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Langmuir 2001, 17, 6426-6432
Preparation and Properties of Vesicles Formed from Phospholipid Analogues of N-(Phosphonoacetyl)-L-aspartate (PALA) by Sonication or Extrusion: Transition Temperature, Particle Size, Glucose Entrapment, and 31P NMR Patrick Oliger,† Marc Hebrant,† Claude Grison,‡ Philippe Coutrot,‡ and Christian Tondre*,† Laboratoire de Chimie Physique Organique et Colloı¨dale and Laboratoire de Chimie Organique II, UMR CNRS 7565, Institut Nance´ ien de Chimie Mole´ culaire, Universite´ Henri Poincare´ -Nancy I, BP 239, 54506 Nancy-Vandoeuvre Cedex, France Received March 5, 2001. In Final Form: May 28, 2001 We characterize here the ability of new amphiphilic compounds to form vesicles that could potentially be used as vectors for therapeutical applications. These compounds are derived from N-phosphonoacetylL-aspartate (PALA), a potential antitumoral agent. Vesicular dispersions of phospholipid analogues of PALA with different alkyl chain lengths (diC12-, diC14-, diC16-, and diC18-PALA), which had been previously synthesized, are tested from different points of view. Two kinds of preparation methods are compared: sonication; extrusion. The two preparation methods resulted in important differences in the properties of the dispersions which confirmed the conclusions of a previous investigation from cryo-TEM imaging. The results of fluorescence polarization experiments, performed either with the pure compounds or their mixtures with lecithins or cholesterol, indicated that the transition temperature Tm can be modulated if it is required for the applications. The sonicated particles showed smaller size and much lower glucose encapsulation than the extruded particles. In both cases these properties are only weakly depending on the amphiphile alkyl chain lengths. The glucose permeability has appeared to considerably increase (lower encapsulation) at T > Tm. The effect of Mn2+ on the 31P NMR signal intensity suggested that unilamellar vesicles are coexisting with other types of particle in case of extrusion, whereas in case of sonication only few and less stable vesicles are present, in agreement with the preceding observations.
Introduction Many double-chain amphiphile molecules form lamellar phases. This is a well-known property of phospholipids, which has been explained on the basis of the amphiphile packing concept.1-3 From this concept, resting on some geometric characteristics of the amphiphile molecule, it can be predicted that when the V/(al) ratio (where V is the volume occupied by the alkyl chains, l their length in fully extended configuration, and a the surface occupied by the polar head) is close to unity (or at least comprised between 0.5 and 1), bilayers are likely to form because they correspond to the self-organized structures which will be the less costly on energetic grounds. Under well-defined conditions these flat bilayers may close up to form vesicles which are spherical structures having an inner aqueous core, which is separated from the external water by the amphiphile membrane. The obtaining of such closed structures usually requires an energy supply. Sonication or extrusion under pressure through filtration membranes are two methods commonly used for this purpose.4 On the basis of the preceding concept, many synthetic lipids have been shown to self-organize in bilayers and consequently † ‡
Laboratoire de Chimie Physique Organique et Colloı¨dale. Laboratoire de Chimie Organique II.
(1) Israelachvili, J. N.; Mitchell, D. J.; Ninham, B. W. J. Chem. Soc., Faraday Trans. 2 1976, 72, 1525. (2) Mitchell, D. J.; Ninham, B. W. J. Chem. Soc., Faraday Trans. 2 1981, 77, 601. (3) Evans, D. F.; Ninham, B. W. J. Phys. Chem. 1986, 90, 226. (4) Les Liposomes, Aspects Technologiques, Biologiques et Pharmacologiques; Delattre, J., Couvreur, P., Puisieux, F., Philippot, J.-R., Schuber, F., Eds.; Edition INSERM and Tec & Doc-Lavoisier: Paris, 1993.
to have vesicle-forming capacities. An abundant literature exists on this topic, and only typical examples5-7 or review articles8,9 will be found in the references quoted here. Vesicles (or liposomes) can interact with cell membranes in different manners depending on their charge and on their fluidity, which can be modulated by addition of cholesterol. Different mechanisms were reported to possibly occur going from simple adhesion to the cell wall to fusion or even endocytosis.4 For this reason vesicular particles have been considered as drug-delivery systems for pharmacological or medical applications.10 The incorporation of a drug in vesicles (or liposomes) can be achieved in different ways, and its localization will depend on the nature of the drug. If it is hydrosoluble, it will be located in the inner aqueous core (assuming the free drug was removed); if it is hydrophobic, it will solubilize in the lipid bilayer. In case it is electrically charged it may interact with the amphiphile polar heads both in the inner layer and in the outer layer. A new concept of drug incorporation was recently proposed in which the active molecule is a part of the amphiphile itself.11 This approach is based on (5) Blandamer, M. J.; Briggs, B.; Cullis, P. M.; Engberts, J. B. F. N.; Wagenaar, A.; Smits, E.; Hoekstra, D.; Kacperska, A. J. Chem. Soc., Faraday Trans. 1994, 90, 2703 and 2709. (6) Neumann, R.; Ringsdorf, H. J. Am. Chem. Soc. 1986, 108, 487. (7) Walde, P.; Wessicken, M.; Ra¨dler, U.; Berclaz, N.; Conde-Frieboes, K.; Luisi, P. L. J. Phys. Chem. B 1997, 101, 7390. (8) Kunitake, T. Angew. Chem., Int. Ed. Engl. 1992, 31, 709. (9) Engberts, J. B. F. N.; Hoekstra, D. Biochim. Biophys. Acta 1995, 1241, 323. (10) Les Liposomes, Applications The´ rapeutiques; Puisieux, F., Delattre, J., Eds.; Technique et Documentation, Lavoisier: Paris, 1985. (11) Coutrot, P.; Oliger, P.; Grison, C.; Joliez, S.; He´brant, M.; Tondre, C. New J. Chem. 1999, 23, 981.
10.1021/la010332x CCC: $20.00 © 2001 American Chemical Society Published on Web 09/15/2001
Vesicles Formed from Analogues of PALA Chart 1
the development of new phospholipid analogues in which the polar head mimic a molecule which was recognized as having anticancer properties. In the ideal case, which still has to be proved, the active molecule would be released inside the cells by an esterolysis reaction catalyzed by enzymes present in situ. Many efforts will be necessary to check the feasibility of this new approach. This implies cooperation among organic chemists (synthesis of new amphiphile molecules), physical chemists (characterization of the self-assembling properties of these new molecules), and biologists (study of the interactions with, and effects on, cell cultures). In the two preceding publications11,12 we have examined the potential of new amphiphile compounds derived from N-phosphonoacetyl-L-aspartate (PALA), which is an inhibitor of the enzyme aspartate transcarbamylase (ATCase).13,14 This enzyme plays a key role in the biosynthesis of pyrimidic nucleotides (see ref 15 and references therein). By inhibiting the formation of carbamyl aspartate from carbamyl phosphate and aspartic acid, PALA was shown to block the proliferation of certain cell cultures14,16 and thus to have some antitumoral properties. Recent investigations suggest that its association with 5-fluorouracil may result in greater antitumor activity.17,18 We previously reported11,12 the synthesis of diCn-PALA with n varying from 12 to 18 (see Chart 1). The first publication11 was restricted to the case of diC18PALA, and sonication was used to prepare the dispersions. Glucose entrapment experiments demonstrated the presence of closed vesicles, but the low value of the encapsulation rate (maximum value of 0.32% for an amphiphile concentration of 11.3 mM) suggested that only a small portion of the amphiphile was in the form of vesicles. The second paper12 confirmed this prediction for the case of diC18-PALA and showed that the tendency to form vesicles is greatly improved when shorter alkyl chains are used (cases of diC12- and diC14-PALA). It also reported for the first time the morphological evolution of the particles formed when the amphiphile chain lengths were varied from C12 to C18 (cryo-TEM imaging). This evolution was shown to be correlated with the value of the transition (12) Oliger, P.; Schmutz, M.; He´brant, M.; Grison, C.; Coutrot, P.; Tondre, C. Langmuir 2001, 17, 3893. (13) Collins, K. D.; Stark, G. R. J. Biol. Chem. 1971, 246, 6599. (14) Swyryd, E. A.; Seaver, S. S.; Stark, G. R. J. Biol. Chem. 1974, 249, 6945. (15) Tondre, C.; Hammes, G. G. Biochemistry 1974, 13, 3131. (16) Sharma, A.; Straubinger, N. L.;. Straubinger, R. M. Pharm. Res. 1993, 10, 1434. (17) Nassim, M. A.; Rouini, M. R.; Cripps, M. C.; Shirazi, F. H.; Veerasinghan, S.; Molepo, J. M.; Obrocea, M.; Redmond, D.; Bates, S.; Fry, D.; Stewart, D. J.; Goel, R. Oncol. Rep. 1998, 5, 217. (18) Fleming, R. A. Capizzi, R. L.; Muss, H. B.; Smith, S.; Fernandes, D. J.; Homesley, H.; Loggie, B. W.; Case, L. D.; Morris, R.; Russel, G. B.; Richards, F. Clin. Cancer Res. 1996, 2, 1107.
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temperature Tm from the gel phase (Lβ) to the liquid crystalline phase (LR). In addition, the results demonstrated that the mode of preparation of the dispersions was extremely important: the extrusion method gave much larger and better defined objects than the sonication method which led more systematically to intense fragmentation of the dispersed phase. Depending on the cases considered spherical unilamellar vesicles can coexist with flat circular disks, bilayer fragments, or other types of distorted vesicles. The physical-chemical characterizations of the particles need to be performed as thoroughly as possible before testing their biological activity. It is well-known that the interactions between the vesicles and the cell membranes may be affected by the fluidity of the bilayer.4,10 Depending on the lengths of the alkyl chains, which change the transition temperature Tm, we may be or not in favorable conditions to induce fusion with the cell membrane or to facilitate endocytosis. For this reason we search here more information on the Tm values and on the effect of additives to eventually modulate these values. Even though with the new drug transport concept tested here we do not really need to encapsulate the drug in the internal aqueous compartment of the vesicles, nevertheless we want to know if closed vesicular structures are formed and what are their characteristic sizes. This has prompted us to carry out different kinds of measurements aimed at (i) precising the external conditions controlling the encapsulation/ permeation of test molecules, (ii) giving information on the sizes of the particles, and (iii) checking the unilamellarity of the vesicles, i.e., their constitution by a single bilayer of amphiphile molecules. We report here the results obtained from fluorescence polarization, size exclusion chromatography, glucose entrapment, quasielastic light scattering, and 31P NMR. They strengthen the conclusions reached from our previous investigations as regards the effects of the alkyl chain length and of the method adopted to prepare the vesicle dispersions. The general observations are consistent with those reported before in the case of classical phospholipids.4,10 Experimental Section Chemicals and Vesicle Preparations. Didodecyl-, ditetradecyl-, dihexadecyl-, and dioctadecyl-N-(phosphonoacetyl)-Laspartate, the dialkyl derivatives of PALA (abbreviated diC12-, diC14-, diC16-, and diC18-PALA), were obtained from original synthesis, as reported in previous publications.11,12 Their purity was checked by 1H, 13C, and 31P NMR, IR, elemental analysis, and melting point. The L-R-phosphatidylcholine-dilauroyl (DLPC), -dimyristoyl (DMPC), -dipalmitoyl (DPPC), and -distearoyl (DSPC) were synthetic Sigma products with approximate purity 99%. 1,6-Diphenyl-1,3, 5-hexatriene (DPH) was purchased from Fluka. d-Glucose, cholesterol, and HEPES buffer were obtained form Sigma. All the other chemicals were reagent grade and commercially available. Their origin was documented in the previous publications. Pure deionized water was obtained from reverse osmosis (Millipore Elix 3). Two different methods were used to prepare the vesicle dispersions: sonication; extrusion. The sonications were performed with a Branson sonifier 200 w (tip 13 mm, 40-45% power output, 5 min sonication at 40 °C). The procedure was derived from that described by Cleij et al.19 as reported before. The dispersions were prepared in 10 mM HEPES, pH 7 ((0.1). They were filtered at room temperature through 0.45 µm Millipore membranes to remove titanium particles detached from the immersion probe. Extruded vesicles were prepared using a 10 mL extruder (Lipex Biomembranes, Vancouver, Canada), ac(19) Cleij, M. C.; Scrimin, P.; Tecilla, P.; Tonellato, U. Langmuir 1996, 12, 2956.
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cording to the procedure described by Hope et al.20 Five freezethaw cycles were usually performed to obtain an initial dispersion in HEPES/NaOH. The extrusions were carried out at temperatures well above the gel to liquid-crystal transition temperature (60 °C for diCn-PALA with n e 16 and 68 °C for diC18-PALA). The dispersions were passed 10 times through two stacked filters (Nuclepore Corp., 0.1 µm pore size, nitrogen applied pressure 40 bar). We checked that a stable and reproducible size was rapidly attained. Particle size measurements with diC16-PALA indicated a hydrodynamic diameter of 98.9 nm after the first passage and 86.6 nm after the second passage. After 10 passages the size had only changed by a few percent to attain 83.1 nm. Techniques. The fluorescence polarization measurements were carried out on a Shimadzu FR 540 spectrofluorometer in which polarizers were added before and after the fluorescence cell compartment. DPH at concentration 3 × 10-5 M was weighted, with the amphiphile, before preparing the vesicle dispersion. The excitation and emission wavelengths were 360 and 430 nm, respectively. The fluorescence polarization was calculated according to21
P ) (IVV - IVHG)/(IVV + IVHG) where IVV and IVH are observed intensities measured with polarizers parallel and perpendicular to the vertically polarized exciting beam, respectively. G is the grating correction factor equal to IHV/IHH.22 The transition temperature was taken equal to the temperature at midtransition. For the glucose entrapment experiments, glucose at concentration 0.5 M was introduced into the dispersions before sonication or extrusion. The free glucose was separated from the encapsulated one by size exclusion chromatography (see below). The glucose content was analyzed through a classical procedure23 based on the use of glucose oxidase (Sigma diagnostics) coupled with a colored reaction with o-dianisidine (Sigma). A calibration curve was determined at 435 nm. Triton X-100 was added to the dispersions in order to break up the vesicles. Size exclusion chromatography was performed on Sephadex G-50 (Sigma) with a Biologic LP apparatus (Bio-Rad) equipped with a model 2128 fraction collector and a double trace recorder (Tracelab BD 41). Both the absorbance (254 nm) and the conductivity of the eluted solutions were measured. The eluent was a 10 mM aqueous solution of HEPES buffer (pH 7.0). For the experiments at temperatures different from room temperature, the column was wrapped up with tubing allowing the circulation of thermostated water. Analysis of Phosphorus Content. The determination of the total phosphorus present in the eluted fractions was carried out by following the procedure described by Bartlett.24 A standard curve giving the absorbance (830 nm) as a function of weighted quantities of phosphorus was used for that purpose. Particle size measurements were carried out at 25 °C with a home-assembled quasielastic light scattering apparatus coupled with a Malvern autocorrelator. Filtration of the dispersions through 0.45 µm Millipore membranes was systematically performed prior to the measurements. The scattering intensity was adjusted by dilution of the dispersions with the HEPES buffer. The data treatment was carried out with a standard Malvern software, which makes use of the Stokes-Einstein relation to calculate the hydrodynamic diameter of the particles assumed to have a spherical shape. 31P NMR spectroscopy was employed to provide an indication of the extent to which the vesicle preparations were unilamellar. The principle was previously described:20 addition of Mn2+ in the external medium will induce broadening of the 31P NMR signal from the phospholipids facing the external medium and only from those ones. In the ideal case only 50% of the signal should remain for large vesicles, for which an equipartition of the (20) Hope, M. J.; Bally, M. B.; Webb, G.; Cullis, P. R. Biochim. Biophys. Acta 1985, 812, 55. (21) Andrich, M. P.; Vanderkoi, J. M. Biochemistry 1976, 15, 1257. (22) Chen, R. F.; Edelhoch, H.; Steiner R. F. In Physical Principles and Techniques of Protein Chemistry; Leach, S. J., Ed.; Academic Press, Inc.: New York, 1969; Part A, p 209. (23) Hugget, A.; Nixon, D. Biochem. J. 1957, 66, 12. (24) Bartlett, G. R. J. Biol. Chem. 1959, 234, 466.
Oliger et al.
Figure 1. Fluorescence polarization P of DPH versus temperature for the following: (0) diC18-PALA; (O) diC14-PALA; (∆) 1:1 mixture of diC14-PALA and diC18-PALA. [diCn-PALA] ) 1 mM, [HEPES] ) 10 mM, and pH ) 7.0. phospholipids between the inner and outer layers exists. For smaller particles due to the dissymmetry of these two layers, this method may be exploited so as to give information on the particle size.25,26 The NMR spectra were obtained with a AM 400 Bruker spectrometer working at 161 MHz. Hexamethylphosphoramide (HMPA) was used as an external reference. Experiments were run at different temperatures as indicated in the text and figures.
Results and Discussion In a previous paper we measured the transition temperatures Tm for the dispersions of pure diCn-PALA obtained by sonication.12 We have checked that similar results are obtained when the extrusion method is used to prepare the dispersions. Here we show how Tm can be modulated by using mixtures of amphiphiles. In Figure 1 is represented the degree of polarization of DPH as a function of temperature for an equimolar mixture of diC14PALA and diC18-PALA. The results obtained for the pure products are also shown for the sake of comparison. The melting temperature of the mixture is intermediate between the Tm values of the pure compounds. The transition takes place over a much larger temperature domain than for the latter compounds. Note that the two amphiphiles were intimately mixed before sonicating the dispersion; for this reason we do not expect the coexistence of two kinds of particles as observed in other circumstances.21 In Figure 2 we have done the same kind of experiment mixing now diC18-PALA with DMPC again in equimolar proportion. The transition temperature of the mixture appears to be much closer to the Tm of diC18PALA than to the Tm of DMPC. So in that case the effect of adding an amphiphile with a shorter chain is weaker than expected. Addition of cholesterol is known to make the bilayer more rigid.27 This is indeed what is observed in Figure 3 where we compare the behavior of pure diC14PALA with the same compound after addition of cholesterol in proportion of 1 mol of cholesterol for 2 mol. Obviously in that case there is no well-defined transition. The mobility of the DPH probe, according to its degree of polarization, is only slowly changing with increasing the temperature, and it remains much weaker than in the pure compound. (25) Fendler J. H. In Membrane Mimetic Chemistry; John Wiley & Sons: New York, 1982; p 129. (26) De Kruijff, B.; Cullis, P. R.; Radda, G. K. Biochim. Biophys. Acta 1975, 406, 6. (27) Shinitzky, M.; Barenholz, Y. Biochim. Biophys. Acta 1978, 515, 367.
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Langmuir, Vol. 17, No. 21, 2001 6429
Figure 2. Fluorescence polarization P of DPH versus temperature for the following: (O) diC18-PALA; ()) DMPC; (∆) 1:1 mixture of diC18-PALA and DMPC. [diC18-PALA] ) 1 mM, [DMPC] ) 1 mM, [HEPES] ) 10 mM, and pH ) 7.0.
Figure 4. Gel permeation chromatographic separation of glucose-containing vesicles of diC14-PALA from free glucose, depending on the preparation technique. [diC14-PALA] ) 7 mM, eluent ) 10 mM HEPES, and pH ) 7.0.
Figure 3. Fluorescence polarization P of DPH versus temperature for diC14-PALA (1 mM) (∆) without or (0) with addition of cholesterol (0.5 mM). [HEPES] ) 10 mM, and pH ) 7.0.
Figure 5. Gel permeation chromatographic separation of glucose-containing vesicles, prepared by extrusion, from free glucose: comparison of diC14-PALA and diC18-PALA (concentration 7 mM). Eluent ) 10 mM HEPES, and pH ) 7.0.
Other mixtures of amphiphiles, not reported here, were also investigated. All the results demonstrate the possibility of modifying the transition temperature by adequately choosing the additive (we recall that, among the series of diCn-PALA investigated, only diC12-PALA has a Tm value lower than the physiological temperature). Glucose encapsulation was found to be systematically larger for the extruded dispersions compared to the sonicated ones. This is illustrated in Figure 4 for the case of diC14-PALA at concentration 7 mM. This figure shows the chromatograms obtained at room temperature from size exclusion chromatography and glucose analysis. The encapsulated glucose is well separated from the free one. It is found, as expected, in the eluted fractions corresponding to the turbidity peaks recorded at 254 nm. The encapsulated volume was found to be 0.93% (or 1.33 L mol-1) for the extruded dispersion and only 0.1% (or 0.14 L mol-1) for the sonicated system. Only small changes were observed when the lengths of the amphiphile alkyl chains were changed. Figure 5 shows a comparison between vesicles of diC18-PALA (encapsulated volume 1.1% or 1.57 L mol-1) and of diC14-PALA (see values given above), both obtained by the extrusion method. The variations of the percent of trapped glucose versus the length of the alkyl chains of diCn-PALA are represented in Figure 6 for the two preparation methods considered here. The much lower values obtained when using sonication are in agreement with the results of cryo-TEM
Figure 6. Amount of encapsulated glucose (%) as a function of alkyl chain length of diCn-PALA (7 mM), with vesicles prepared by ([) sonication and (9) extrusion.
imaging,12 which indicated a spectacular difference between the two kinds of preparation: the population of dispersed objects, whatever they are (vesicles, flat disks, bilayer fragments, etc., depending on the chain lengths), was found to be in a much more fragmented state in the case of sonicated systems compared to extruded systems. A similar observation was reported by Pansu et al.28 when vesicles of DODAC (N,N-dimethyl-N,N-dioctadecylam(28) Pansu, R. B.; Arrio, B.; Roncin, J.; Faure, J. J. Phys. Chem. 1990, 94, 796.
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Oliger et al.
Table 1. Influence of Temperature (Thermostated Chromatographic Column) on Glucose Encapsulation by Different Types of Dispersions Obtained by Extrusiona glucose encapsulation (%) for fixed temp during elution dispersion composn
Tm (°C)
diC12-PALA diC14-PALA diC16-PALA diC18-PALA diC16-PALA + DPPC diC16-PALA + cholesterol
25 41 54 64
a
15 °C 0.99
25 °C 0.67 1.07 0.81 1.1 1
35 °C
45 °C
55 °C
0.98 0.66
0.09 0.74
0.09
0.93 1.54
0.2 1.34
65 °C
0.07 0.57
0.23
Total amphiphile concentration ) 7 mM, cholesterol ) 3.5 mM, [HEPES] ) 10 mM, and pH ) 7.0.
monium chloride) were prepared by sonication. Many parameters are known to affect the entrapment efficiency, including the preparation method, as reported in other circumstances.29 In addition we can find a second reason to explain the low glucose encapsulation by the sonicated dispersions: the phosphorus analyses made on the eluted fractions have shown that only 40% of the total amphiphile was recovered in case of sonicated diC18-PALA, whereas 90% or more was recovered for the extruded systems, whatever the chain lengths. The fact that a nonnegligible part of the amphiphile was lost on the chromatographic column when sonication was used is obviously contributing to the diminution of the entrapment capacity. The permeability of vesicles is known to be affected by their fluidity (at T > Tm, the addition of cholesterol, which makes the amphiphile membrane more rigid, brings about a decrease of the permeability).30 We have investigated the extent of glucose entrapment as a function of temperature for the different chain lengths. The gel chromatography separation technique was used, with a temperature control of the column. The results of glucose encapsulation have been collected in Table 1, where are also shown for the sake of comparison some results obtained with phosphatidylcholines and with addition of cholesterol. All the dispersions were prepared by the extrusion method. The data clearly show a relationship between the variation of the percent of encapsulation and the transition temperature, Tm. Bringing the temperature above Tm is systematically associated with a significant drop of the extent of encapsulation. As expected the addition of cholesterol results in an increase of the rate of encapsulation and to a decrease of the sensitivity to temperature. If a temperature above Tm is clearly increasing the permeability of the vesicles, which could explain by itself the weaker retention of glucose, one should not forget the possible contribution of a second effect. Indeed one of the drawbacks of gel chromatography for separating the free probe from the encapsulated one is the risk of adsorption of the lipids on the gel. We may assume that this risk is increased in the fluid state of the membrane as compared to its rigid state, with potential destabilization of some vesicles. We have also checked that a dispersion of diC18-PALA, initially prepared in the absence of glucose, can incorporate some glucose when it is equilibrated for 2 h with a 0.5 M concentration of this probe. However the penetration occurs only when the equilibration is carried out at 60 °C (temperature close to Tm) and not at 25 °C. In addition the final content was found to be much higher for extruded dispersions compared to sonicated ones. These results demonstrate that the interaction of the amphiphiles with the Sephadex gel can only marginally be invoked to explain (29) Monnard, P.-A.; Oberholzer, T.; Luisi, P. L. Biochim. Biophys. Acta 1997, 1329, 39. (30) See ref 10, p 117.
Figure 7. Variation of the amount of encapsulated glucose (%) versus diC14-PALA concentration. Dashed line: theoretical prediction from eq (1) with a0 ) 61 Å2 and Dw ) 73 nm (see text).
the preceding observations. The fact that no glucose was detected in the particles cleared of the free glucose when the equilibration was carried out at 25 °C is an indication that glucose adsorption on the exterior of the particles can be totally neglected. The thermosensitivity of the entrapment efficiency has been observed in several instances31-34 (sometimes in an unexpected direction34), and it is an important factor which has been considered to improve therapeutic treatments and the antitumor efficacy of encapsulated drugs.32,33 Particle size measurements were performed both for the extruded and for the sonicated dispersions, varying the chain lengths. Surprisingly the hydrodynamic diameter was found almost independent of the length of the alkyl chains,12 with an average value of 81 nm for the extruded particles and of 61 nm for the sonicated particles. In the first case the values are in satisfactory agreement with the results of cryo-TEM imaging. The agreement is less good in the second case. The size measurements from quasielastic light scattering (QELS) should be considered with caution, considering that the data treatment assumes the presence of spherical particles, whereas different types of objects, including flat disks, are present in the dispersions.12 We have measured the variation of the extent of glucose entrapment by extruded dispersions when the concentration of diC14-PALA was increased. The results are presented in Figure 7. They show a nice linear dependence. This behavior is totally different from that reported before for the case of sonicated diC18-PALA dispersions:11 not only the extent of encapsulation was much lower (in (31) Sumida, Y.; Masuyama, A.; Takasu, M.; Kida, T.; Nakatsuji, Y.; Ikeda, I.; Nojima, M. Langmuir 2000, 16, 8005. (32) Merlin, J.-L. Eur. J. Cancer 1991, 27, 1026. (33) Sandip, B.Tiwari; Udupa, N.; Rao, B. S. S.; Devi, P. Uma Indian J. Pharmacol. 2000, 32, 214. (34) Gaber, M. H. J. Microencapsulation 1998, 15, 207.
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Figure 8. 31P NMR signal intensity (in % of its initial value) as a function of the molar ratio between Mn2+ concentration and amphiphile concentration (10 mM): ([) diC14-PALA, vesicles prepared by extrusion; (2) diC18-PALA, vesicles prepared by sonication. Temperature of the experiments: 25 °C.
accordance with all the observations reported above or in the previous publications) but the percent encapsulation Ev was tending toward a saturation value at high amphiphile concentration. This was put in relation with the change of the particle size independently determined, according to11
Ev (%) )
25 cNAa0Dw 3
(1)
where c is the amphiphile concentration, NA the Avogadro number, a0 the surface/polar head, and Dw the diameter of the water core of the particles, i.e., the hydrodynamic diameter decreased by two times the thickness of the bilayer. With the extrusion method, where the dispersion is forced through membrane pores of defined diameters, the particle diameter remains practically unchanged and a linear variation of Ev versus c is resulting as observed in Figure 7. The dashed line represents the theoretical expected variation from the preceding equation (assuming Dw ) 73 nm and a0 ) 61 Å2, as previously reported for phosphatidylcholine at the inner surface of vesicles).35 The gap between the experimental and theoretical curves can readily be explained from the observation that a part of the amphiphile contributes to the formation of flat disk structures, which does not have an inner aqueous core. We should like to mention that we were surprised to find that the size of the extruded particles measured by QELS was almost independent of the alkyl chain lengths of diCn-PALA, whereas the turbidity (at room temperature) strongly increased when increasing the n value. In fact we came to the conclusion that this was due, at least in part, to the progressive modification of the state of the bilayer (from fluid to rigid when increasing n), since the fact of bringing the temperature of the diC18-PALA from room temperature to 60 °C considerably decreased the turbidity. Finally we attempted to use 31P NMR spectroscopy in view of demonstrating the unilamellarity of the vesicles (see Experimental Section). Here again we were faced with important differences in behavior depending on whether the dispersions were obtained by sonication or extrusion. The 31P NMR signal intensity has been measured as a function of the concentration of Mn2+ added in the dispersion. In Figures 8 and 9 we have plotted the (35) See ref 25, p 130.
Figure 9. 31P NMR signal intensity (in % of its initial value) as a function of the Mn2+/amphiphile molar ratio for diC14PALA (10 mM), with vesicles prepared by extrusion. Temperature of the experiments: (O) 25 °C; (9) 45 °C.
ratios of the signal intensities measured with and without Mn2+, respectivelly, as a function of the Mn2+/amphiphile molar ratio (amphiphile concentration 10 mM). The maximum Mn2+ concentration used never passed 10% of the amphiphile, but it is expected that the metal ions are rapidly exchanging between the polar heads. A too large concentration would have destabilized the vesicles by osmotic shock, and for this reason, we used as low concentrations as possible. We compare in Figure 8 the results obtained at 25 °C for two kinds of dispersions, namely a dispersion of diC14PALA obtained by extrusion and a dispersion of diC18PALA obtained by sonication. The results are in line with what could be expected from the previously reported experiments, such as glucose entrapment or cryo-TEM imaging.11,12 In sonicated diC18-PALA we know that only few vesicles are present, coexisting with a population of flat disks or bilayer fragments.12 A large number of polar heads are thus in contact with Mn2+, which explains the rapid decrease of the NMR signal from the first additions of Mn2+. A plateau is found at a Mn2+/amphiphile molar ratio around 0.02-0.03, which corresponds to a remaining signal of about 15%. This would be consistent with the existence of a reduced number of vesicles, but they would be destroyed by further additions of Mn2+, being less stable than those obtained by extrusion. With diC14-PALA the signal decrease is slower and a stable asymptote is obtained at about 25%, indicating that one-quarter of the amphiphile polar heads have no contact with Mn2+. If one takes the size of the particles measured by QELS, i.e., ∼80 nm, and takes an approximate value of 40 Å for the thickness of the bilayer, a simple calculation25 indicates that the number of amphiphiles in the outer layer of unilamellar spherical vesicles should be approximately 1.25 times larger than the number in the inner layer. If all the amphiphile was included in such vesicles, we would expect a remaining NMR signal of 44% of its value in the absence of Mn2+. As we know from cryo-TEM experiments that the vesicles are coexisting with circular flat disk (which are accessible to Mn2+ on both sides), the experimental value of 25% seems in reasonable agreement. Remembering that at the temperature of 45 °C the encapsulation of glucose by diC14-PALA vesicles drops from about 1% to 10 times less (see Table 1), we wanted to check if the change of state of the bilayer (Tm ) 41 °C) is also accompanied by a permeability of the membrane to Mn2+ ions. In that case the NMR signal should completely vanish. This is not the case according to the results reported in Figure 9, where we compare the effect of Mn2+ on diC14-PALA dispersions at 25 and 45 °C, respectively.
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The change of the equilibrium temperature has no effect on the asymptotic behavior. These results tend to indicate that, contrary to glucose which is a neutral probe, the Mn2+ ion is not easily transferred across the fluid membrane. The electrostatic attractions between Mn2+ and the negatively charged amphiphile headgroups may explain that the metal ions remain at the surface of the particles even if chain fluctuations can favor the permeation of a neutral molecule. Another explanation could have been an increase of Tm induced by the presence of the divalent metal ions as reported in other circumstances.36 We can definitely rule out the latter hypothesis since we checked that in our experimental conditions the Tm value remains practically unaffected by the addition of Mn2+. Conclusion The general trends observed in the experiments reported here are consistent with the current knowledge on the behavior of classical phospholipid vesicles. However the dispersions studied here are of a complex nature since different types of particles are coexisting (true vesicles, (36) Kwon, K. O.; Kim, M. J.; Abe, M.; Ishinomori, T.; Ogino, K. Langmuir 1994, 10, 1415.
Oliger et al.
flat disks, bilayer fragments), as confirmed both by the glucose entrapment and the 31P NMR experiments. For this reason different techniques have appeared necessary to be able to propose a comprehensive description of these systems. We can conclude from the experiments that sonicated and extruded dispersions of diCn-PALA have quite different properties, with the latter ones presenting more of the characteristics usually associated with closed vesicular objects. This confirms our previous investigation using cryo-TEM imaging.12 If the alkyl chain length has no important effect on these properties, however, the permeability of the vesicles is strongly dependent on the transition temperature, Tm, itself closely related with the alkyl chain length. We have also shown in this work that Tm can be modulated by addition of lecithins or cholesterol. This may be important to optimize the intracellular transport of the PALA derivatives. Biological tests are now in progress to test the activity of the dispersions on cell cultures. Acknowledgment. The 31P NMR measurements were performed using the NMR facilities of Universite´ Henri Poincare´-Nancy I. The technical assistance of Mrs. E. Eppiger is greatly acknowledged. LA010332X
