophysical aspects of using liposomes as delivery vehicles. Biosci. Rep. 2002, 52, 129. (9) Koltover, I.; Salditt, T.; Radler, J. O.; Safinya, C. R. An inverted hexagonal phase of cationic liposome-DNA complexes related to DNA release and delivery. Science 1998, 281, 78. (10) Kao, Y. J.; Juliano, R. L. Interaction of liposomes with the reticuloendothelial system. Effects of reticuloendothelial blockade on the clearance of large unilamellar vesicles. Biochim. Biophys. Acta 1981, 677, 453. (11) Martin, B.; Sainlos, S.; Aissaoui, A.; Oudrhiri, N.; Hauchecorne, M.; Vigneron, J.-P.; Lehn, J.-P.; Lehn, P. The design of cationic lipids for gene delivery. Curr. Pharm. Design 2005, 11, 375. (12) Kazakov, S.; Levon, K. Liposome-nanogel structures for future pharmacological applications. Curr. Pharm. Des. 2006, 36, 4713. (13) Elhaney, R. N. The use of differential scanning calorimetry and differential thermal analysis in studies of model and biological membranes. Chem. Phys. Lipids 1982, 30, 229. (14) Schmitz, K. S. An Introduction to Dynamic Light Scattering by Macromolecules; Academic Press: San Diego, CA, 1990. (15) Chonm, A.; Cullis, P. R. Recent advances in liposome technology and their applications for systemic gene delivery. AdV. Drug Del. ReV. 1998, 30, 73.
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