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The effect of tocopherol on the structure and permeability of phosphatidylcholine liposomes
Journal of Controlled Release 160 (2012) 158–163
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The effect of tocopherol on the structure and permeability of phosphatidylcholine liposomes Peter J. Quinn ⁎ Department of Biochemistry, King's College London, 150 Stamford Street, London SE1 9NH, UK
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Article history: Received 29 July 2011 Accepted 21 December 2011 Available online 30 December 2011 Keywords: Phospholipid α-tocopherol X-ray diffraction Bilayer structure Permeability of liposomes
a b s t r a c t There are numerous phospholipid formulations that incorporate α-tocopherol as a stabilizing agent but there are few studies of the effect of α-tocopherol on phospholipid structure and bilayer permeability. This study uses synchrotron X-ray powder diffraction methods to investigate how α-tocopherol changes the structure of distearoylphosphatidylcholines bilayers. Increasing proportions of α-tocopherol up to 20 mol% induces ripple structures in the bilayers. Two types of ripple structure are produced which are seen in electron micrographs of freeze-fracture replicas with periodicities of 16 and 12 nm, respectively. The stoichiometry of phospholipid: α-tocopherol in the ripple structures at 37 °C is 8:1. The presence of α-tocopherol tends to reduce the angle of tilt of the hydrocarbon chains of the phospholipid in the gel phase from about 34° to the bilayer normal at 20 °C into a more vertical orientation. Increasing proportions of α-tocopherol progressively decrease the temperature of the gel to liquid-crystal phase transition of the phospholipid. The presence of up to 20 mol% α-tocopherol in 1-palmitoyl-2-oleoyl-phosphocholine inhibits leakage of phenol red dye from liposomes. The effect of 7 mol% α-tocopherol on leakage was compared with phospholipid liposomes containing 50 mol% cholesterol. The cholesterol-containing liposomes inhibited leakage to a greater extent than the vesicles incorporating α-tocopherol but the effect of α-tocopherol at equivalent molar proportions was comparable to cholesterol. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Tocopherols have been incorporated into phospholipid liposomes as a method for delivery of the vitamin to cells and tissues [1] and as a component of liposomal formulations to act as a membrane stabilizing agent [2,3]. Thus inclusion of α-tocopherol in liposomal dispersions has long been known to reduce lipid oxidation that result in changes in bilayer permeability and to prolong storage life [4]. Oxidative damage to tissues such as lung exposed to 2-chloroethyl ethyl sulphide has been shown to be ameliorated by intratracheally administered liposomes of dipalmitoylphosphatidylcholine containing α-tocopherol [5]. Furthermore, intravenous administration of dipalmitoylphosphatidylcholine liposomes containing 30 mol% αtocopherol was reported to protect against lipopolysaccharideinduced liver injury in rats [6]. Controlled release formulations of pH-sensitive liposomes comprised of palmitoleoylphosphatidylethanolamine containing 20 mol% α-tocopherol hemisuccinate have been reported in which the tocopherol derivative appears to stabilize the bilayer and reduce the permeability to dyes [7] or contrast reagents [8]. Indeed α-tocopherol was found to retard the release of entrapped carboxyfluorescein from dimyristoylphosphatidylcholine vesicles to a
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greater extent than cholesterol [9]. In vivo experiments of liposomes containing α-tocopherol have been reported in rats [10]. The presence of 2 mol% α-tocopherol in small unilamellar vesicles resulted in a decreased clearance rate from the blood, prolonged blood levels and reduced uptake by the liver compared with liposomes prepared in the absence of α-tocopherol. The incorporation of α-tocopherol, however, appeared to increase the permeability of the liposomes to [3H]-inulin which was recovered in the urine. Other derivatives of tocopherol have also been extensively used in lipid drug delivery vectors. The succinyl derivative of α-tocopherol is known to be an efficient anti-cancer agent and, when administered in liposomal formulation to transgenic mice bearing spontaneous breast cancers, was able to inhibit growth of the tumours [11]. Inclusion of the vitamin in lipoplexes has also been shown to prevent oxidative damage to DNA in dry formulations [12]. A number of cationic derivatives of tocopherol have been incorporated into phospholipid lipoplexes and showed four-fold improvement of transfection efficiency in a variety of cell types over conventional formulations [13]. Furthermore, the derivatives have relatively low toxicity and there is evidence that they may be preferentially targeted to the liver. The presence of α-tocopherol in phospholipid bilayers has been reported to influence permeability to small solutes. Earlier studies reported that incorporation of 20 mol% [14] or 33 mol% [15] αtocopherol into small vesicles of phosphatidylcholine results in increased permeability of the bilayers to small solutes. The methods
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employed in these studies involved permeation of reducing or chemical shift reagents into the inner monolayer of unilamellar vesicles and the presence of α-tocopherol may alter the packing of the lipid in a manner different from that in planar lipid bilayers. The amount of α-tocopherol incorporated into phospholipid dispersions is also a factor in governing permeability of bilayers to small solutes. Thus the permeability of liposomes of dipalmitoylphosphatidylcholine to glucose at 37 °C was found to be increased when the proportion of α-tocopherol was less than 5 mol% but bilayers with greater than 5 mol% showed decreased permeability to the sugar [16]. This is consistent with reports that incorporation of 20 mol% α-tocopherol into bilayers of egg-phosphatidylcholine [17] or 5 mol% α-tocopherol into bilayers of digalactosyldiacylglycerol [18] decreased permeability of glucose. Egg-phosphatidylcholine can incorporate a maximum of 33 mol% α-tocopherol the effect of which is to increase the size of liposomes that appear to trap dyes more efficiently in a manner similar to the effect of cholesterol [19]. There have been several reviews of the effect of α-tocopherol on the structure and phase behaviour of phospholipid bilayers [20,21]. Proton-NMR studies of the effect of α-tocopherol on multilamellar dispersions of dipalmitoylphosphatidylcholine containing up to 20 mol% α-tocopherol showed that the acyl chains of the phospholipid were progressively perturbed at temperatures below the gel to liquid-crystal phase transition temperature of the phospholipid (41 °C) and more ordered at higher temperatures with increasing proportions of α-tocopherol in the bilayers [22]. Fluorescence quenching methods have been used to assess the lateral diffusion rates of α-tocopherol in bilayers of dipalmitoylphosphatidylcholine [23] and palmitoleoylphosphatidylcholine [24]. These show that αtocopherol diffuses rapidly in the plane of the phospholipid bilayer at a rate which is expected from the relative size of the molecule and the bilayer viscosity. There is apparently some lateral diffusion of α-tocopherol in ripple phase of the phospholipid but no fluorescence quenching was observed with phospholipids in gel phase. There are fewer data on the effect of α-tocopherol on the structure and phase behaviour of phospholipid bilayers. α-tocopherol has hitherto been included in liposomal formulations as an antioxidant particularly in liposomes containing unsaturated phospholipids but there is evidence that it has favourable properties on bilayer structure of saturated phospholipids. Preliminary studies of the incorporation of α-tocopherol into bilayers formed by saturated phosphatidylcholines have indicated that the vitamin stabilizes the ripple structure at temperatures between gel and liquid-crystal phases [25]. The present study was undertaken to examine in more detail using synchrotron X-ray diffraction and freeze-fracture electron microscopic methods how α-tocopherol changes the structure and phase behaviour of distearoylphosphatidylcholine. 2. Materials and methods
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needle and dispersed using a rotamixer at 70°–80 °C until a homogeneous dispersion was obtained. The samples were stored under argon at a temperature not below 4 °C for at least 1 week before examination. X-ray diffraction examination was performed on samples equilibrated at 20 °C for 5 h before transfer into the measurement cell. In order to check for possible dehydration or demixing of the components various control measurements were undertaken such as checking for reversibility of phase behaviour during subsequent heating and cooling cycles. The samples were also checked for the absence in small- and wide-angle X-ray scattering regions for diffraction peaks from subgel phases of DSPC. 2.2. Synchrotron X-ray diffraction methods X-ray diffraction measurements were performed at Station 8.2 of the Daresbury Synchrotron Radiation Source (Cheshire, UK). The Xray wavelength was 0.141 nm with a beam geometry of ~1 × 2.5 mm. Simultaneous small-angle (SAXS; 2θ = 0.043–7.9°) and wide-angle X-ray scattering (WAXS; 2θ = 8–60°) intensities were recorded so that a correlation could be established between the mesophase repeat spacings and the packing arrangement of acyl chains. The SAXS intensity was recorded on a quadrant detector calibrated using wet rat-tail collagen (67 nm [26]). The sample to quadrant detector distance was 1 m. The WAXS intensity was recorded with a curved INEL detector (Instrumentation Electronique, Artenay, France). The wide-angle X-ray scattering intensity profiles were calibrated using the diffraction peaks from high-density polyethylene [27]. Lipid dispersions (20 μl) were sandwiched between two thin mica windows 0.5 mm apart and the measurement cell was mounted on a programmable temperature stage (Linkam, Surrey, UK). The temperature was monitored by a thermocouple inserted directly into the lipid dispersion (Quad Service, Poissy, France). The setup, calibration, and facilities available on Station 8.2 are described elsewhere [28]. Data reduction and analysis were performed using OriginPro8 software (OriginLab Corp.). 2.3. Analysis of X-ray diffraction data The small angle X-ray scattering intensity profiles were analyzed using standard procedures [29]. Polarization and geometric corrections for line-width smearing were assessed by checking the symmetry of diffraction peaks in the present camera configuration using a sample of silver behenate (d = 5.838 nm). The orders of reflection could all be fitted by Voigt functions with fitting coefficients greater than R 2 = 0.99. Deconvolution is consistent with the sample to detector distance used [30]. Deconvolution of the SAXS and WAXS intensity peaks was undertaken using PeakFit (v4.12; Systat Software Inc.) software. Background subtraction was performed on each diffraction band but no corrections for polarization or geometric factors were necessary with the camera configuration employed.
2.1. Sample preparation 2.4. Freeze-fracture electron microscopy 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1palmityl-2-oleoyl-sn-phosphocholine (POPC) were purchased from Avanti Polar Lipids (Alabaster, AL). Cholesterol was purchased from Sigma–Aldrich (UK) and α-tocopherol (99%) from Acros Organics (Geel, Belgium). The lipids were used without further purification. Samples for X-ray diffraction examination were prepared by dissolving lipids in warm (45 °C) chloroform and mixing them in the desired proportions (denoted as moles α-tocopherol/100 mol DSPC + α-tocopherol). The organic solvent was subsequently evaporated under a stream of oxygen-free dry nitrogen at 45 °C and any remaining traces of solvent were removed by storage under high vacuum for two days at 20 °C. The dry lipids were hydrated with deionized water to give a dispersion of 25 wt.% lipid. This was sufficient for full hydration of the lipids. The lipids were stirred thoroughly with a thin
Lipid dispersions were equilibrated at the desired temperature for 15 min before cryofixation. This was performed by placing a small drop of the dispersion between two copper plates and plunging the sandwich into liquid propane. The frozen specimen was fractured and shadowed at −150 °C in a BAF 400D freeze-etch device (Balzers, Lichtenstein). The replicas were cleaned with chloroform and examined under a CEM 902A electron microscope (Zeiss, Germany). 2.5. Permeability measurements The dry lipids for permeability measurements were dispersed in buffer consisting of 10 mM glycine (pH 9.5), 100 mM NaCl containing 10 mM phenol red indicator and dispersed by ultrasonic irradiation
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Fig. 1. SAXS (left) and WAXS (right) intensity profiles recorded from an aqueous dispersion of DSPC heated from 20° to 60 °C at 3°/min.
for 70 s at 0 °C using a probe sonicator. The dispersion was equilibrated for 2 h at 22 °C and non-trapped dye removed from the liposomes by gel filtration through Sephadex G75 eluting with glycine buffer isotonic with the trapping medium. A value of ε558 = 52,700 was obtained for phenol red. Using this value an entrapped volume of the phospholipid liposomes was calculated to be 4.9 L.mol − 1 of phospholipid for phenol red-containing liposomes. This is in reasonable agreement with published values of 5.3 L.mole − 1 [31]. Leakage was determined after incubation under specified conditions by subjecting the suspension to gel filtration on Sephadex G75 amd measuring phenol red concentrations remaining in the liposomes and retarded in the gel. 3. Results Initial experiments were undertaken to establish the parameters of structural phases and transitions in pure DSPC. Fig. 1 shows a sequence of SAXS/WAXS patterns recorded from an aqueous dispersion of DSPC during a heating scan from 22° to 60 °C at 3°/min. 5-orders of a lamellar gel phase (d = 5.02 nm) can be seen in the SAXS region
Fig. 2. SAXS (left) and WAXS (right) intensity profiles recorded from an aqueous dispersion of DSPC containing 10 mol% α-tocopherol heated from 25° to 65 °C at 3°/min.
Fig. 3. WAXS d-spacings of (A) an aqueous dispersion of DSPC and (B) a codispersion of DSPC with 10 mol% α-tocopherol as a function of temperature.
which undergoes a transition to a liquid-crystal structure (d = 5.09 nm) at 54.5 °C evidenced by changes in the position of the WAXS intensity peak. An intermediate ripple phase with a periodicity of 11.62 nm can also be detected at an onset temperature of 51 °C and is characterised by lamellar structures with d-spacings of 5.42 and 4.69 nm. The effect of codispersion of DSPC with 10 mol% α-tocopherol on thermotropic structural changes is shown in Fig. 2. There are a number of Bragg reflections in the SAXS region at temperatures in which the phospholipid is in gel phase evidenced by the sharp reflection in the WAXS region. There is a transition to a lamellar disordered structure at about 53 °C which is characterised by a single lamellar structure of d-spacing 5.13 nm at 65 °C. On cooling the sample illustrated in Fig. 2 and reheating immediately under the same conditions showed that there was no significant temperature hysteresis in the structural transitions (Fig. S1 of the Supplementary Information). It can be seen from the WAXS profiles of Fig. 1 that the hydrocarbon chains of the pure DSPC are tilted with respect to the bilayer normal evidenced by the broad shoulder on the high-angle side of the sharp diffraction peak. These correspond to the dII and d20 reflections,
Fig. 4. SAXS intensity profiles of DSPC codispersed with the indicated mol% αtocopherol recorded at 37 °C. Peaks assigned as pure DSPC and DSPC-α-tocopherol complex are indicated.
P.J. Quinn / Journal of Controlled Release 160 (2012) 158–163
respectively, of the tilted chains [32]. The tilt angle of the hydrocarbon chains at 20 °C was calculated to be 34.8° in reasonable agreement with values reported of 32.5° at 25 °C [33]. Comparison of the WAXs profiles of the pure phospholipid with those of the codispersion with 10 mol% α-tocopherol indicates that the presence of αtocopherol has a significant effect on the tilt angle of the hydrocarbon chains. This effect is illustrated in Fig. 3. which shows the temperature dependence of the d-spacing of WAXS intensity peaks of DSPC compared with DSPC codispersed with 10 mol% α-tocopherol. The d20 reflection remains relatively constant at 0.425 and 0.422 nm for the dispersions of pure DSPC and the dispersion containing αtocopherol, respectively, up to the pre-transition temperature (~50 °C) but the d-spacing of the dII reflection increased with increasing temperature as the chain tilt decreases and the chains approach a vertical orientation with respect to the bilayer plane. This process appears to take place in two stages in the pure phospholipid with a plateau region at about 40 °C. In the presence of α-tocopherol the chain tilt decreases to zero between 20° and 40 °C and the chains remain perpendicular to the bilayer plane at higher temperatures. To investigate the structural changes associated with the presence of α-tocopherol in bilayers of DSPC SAXS intensity profiles were recorded from samples of DSPC codispersed with different proportions of α-tocopherol and the results obtained at 37 °C are presented in Fig. 4. This shows that the intensity of Bragg reflections assigned as bilayers of pure DSPC decrease relative to peaks at lower angles as the proportion of α-tocopherol in the phospholipid increases. In the presence of 20 mol% α-tocopherol no bilayer structures of pure DSPC can be observed. On the assumption that all the α-tocopherol present in the dispersion is present in structures other than those assigned as pure DSPC the relationship between the scattering intensity of pure DSPC and the proportion of α-tocopherol in the mixture gives the amount of DSPC in the structures containing α-tocopherol. This relationship is plotted in Fig. 5 and shows that the ratio of DSPC: αtocopherol in the α-tocopherol-rich structures is 8:1. The structure of DSPC dispersions containing α-tocopherol were examined by freeze-fracture electron microscopy. The replicas prepared from dispersions of the pure phospholipid thermally quenched from 25 °C showed multilamellar structures with smooth untextured surfaces (data not shown). The presence of α-tocopherol induced the formation of ripple structures. This is illustrated in Fig. 6 which is an electron micrograph of a replica prepared from a codispersion of DSPC with 10 mol% α-tocopherol. Consistent with the WAXS data for this dispersion showing gel phase structure the arrangement is multilamellar. Two types of ripple structure can be seen. The more
Fig. 5. Relationship between the relative scattering intensity from the first-order Bragg reflection assigned as DSPC and the amount of α-tocopherol in the mixture. The best fit regression has the form y = − 0.0854*x + 1.086 (R2 = 0.932).
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Fig. 6. Electron micrograph of a freeze-fracture replica prepared from a codispersion of DSPC with 10 mol% α-tocopherol thermally quenched from 25 °C after heating from 4 °C. The scale bar = 100 nm. The large ripple has a periodicity of about 16 nm and the * indicates domains of bilayer with ripple structures of periodicity of about 12 nm.
prominent ripple structure has a periodicity of about 16 nm. Similar structure is also seen in mixtures containing 5 mol% α-tocopherol (data not shown). Another ripple structure can also be seen in Fig. 6 and the periodicity of this structure is about 12 nm. The ripple structures apparently give rise to the additional Bragg reflections observed in the SAXS intensity profiles at temperatures up to the gel to liquidcrystal phase transition of the phospholipid. Experiments were performed to investigate the effect of αtocopherol on the passive permeability of phospholipid bilayers to solutes and to compare this with cholesterol which is known to increase the barrier properties of phospholipid structures. Fig. 7 shows the rate of leakage of phenol red from POPC liposomes containing different proportions of α-tocopherol or cholesterol during 5 h incubation at 4 °C. The presence of up to 20 mol% α-tocopherol in the phospholipid resulted in a proportionate decrease in permeability of the dye but incorporation of greater proportions of αtocopherol tended to reverse this trend. The inhibition of leakage by α-tocopherol was comparable to that of cholesterol at equivalent amounts but higher proportions of cholesterol, up to 50 mol%, caused even greater inhibition of dye leakage. The leakage parameters were examined as a function of temperature and over time in POPC liposomes containing 7 mol% α-tocopherol or 50 mol% cholesterol and the results are shown in Fig. 8. It can be seen that 50 mol% cholesterol inhibits passive leakage of phenol red to a greater extent than 7 mol% α-tocopherol at all temperatures up to 45 °C and over 75 h at 4 °C but
Fig. 7. Percent leakage of phenol red from POPC liposomes containing varying proportions of α-tocopherol (●) or cholesterol (○) after 5 h incubation at 4 °C.
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Fig. 8. Percent leakage of phenol red from POPC liposomes containing 7 mol% αtocopherol (●) or 50 mol% cholesterol (○), A; after incubation at 4 °C for 2 h at different temperatures, B; after incubation for different times at 4 °C. The regression lines are of an expression y = 0.31*x + 5.9 for liposomes containing α-tocopherol and y = 0.06*x + 5.8 for cholesterol-containing liposomes.
the inhibition by α-tocopherol is nevertheless significant and comparable to that of cholesterol on an equimolar basis. 4. Discussion The inclusion of cholesterol in phospholipid formulations for drug delivery is a widespread practice. An advantage of cholesterol is that it abolishes both the pre- and main gel to liquid-crystal phase transition of phospholipids when present in proportions exceeding about 30 mol% and hence reduces the sensitivity of liposomes to temperature perturbations. By contrast tocopherol tends to stabilize ripple phases at temperatures below the main transition temperature and form complexes of defined stoichiometry with phospholipids. There are, however, some undesirable effects of using cholesterolcontaining drug delivery systems including an increased risk factor of cardiovascular disease in humans and changes in lipoprotein synthesis. Another case in point is the effect of cholesterol on stimulating the activity of P-glycoprotein, an ATP-dependent membrane transporter responsible for efflux of a variety of drugs from cells. Activation of the transporter not only interferes with drug absorption and distribution amongst different tissues but often leads to detrimental side effects from the interaction between drugs under co-medication [34,35]. Moreover, therapies involving cytotoxic drugs induce overexpression of P-glycoprotein in tumor cells resulting in reduced sensitivity to the anticancer therapy due to development of a multidrugresistant phenotype [36]. Cholesterol is reported to stimulate the basal activity of the ATPase an effect that was avoided by substituting the 20 mol% cholesterol in the bilayer phospholipid with the same proportion of α-tocopherol [37].
The present results on the effect of tocopherol on the structure and permeability of DSPC bilayers indicate that α-tocopherol may be an efficient substitute for cholesterol in liposome drug vectors. A notable feature of the presence of α-tocopherol in DSPC bilayers is the induction of ripple structures. The stabilization of ripple structures by α-tocopherol has also been reported for other saturated phosphatidylcholines [25]. Calculations based on both calorimetry and X-ray diffraction studies of codispersions of dipalmitoylphosphatidylcholine and α-tocopherol showed a stoichiometry of 9.6:1 of the respective components in the ripple structures formed in these mixtures [38]. This ratio is close to that observed in the ripple structure induced in bilayers of DSPC, 8:1, reported here. Stabilization of ripple structures by tocopherol is not only observed in saturated phosphatidylcholines. Studies of binary mixtures of 1-palmitoyl-2-oleoylphosphatidylcholine indicate formation of a ripple structure that is distinct from that formed by aqueous dispersions of the pure phospholipid [39]. Furthermore, increasing proportions of tocopherol result in a progressive decrease in the gel to liquid-crystal phase transition temperature of the unsaturated phospholipid. A structural effect on phospholipid bilayers that is shared by cholesterol and α-tocopherol is a tendency to prevent tilting of the hydrocarbon chains with respect to the bilayer normal. It is well known that relatively small proportions of cholesterol in phospholipid bilayers results in orientation of the chains perpendicular to the bilayer plane [40]. Whilst a similar effect is observed with α-tocopherol in bilayers of DSPC it is not as pronounced as that with cholesterol. This difference may be due to the more rigid structure of the sterol rings compared with a more flexible configuration of the polyisoprene chain of α-tocopherol. Another effect of α-tocopherol on bilayers of DSPC that has consequences similar to that of cholesterol is on the stability of the gel phase of the phospholipid and on the presence of a pre-transition that precedes the main gel to liquid-crystal phase transition. Differential scanning calorimetry of fully hydrated mixtures of α-tocopherol with saturated molecular species of phosphatidylcholines have been reported showing that α-tocopherol results in disappearance of the pre-transition endothermic peak, a broadening of the main gel to liquid-crystal phase transition which occurs at a lower temperature and with a reduced enthalpy [41,42]. The interpretation of the calorimetric results can be made more confidently on the basis of the diffraction obtained in the present experiments. The loss of the pre transition structure is due to the formation of a stable ripple structure at temperatures where the pure phospholipid is in a gel phase with tilted hydrocarbon chains. The broadening of the main phase transition and reduction in transition temperature is likely to be due to a weakening of the intermolecular van der Waals interactions between the phospholipid molecules by interpolation of α-tocopherol molecules. The leakage of phenol red from liposomes prepared from DSPC with α-tocopherol was comparable with that of liposomes containing equivalent amounts of cholesterol. However, much higher proportions of cholesterol could be incorporated into the phospholipid without destroying the integrity of the structure. Accelerated leakage occurs with proportions of α-tocopherol exceeding about 20 mol% whereas up to 50 mol% of cholesterol can be incorporated into DSPC liposomes before increased permeability is observed. Comparisons of the effects of up to 30 mol% cholesterol or α-tocopherol on the proton permeability of bilayers of phosphatidylcholine molecular species comprised of C-18:0 in the sn-1 position of the glycerol and either C18:3 or C-22:6 in the sn-2 position [43]. It was demonstrated that cholesterol inhibits proton permeability to a significantly greater extent in liposomes comprised of the C-18:3 molecular species than those with C-22:6 fatty acid located in the sn-2 position. By contrast, α-tocopherol inhibited proton permeability to an equal extent in liposomes comprised of either molecular species.
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The data presented in this study indicates that α-tocopherol may be a useful substitute for cholesterol in phospholipid drug delivery vehicles. The addition of α-tocopherol to such formulations could also be made in conjunction with cholesterol to complement the beneficial effects of the sterol.
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Acknowledgements The work was aided by the assistance of Drs. Xiaoyuan Wang, Tony Brain and Wei Luo.
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Appendix A. Supplementary data Supplementary data to this article can be found online at doi:10. 1016/j.jconrel.2011.12.029.
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The effect of tocopherol on the structure and permeability of phosphatidylcholine liposomes Peter J. Quinn ⁎ Department of Biochemistry, King's College London, 150 Stamford Street, London SE1 9NH, UK
a r t i c l e
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Article history: Received 29 July 2011 Accepted 21 December 2011 Available online 30 December 2011 Keywords: Phospholipid α-tocopherol X-ray diffraction Bilayer structure Permeability of liposomes
a b s t r a c t There are numerous phospholipid formulations that incorporate α-tocopherol as a stabilizing agent but there are few studies of the effect of α-tocopherol on phospholipid structure and bilayer permeability. This study uses synchrotron X-ray powder diffraction methods to investigate how α-tocopherol changes the structure of distearoylphosphatidylcholines bilayers. Increasing proportions of α-tocopherol up to 20 mol% induces ripple structures in the bilayers. Two types of ripple structure are produced which are seen in electron micrographs of freeze-fracture replicas with periodicities of 16 and 12 nm, respectively. The stoichiometry of phospholipid: α-tocopherol in the ripple structures at 37 °C is 8:1. The presence of α-tocopherol tends to reduce the angle of tilt of the hydrocarbon chains of the phospholipid in the gel phase from about 34° to the bilayer normal at 20 °C into a more vertical orientation. Increasing proportions of α-tocopherol progressively decrease the temperature of the gel to liquid-crystal phase transition of the phospholipid. The presence of up to 20 mol% α-tocopherol in 1-palmitoyl-2-oleoyl-phosphocholine inhibits leakage of phenol red dye from liposomes. The effect of 7 mol% α-tocopherol on leakage was compared with phospholipid liposomes containing 50 mol% cholesterol. The cholesterol-containing liposomes inhibited leakage to a greater extent than the vesicles incorporating α-tocopherol but the effect of α-tocopherol at equivalent molar proportions was comparable to cholesterol. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Tocopherols have been incorporated into phospholipid liposomes as a method for delivery of the vitamin to cells and tissues [1] and as a component of liposomal formulations to act as a membrane stabilizing agent [2,3]. Thus inclusion of α-tocopherol in liposomal dispersions has long been known to reduce lipid oxidation that result in changes in bilayer permeability and to prolong storage life [4]. Oxidative damage to tissues such as lung exposed to 2-chloroethyl ethyl sulphide has been shown to be ameliorated by intratracheally administered liposomes of dipalmitoylphosphatidylcholine containing α-tocopherol [5]. Furthermore, intravenous administration of dipalmitoylphosphatidylcholine liposomes containing 30 mol% αtocopherol was reported to protect against lipopolysaccharideinduced liver injury in rats [6]. Controlled release formulations of pH-sensitive liposomes comprised of palmitoleoylphosphatidylethanolamine containing 20 mol% α-tocopherol hemisuccinate have been reported in which the tocopherol derivative appears to stabilize the bilayer and reduce the permeability to dyes [7] or contrast reagents [8]. Indeed α-tocopherol was found to retard the release of entrapped carboxyfluorescein from dimyristoylphosphatidylcholine vesicles to a
⁎ Tel.: + 44 2078484408; fax: + 44 8484500. E-mail address: [email protected]. 0168-3659/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2011.12.029
greater extent than cholesterol [9]. In vivo experiments of liposomes containing α-tocopherol have been reported in rats [10]. The presence of 2 mol% α-tocopherol in small unilamellar vesicles resulted in a decreased clearance rate from the blood, prolonged blood levels and reduced uptake by the liver compared with liposomes prepared in the absence of α-tocopherol. The incorporation of α-tocopherol, however, appeared to increase the permeability of the liposomes to [3H]-inulin which was recovered in the urine. Other derivatives of tocopherol have also been extensively used in lipid drug delivery vectors. The succinyl derivative of α-tocopherol is known to be an efficient anti-cancer agent and, when administered in liposomal formulation to transgenic mice bearing spontaneous breast cancers, was able to inhibit growth of the tumours [11]. Inclusion of the vitamin in lipoplexes has also been shown to prevent oxidative damage to DNA in dry formulations [12]. A number of cationic derivatives of tocopherol have been incorporated into phospholipid lipoplexes and showed four-fold improvement of transfection efficiency in a variety of cell types over conventional formulations [13]. Furthermore, the derivatives have relatively low toxicity and there is evidence that they may be preferentially targeted to the liver. The presence of α-tocopherol in phospholipid bilayers has been reported to influence permeability to small solutes. Earlier studies reported that incorporation of 20 mol% [14] or 33 mol% [15] αtocopherol into small vesicles of phosphatidylcholine results in increased permeability of the bilayers to small solutes. The methods
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employed in these studies involved permeation of reducing or chemical shift reagents into the inner monolayer of unilamellar vesicles and the presence of α-tocopherol may alter the packing of the lipid in a manner different from that in planar lipid bilayers. The amount of α-tocopherol incorporated into phospholipid dispersions is also a factor in governing permeability of bilayers to small solutes. Thus the permeability of liposomes of dipalmitoylphosphatidylcholine to glucose at 37 °C was found to be increased when the proportion of α-tocopherol was less than 5 mol% but bilayers with greater than 5 mol% showed decreased permeability to the sugar [16]. This is consistent with reports that incorporation of 20 mol% α-tocopherol into bilayers of egg-phosphatidylcholine [17] or 5 mol% α-tocopherol into bilayers of digalactosyldiacylglycerol [18] decreased permeability of glucose. Egg-phosphatidylcholine can incorporate a maximum of 33 mol% α-tocopherol the effect of which is to increase the size of liposomes that appear to trap dyes more efficiently in a manner similar to the effect of cholesterol [19]. There have been several reviews of the effect of α-tocopherol on the structure and phase behaviour of phospholipid bilayers [20,21]. Proton-NMR studies of the effect of α-tocopherol on multilamellar dispersions of dipalmitoylphosphatidylcholine containing up to 20 mol% α-tocopherol showed that the acyl chains of the phospholipid were progressively perturbed at temperatures below the gel to liquid-crystal phase transition temperature of the phospholipid (41 °C) and more ordered at higher temperatures with increasing proportions of α-tocopherol in the bilayers [22]. Fluorescence quenching methods have been used to assess the lateral diffusion rates of α-tocopherol in bilayers of dipalmitoylphosphatidylcholine [23] and palmitoleoylphosphatidylcholine [24]. These show that αtocopherol diffuses rapidly in the plane of the phospholipid bilayer at a rate which is expected from the relative size of the molecule and the bilayer viscosity. There is apparently some lateral diffusion of α-tocopherol in ripple phase of the phospholipid but no fluorescence quenching was observed with phospholipids in gel phase. There are fewer data on the effect of α-tocopherol on the structure and phase behaviour of phospholipid bilayers. α-tocopherol has hitherto been included in liposomal formulations as an antioxidant particularly in liposomes containing unsaturated phospholipids but there is evidence that it has favourable properties on bilayer structure of saturated phospholipids. Preliminary studies of the incorporation of α-tocopherol into bilayers formed by saturated phosphatidylcholines have indicated that the vitamin stabilizes the ripple structure at temperatures between gel and liquid-crystal phases [25]. The present study was undertaken to examine in more detail using synchrotron X-ray diffraction and freeze-fracture electron microscopic methods how α-tocopherol changes the structure and phase behaviour of distearoylphosphatidylcholine. 2. Materials and methods
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needle and dispersed using a rotamixer at 70°–80 °C until a homogeneous dispersion was obtained. The samples were stored under argon at a temperature not below 4 °C for at least 1 week before examination. X-ray diffraction examination was performed on samples equilibrated at 20 °C for 5 h before transfer into the measurement cell. In order to check for possible dehydration or demixing of the components various control measurements were undertaken such as checking for reversibility of phase behaviour during subsequent heating and cooling cycles. The samples were also checked for the absence in small- and wide-angle X-ray scattering regions for diffraction peaks from subgel phases of DSPC. 2.2. Synchrotron X-ray diffraction methods X-ray diffraction measurements were performed at Station 8.2 of the Daresbury Synchrotron Radiation Source (Cheshire, UK). The Xray wavelength was 0.141 nm with a beam geometry of ~1 × 2.5 mm. Simultaneous small-angle (SAXS; 2θ = 0.043–7.9°) and wide-angle X-ray scattering (WAXS; 2θ = 8–60°) intensities were recorded so that a correlation could be established between the mesophase repeat spacings and the packing arrangement of acyl chains. The SAXS intensity was recorded on a quadrant detector calibrated using wet rat-tail collagen (67 nm [26]). The sample to quadrant detector distance was 1 m. The WAXS intensity was recorded with a curved INEL detector (Instrumentation Electronique, Artenay, France). The wide-angle X-ray scattering intensity profiles were calibrated using the diffraction peaks from high-density polyethylene [27]. Lipid dispersions (20 μl) were sandwiched between two thin mica windows 0.5 mm apart and the measurement cell was mounted on a programmable temperature stage (Linkam, Surrey, UK). The temperature was monitored by a thermocouple inserted directly into the lipid dispersion (Quad Service, Poissy, France). The setup, calibration, and facilities available on Station 8.2 are described elsewhere [28]. Data reduction and analysis were performed using OriginPro8 software (OriginLab Corp.). 2.3. Analysis of X-ray diffraction data The small angle X-ray scattering intensity profiles were analyzed using standard procedures [29]. Polarization and geometric corrections for line-width smearing were assessed by checking the symmetry of diffraction peaks in the present camera configuration using a sample of silver behenate (d = 5.838 nm). The orders of reflection could all be fitted by Voigt functions with fitting coefficients greater than R 2 = 0.99. Deconvolution is consistent with the sample to detector distance used [30]. Deconvolution of the SAXS and WAXS intensity peaks was undertaken using PeakFit (v4.12; Systat Software Inc.) software. Background subtraction was performed on each diffraction band but no corrections for polarization or geometric factors were necessary with the camera configuration employed.
2.1. Sample preparation 2.4. Freeze-fracture electron microscopy 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1palmityl-2-oleoyl-sn-phosphocholine (POPC) were purchased from Avanti Polar Lipids (Alabaster, AL). Cholesterol was purchased from Sigma–Aldrich (UK) and α-tocopherol (99%) from Acros Organics (Geel, Belgium). The lipids were used without further purification. Samples for X-ray diffraction examination were prepared by dissolving lipids in warm (45 °C) chloroform and mixing them in the desired proportions (denoted as moles α-tocopherol/100 mol DSPC + α-tocopherol). The organic solvent was subsequently evaporated under a stream of oxygen-free dry nitrogen at 45 °C and any remaining traces of solvent were removed by storage under high vacuum for two days at 20 °C. The dry lipids were hydrated with deionized water to give a dispersion of 25 wt.% lipid. This was sufficient for full hydration of the lipids. The lipids were stirred thoroughly with a thin
Lipid dispersions were equilibrated at the desired temperature for 15 min before cryofixation. This was performed by placing a small drop of the dispersion between two copper plates and plunging the sandwich into liquid propane. The frozen specimen was fractured and shadowed at −150 °C in a BAF 400D freeze-etch device (Balzers, Lichtenstein). The replicas were cleaned with chloroform and examined under a CEM 902A electron microscope (Zeiss, Germany). 2.5. Permeability measurements The dry lipids for permeability measurements were dispersed in buffer consisting of 10 mM glycine (pH 9.5), 100 mM NaCl containing 10 mM phenol red indicator and dispersed by ultrasonic irradiation
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Fig. 1. SAXS (left) and WAXS (right) intensity profiles recorded from an aqueous dispersion of DSPC heated from 20° to 60 °C at 3°/min.
for 70 s at 0 °C using a probe sonicator. The dispersion was equilibrated for 2 h at 22 °C and non-trapped dye removed from the liposomes by gel filtration through Sephadex G75 eluting with glycine buffer isotonic with the trapping medium. A value of ε558 = 52,700 was obtained for phenol red. Using this value an entrapped volume of the phospholipid liposomes was calculated to be 4.9 L.mol − 1 of phospholipid for phenol red-containing liposomes. This is in reasonable agreement with published values of 5.3 L.mole − 1 [31]. Leakage was determined after incubation under specified conditions by subjecting the suspension to gel filtration on Sephadex G75 amd measuring phenol red concentrations remaining in the liposomes and retarded in the gel. 3. Results Initial experiments were undertaken to establish the parameters of structural phases and transitions in pure DSPC. Fig. 1 shows a sequence of SAXS/WAXS patterns recorded from an aqueous dispersion of DSPC during a heating scan from 22° to 60 °C at 3°/min. 5-orders of a lamellar gel phase (d = 5.02 nm) can be seen in the SAXS region
Fig. 2. SAXS (left) and WAXS (right) intensity profiles recorded from an aqueous dispersion of DSPC containing 10 mol% α-tocopherol heated from 25° to 65 °C at 3°/min.
Fig. 3. WAXS d-spacings of (A) an aqueous dispersion of DSPC and (B) a codispersion of DSPC with 10 mol% α-tocopherol as a function of temperature.
which undergoes a transition to a liquid-crystal structure (d = 5.09 nm) at 54.5 °C evidenced by changes in the position of the WAXS intensity peak. An intermediate ripple phase with a periodicity of 11.62 nm can also be detected at an onset temperature of 51 °C and is characterised by lamellar structures with d-spacings of 5.42 and 4.69 nm. The effect of codispersion of DSPC with 10 mol% α-tocopherol on thermotropic structural changes is shown in Fig. 2. There are a number of Bragg reflections in the SAXS region at temperatures in which the phospholipid is in gel phase evidenced by the sharp reflection in the WAXS region. There is a transition to a lamellar disordered structure at about 53 °C which is characterised by a single lamellar structure of d-spacing 5.13 nm at 65 °C. On cooling the sample illustrated in Fig. 2 and reheating immediately under the same conditions showed that there was no significant temperature hysteresis in the structural transitions (Fig. S1 of the Supplementary Information). It can be seen from the WAXS profiles of Fig. 1 that the hydrocarbon chains of the pure DSPC are tilted with respect to the bilayer normal evidenced by the broad shoulder on the high-angle side of the sharp diffraction peak. These correspond to the dII and d20 reflections,
Fig. 4. SAXS intensity profiles of DSPC codispersed with the indicated mol% αtocopherol recorded at 37 °C. Peaks assigned as pure DSPC and DSPC-α-tocopherol complex are indicated.
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respectively, of the tilted chains [32]. The tilt angle of the hydrocarbon chains at 20 °C was calculated to be 34.8° in reasonable agreement with values reported of 32.5° at 25 °C [33]. Comparison of the WAXs profiles of the pure phospholipid with those of the codispersion with 10 mol% α-tocopherol indicates that the presence of αtocopherol has a significant effect on the tilt angle of the hydrocarbon chains. This effect is illustrated in Fig. 3. which shows the temperature dependence of the d-spacing of WAXS intensity peaks of DSPC compared with DSPC codispersed with 10 mol% α-tocopherol. The d20 reflection remains relatively constant at 0.425 and 0.422 nm for the dispersions of pure DSPC and the dispersion containing αtocopherol, respectively, up to the pre-transition temperature (~50 °C) but the d-spacing of the dII reflection increased with increasing temperature as the chain tilt decreases and the chains approach a vertical orientation with respect to the bilayer plane. This process appears to take place in two stages in the pure phospholipid with a plateau region at about 40 °C. In the presence of α-tocopherol the chain tilt decreases to zero between 20° and 40 °C and the chains remain perpendicular to the bilayer plane at higher temperatures. To investigate the structural changes associated with the presence of α-tocopherol in bilayers of DSPC SAXS intensity profiles were recorded from samples of DSPC codispersed with different proportions of α-tocopherol and the results obtained at 37 °C are presented in Fig. 4. This shows that the intensity of Bragg reflections assigned as bilayers of pure DSPC decrease relative to peaks at lower angles as the proportion of α-tocopherol in the phospholipid increases. In the presence of 20 mol% α-tocopherol no bilayer structures of pure DSPC can be observed. On the assumption that all the α-tocopherol present in the dispersion is present in structures other than those assigned as pure DSPC the relationship between the scattering intensity of pure DSPC and the proportion of α-tocopherol in the mixture gives the amount of DSPC in the structures containing α-tocopherol. This relationship is plotted in Fig. 5 and shows that the ratio of DSPC: αtocopherol in the α-tocopherol-rich structures is 8:1. The structure of DSPC dispersions containing α-tocopherol were examined by freeze-fracture electron microscopy. The replicas prepared from dispersions of the pure phospholipid thermally quenched from 25 °C showed multilamellar structures with smooth untextured surfaces (data not shown). The presence of α-tocopherol induced the formation of ripple structures. This is illustrated in Fig. 6 which is an electron micrograph of a replica prepared from a codispersion of DSPC with 10 mol% α-tocopherol. Consistent with the WAXS data for this dispersion showing gel phase structure the arrangement is multilamellar. Two types of ripple structure can be seen. The more
Fig. 5. Relationship between the relative scattering intensity from the first-order Bragg reflection assigned as DSPC and the amount of α-tocopherol in the mixture. The best fit regression has the form y = − 0.0854*x + 1.086 (R2 = 0.932).
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Fig. 6. Electron micrograph of a freeze-fracture replica prepared from a codispersion of DSPC with 10 mol% α-tocopherol thermally quenched from 25 °C after heating from 4 °C. The scale bar = 100 nm. The large ripple has a periodicity of about 16 nm and the * indicates domains of bilayer with ripple structures of periodicity of about 12 nm.
prominent ripple structure has a periodicity of about 16 nm. Similar structure is also seen in mixtures containing 5 mol% α-tocopherol (data not shown). Another ripple structure can also be seen in Fig. 6 and the periodicity of this structure is about 12 nm. The ripple structures apparently give rise to the additional Bragg reflections observed in the SAXS intensity profiles at temperatures up to the gel to liquidcrystal phase transition of the phospholipid. Experiments were performed to investigate the effect of αtocopherol on the passive permeability of phospholipid bilayers to solutes and to compare this with cholesterol which is known to increase the barrier properties of phospholipid structures. Fig. 7 shows the rate of leakage of phenol red from POPC liposomes containing different proportions of α-tocopherol or cholesterol during 5 h incubation at 4 °C. The presence of up to 20 mol% α-tocopherol in the phospholipid resulted in a proportionate decrease in permeability of the dye but incorporation of greater proportions of αtocopherol tended to reverse this trend. The inhibition of leakage by α-tocopherol was comparable to that of cholesterol at equivalent amounts but higher proportions of cholesterol, up to 50 mol%, caused even greater inhibition of dye leakage. The leakage parameters were examined as a function of temperature and over time in POPC liposomes containing 7 mol% α-tocopherol or 50 mol% cholesterol and the results are shown in Fig. 8. It can be seen that 50 mol% cholesterol inhibits passive leakage of phenol red to a greater extent than 7 mol% α-tocopherol at all temperatures up to 45 °C and over 75 h at 4 °C but
Fig. 7. Percent leakage of phenol red from POPC liposomes containing varying proportions of α-tocopherol (●) or cholesterol (○) after 5 h incubation at 4 °C.
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Fig. 8. Percent leakage of phenol red from POPC liposomes containing 7 mol% αtocopherol (●) or 50 mol% cholesterol (○), A; after incubation at 4 °C for 2 h at different temperatures, B; after incubation for different times at 4 °C. The regression lines are of an expression y = 0.31*x + 5.9 for liposomes containing α-tocopherol and y = 0.06*x + 5.8 for cholesterol-containing liposomes.
the inhibition by α-tocopherol is nevertheless significant and comparable to that of cholesterol on an equimolar basis. 4. Discussion The inclusion of cholesterol in phospholipid formulations for drug delivery is a widespread practice. An advantage of cholesterol is that it abolishes both the pre- and main gel to liquid-crystal phase transition of phospholipids when present in proportions exceeding about 30 mol% and hence reduces the sensitivity of liposomes to temperature perturbations. By contrast tocopherol tends to stabilize ripple phases at temperatures below the main transition temperature and form complexes of defined stoichiometry with phospholipids. There are, however, some undesirable effects of using cholesterolcontaining drug delivery systems including an increased risk factor of cardiovascular disease in humans and changes in lipoprotein synthesis. Another case in point is the effect of cholesterol on stimulating the activity of P-glycoprotein, an ATP-dependent membrane transporter responsible for efflux of a variety of drugs from cells. Activation of the transporter not only interferes with drug absorption and distribution amongst different tissues but often leads to detrimental side effects from the interaction between drugs under co-medication [34,35]. Moreover, therapies involving cytotoxic drugs induce overexpression of P-glycoprotein in tumor cells resulting in reduced sensitivity to the anticancer therapy due to development of a multidrugresistant phenotype [36]. Cholesterol is reported to stimulate the basal activity of the ATPase an effect that was avoided by substituting the 20 mol% cholesterol in the bilayer phospholipid with the same proportion of α-tocopherol [37].
The present results on the effect of tocopherol on the structure and permeability of DSPC bilayers indicate that α-tocopherol may be an efficient substitute for cholesterol in liposome drug vectors. A notable feature of the presence of α-tocopherol in DSPC bilayers is the induction of ripple structures. The stabilization of ripple structures by α-tocopherol has also been reported for other saturated phosphatidylcholines [25]. Calculations based on both calorimetry and X-ray diffraction studies of codispersions of dipalmitoylphosphatidylcholine and α-tocopherol showed a stoichiometry of 9.6:1 of the respective components in the ripple structures formed in these mixtures [38]. This ratio is close to that observed in the ripple structure induced in bilayers of DSPC, 8:1, reported here. Stabilization of ripple structures by tocopherol is not only observed in saturated phosphatidylcholines. Studies of binary mixtures of 1-palmitoyl-2-oleoylphosphatidylcholine indicate formation of a ripple structure that is distinct from that formed by aqueous dispersions of the pure phospholipid [39]. Furthermore, increasing proportions of tocopherol result in a progressive decrease in the gel to liquid-crystal phase transition temperature of the unsaturated phospholipid. A structural effect on phospholipid bilayers that is shared by cholesterol and α-tocopherol is a tendency to prevent tilting of the hydrocarbon chains with respect to the bilayer normal. It is well known that relatively small proportions of cholesterol in phospholipid bilayers results in orientation of the chains perpendicular to the bilayer plane [40]. Whilst a similar effect is observed with α-tocopherol in bilayers of DSPC it is not as pronounced as that with cholesterol. This difference may be due to the more rigid structure of the sterol rings compared with a more flexible configuration of the polyisoprene chain of α-tocopherol. Another effect of α-tocopherol on bilayers of DSPC that has consequences similar to that of cholesterol is on the stability of the gel phase of the phospholipid and on the presence of a pre-transition that precedes the main gel to liquid-crystal phase transition. Differential scanning calorimetry of fully hydrated mixtures of α-tocopherol with saturated molecular species of phosphatidylcholines have been reported showing that α-tocopherol results in disappearance of the pre-transition endothermic peak, a broadening of the main gel to liquid-crystal phase transition which occurs at a lower temperature and with a reduced enthalpy [41,42]. The interpretation of the calorimetric results can be made more confidently on the basis of the diffraction obtained in the present experiments. The loss of the pre transition structure is due to the formation of a stable ripple structure at temperatures where the pure phospholipid is in a gel phase with tilted hydrocarbon chains. The broadening of the main phase transition and reduction in transition temperature is likely to be due to a weakening of the intermolecular van der Waals interactions between the phospholipid molecules by interpolation of α-tocopherol molecules. The leakage of phenol red from liposomes prepared from DSPC with α-tocopherol was comparable with that of liposomes containing equivalent amounts of cholesterol. However, much higher proportions of cholesterol could be incorporated into the phospholipid without destroying the integrity of the structure. Accelerated leakage occurs with proportions of α-tocopherol exceeding about 20 mol% whereas up to 50 mol% of cholesterol can be incorporated into DSPC liposomes before increased permeability is observed. Comparisons of the effects of up to 30 mol% cholesterol or α-tocopherol on the proton permeability of bilayers of phosphatidylcholine molecular species comprised of C-18:0 in the sn-1 position of the glycerol and either C18:3 or C-22:6 in the sn-2 position [43]. It was demonstrated that cholesterol inhibits proton permeability to a significantly greater extent in liposomes comprised of the C-18:3 molecular species than those with C-22:6 fatty acid located in the sn-2 position. By contrast, α-tocopherol inhibited proton permeability to an equal extent in liposomes comprised of either molecular species.
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The data presented in this study indicates that α-tocopherol may be a useful substitute for cholesterol in phospholipid drug delivery vehicles. The addition of α-tocopherol to such formulations could also be made in conjunction with cholesterol to complement the beneficial effects of the sterol.
[18]
[19] [20]
Acknowledgements The work was aided by the assistance of Drs. Xiaoyuan Wang, Tony Brain and Wei Luo.
[21] [22]
[23]
Appendix A. Supplementary data Supplementary data to this article can be found online at doi:10. 1016/j.jconrel.2011.12.029.
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