Combined Cryogenic Transmission Electron Microscopy and Electron

Synthesis, Liposomal Preparation, and in Vitro Toxicity of Two Novel Dodecaborate Cluster Lipids for Boron Neutron Capture Therapy. Eugen Justus, Doaa...
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Combined Cryogenic Transmission Electron Microscopy and Electron Spin Resonance Studies of Egg Phosphatidylcholine Liposomes Loaded with a Carboranyl Compound Intended for Boron Neutron Capture Therapy Simona Rossi,*,† Go¨ran Karlsson,‡ Giacomo Martini,† and Katarina Edwards‡ Dipartimento di Chimica, Universita` di Firenze, Polo Scientifico, Via della Lastruccia 3, 50019 Sesto Fiorentino, Firenze, Italy, and Department of Physical Chemistry, Uppsala University, P.O. Box 579, S-751 23 Uppsala, Sweden Received December 30, 2002. In Final Form: April 15, 2003 The lactosyl carborane [1,2-dicarba-closo-dodecarboran(12)-1-ylmethyl](β-D-galactopyranosyl)-(1f4)β-gluco-pyranoside (LCOB) is an amphipilic boron-containing compound intended for boron neutron capture therapy. In this study cryogenic transmission electron microscopy was used to investigate the effects of LCOB on the structure and properties of extruded egg phosphatidylcholine liposomes. The results showed that LCOB concentrations up to at least xLCOB ) 0.25 could be included in the preparations without any noticeable effects on the liposomal size or structure. Inclusion of LCOB concentrations corresponding to xLCOB ) 0.44 or more gave, however, rise to significant changes in the liposome size distribution and the overall sample structure. In fresh samples no signs of phospholipid solubilization were detected until the molar fraction of LCOB reached 0.82. At this concentration the boronated compound induced, however, formation of open liposomes and threadlike micelles and, furthermore, with time the sample displayed a macroscopic phase separation. More details on the molecular interaction between LCOB and the liposome membranes were obtained by electron spin resonance spectroscopy. Two different n-doxyl stearic acid spin probes were used to measure the dynamics and the degree of order in the mixed systems. The results indicated that LCOB insertion into the phospholipid bilayer increased the packing order in the vicinity of the water/hydrocarbon interface whereas the inner hydrophobic region of the bilayer was unaffected.

Introduction Boron neutron capture therapy (BNCT) is a binary cancer targeted therapy based on the nuclear reaction that occurs when a stable isotope, 10B, is irradiated with low energy neutrons to yield 4He2+ (R particles) and 7Li3+ ions.1 The nuclear fragments thus produced are highly cytotoxic and move within tissues along distances (5 µm for Li particles and 9 µm for R particles) that are in the order of a cell diameter. As a result, those cells that have bound or taken up a 10B-containing drug may be selectively destroyed. For BNCT to be successful, the boron concentration in the tumor is required to be at least 20 µg of 10B/g of tissue.2 This means that approximately 109 atoms of 10B must be delivered to each tumor cell in order to achieve a significant therapeutic effect.3 Several different approaches have been developed and explored in order to maximize the boron uptake in the tumor cells, while at the same time avoiding negative effects on healthy cells and tissue.4-6 A promising alternative involves the use of macromolecular delivery vehicles, such as low density lipoprotein particles,7 and, † ‡

Universita` di Firenze. Uppsala University.

(1) Hawthorne, M. F. Mol. Med. Today 1998, 4, 174. (2) LaHann, T.; Lu, D. R.; Daniell, G.; Sills. C.; Craft, S.; Gavin, P.; Bauer, W. F. Advances in Neutron Capture Therapy; Barth, R. F., Soloway, A. H., Eds.; Plenum Press: New York, 1993; pp 585-589. (3) Hartman, T.; Carlsson, J. Radiother. Oncol. 1994, 31, 61. (4) Gabel, D. Radiother. Oncol. 1994, 30, 199. (5) Metha, S. C.; Lu, D. R. Pharm. Res. 1996, 13, 344. (6) Soloway, A. H.; Tjarks, W.; Barnum, B. A.; Rong, F.-G.; Barth, R. F.; Codogni, I. M.; Winson, J. G. Chem. Rev. 1998, 98, 1515. (7) Setiawan, Y.; Moore, D. E.; Allen, B. J. Br. J. Cancer 1996, 74, 1705.

in particular, liposomes.8-17 The latter may be filled with large quantities of both hydrophobic and hydrophilic tumoricidal agents, and their ability to accumulate in tumor tissue has been well documented.18 Carboranes, such as dicarba-closo-dodecarborane,19,20 have been evaluated as promising new BNCT agents because of their high boron content and relatively high stability in vivo.6 The boronated compound used in this study (henceforth called LCOB) is a carbohydrate carborane hybrid composed of a highly lipophilic boron icosahedral cage and a hydrophilic lactosyl moiety (1).21 The chemical structure of LCOB, which may be classified (8) Carlsson, J.; Bohl-Kullberg, E.; Capala J.; Sjo¨berg, S.; Edwards, K., Gedda, L. J. Neurooncol., in press. (9) Shelly, K.; Feakes, D. A.; Hawthorne F.; Schmidt P. G.; Krisch, T. A.; Bauer W. F. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 9039. (10) Feakes, D. A.; Shelly, K.; Knobler, B.; Hawthorne F. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 3029. (11) Feakes, D. A.; Shelly, K.; Hawthorne, F. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 1367. (12) Mehta, S. C.; Lai, J. C. K.; Lu, D. R. J. Microencapsulation 1996, 13, 269. (13) Hawthorne, F.; Shelly, K. J. Neurooncol. 1997, 33, 53. (14) Yanagie, H.; Fujii, Y.; Tomita, T.; Sekiguchi, M.; Eriguchi, M.; Kobay, T.; Ono, K.; Kobayashi, H. Advances in Neutron Capture Therapy; Larsson, B., Crawford, J., Weinreich, R., Eds.; Elsevier Science: Amsterdam, 1997; Vol. II, Chemistry and Biology. (15) Perugini, P.; Pavanetto F. J. Microencapsulation 1998, 15, 473. (16) Bergstrand, N.; Johansson, M.; Edwards, K. J. Liposome Res. 1999, 9, 53. (17) Moraes, A. M.; Santana, H. A.; Carbonell, R. G. J. Microencapsulation 1999, 16, 647. (18) Medical Applications of Liposomes; Lasic D. D., Papahadjopoulus, D., Eds.; Elsevier Science: Amsterdam, 1998. (19) Watson-Clark, R. A.; Banquerigo, M. L.; Shelly, K.; Hawthorne, M. F.; Brahn, E. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 2531. (20) Valliant, J. F.; Guenther, K. J.; King A. S.; Morel, P.; Schaffer, P.; Sogbein, O. O.; Stephenson, K. A. Coord. Chem. Rev. 2002, 232, 173. (21) Tietze, L. F.; Bothe, U. Chem. Eur. J. 1998, 4, 1179.

10.1021/la0209841 CCC: $25.00 © 2003 American Chemical Society Published on Web 06/07/2003

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as a “third generation” boronated compound for use in BNCT, suggests that the molecule possesses surfactant properties.

The ortho-carborane has the potential to mediate a firm anchorage of the LCOB molecule in the hydrophobic domain of the bilayer. The function of the carbohydrate moiety is double: to increase the solubility in water and to escort and bind the boronated compound to the tumor cell surface.22,23 The rationale behind the latter is based on the fact that high concentrations of lactose-binding lectins are present on the neoplastic membrane surface and several β-lactosyl derivatives are known to be preferentially retained in tumors.24 In this work we employed cryogenic transmission electron microscopy (cryo-TEM) to carry out systematic studies of the structural effects caused by inclusion of the carboranyl lactoside, LCOB, in egg phosphatidylcholine (EPC) liposomes. To improve the understanding of the perturbations brought about by insertion of the LCOB into the lipid membrane, we complemented the cryo-TEM analysis with electron spin resonance (ESR) spectroscopy using lipophilic spin probes.25,26 The main intention of the present study was to explore the potential of phospholipid liposomes as carriers for LCOB and related carboranyl glycosides. LCOB represents a new and interesting class of surfactants, and information regarding the molecules aggregation behavior and interaction with lipid bilayers is of great value also from a more fundamental point of view. Experimental Section Materials. Egg yolk lecithin (EPC) of grade 1 was purchased from Lipid Products (Nutfield, England). The boronated drug, 1, was a lactosyl-ortho-carborane, LCOB, which was a gift of Professor L. Panza and synthesized as described in ref 27. The compound was chromatographically pure as verified by NMR measurements. Structural and further purity characterizations were carried out with 1H NMR, 11B NMR,28 and mass spectroscopy. Nitroxide radicals n-doxyl stearic acids, n-DSA, 2, were purchased from Sigma Chemicals, Mu¨nchen, Germany, and used (22) Peymann, T. Sauerstoff und Schwefel am Undecahydro-closododecaborat(2-)als Nucleophile. Ph.D. Thesis, University of Bremen, Germany, 1995. (23) Gabel, D.; Harfst, S.; Moller, D.; Ketz, H.; Peymann, T.; Ro¨sler, J. In Current Topics in the Chemistry of Boron; Kabalka, G. W., Ed.; The Royal Society of Chemistry: Cambridge, U.K., 1994; p 161. (24) Raz, A.; Lotan, R. Cancer Res. 1981, 41, 1, 3642. (25) Berliner, L. J., Ed. Biological Magnetic Resonance. Spin Labeling: The Next Millennium; Plenum Press: New York, 1998; Vol. 14. (26) Berliner, L. J., Ed. Spin Labeling. Theory and Applications; Academic Press: New York, 1976, Vol. 1, and 1979, Vol. 2. (27) Giovenzana, G. B.; Lay, L.; Monti, D.; Palmisano, G.; Panza, L. Tetrahedron 1999, 55, 14123. (28) Unpublished data from the Department of Chemistry, University of Florence.

Langmuir, Vol. 19, No. 14, 2003 5609 without further purification. All other salts and reagents were of analytical grade and used as received.

Preparation of the Liposomes. Lipid/LCOB mixtures were prepared by dissolving the appropriate amounts of lipids and lactosyl-carborane 1 in a 1:1 (v/v) mixture of chloroform/methanol. The solvent was removed under a gentle stream of nitrogen, and the remaining solvent traces were evaporated under vacuum. The dried lipid/LCOB film was suspended in HEPES buffer (0.15 M NaCl, 0.02 M HEPES, pH ) 7.4), and the lipid mixtures were subjected to at least eight freeze-thaw cycles (including freezing in liquid nitrogen and heating to above 323 K), whereafter the resulting solutions were extruded (30 times) through polycarbonate filters (pore size 100 nm) mounted in a Liposofast miniextruder from Avestin, Ottawa, Canada. The EPC concentration was invariantly 10-3 M and the total final concentration of LCOB was kept between 0.34 × 10-3 and 4.62 × 10-3 M. In the following the amount of lactosyl-carborane added to the lipids will be given in molar fraction. The samples were thermostated at 298 K before each measurement. Preparation of Pure LCOB Solutions and of Spin-Labeled Liposome Dispersions. Films of the n-DSA spin probes were formed from their chloroform solutions, dried overnight under a nitrogen stream to remove all traces of the organic solvent. Water solutions of the labeled lactosyl-carborane were prepared by dissolving the spin probe film in solutions at LCOB concentrations from 4 × 10-4 to 10-2 M. For equilibration the solutions were shaken several times and maintained at 323 K for at least 24 h before the spectroscopic measurements were carried out. The n-DSA concentration was 2 × 10-5 M, low enough to prevent any line width broadening by spin-spin effects. Spin-labeled dispersions of EPC and EPC/LCOB were prepared by adding the liposome dispersions to the dried film of n-DSA to a final n-DSA concentration of 10-5 M, and the samples were gently stirred for 5 min. The ESR spectra were always taken not later than 1 h after the addition of the spin probes to the dispersions. Methods. Electron Spin Resonance Spectra. The Bruker model 200D ESR spectrometer operating at X band (∼9.5 GHz) was used for ESR measurements. Data acquisition was performed with STELAR software, and temperature was controlled with Bruker VTB 3000 accessory ((0.5 K accuracy). Quartz tubes of inner diameter N-O rather than the actual size of the reorienting object. The restoring potential S20 ) [〈3 cos2 θ - 1〉]/2, where θ is the angle between the molecular axis of the doxyl stearic acid and the normal to the bilayer, used in the calculations, gave information on the spin label microscopic orientational ordering. The macroscopic disorder of the system, due to the random orientation of local bilayer (29) Schneider, D. J.; Freed, J. H. Biological Magnetic Resonance. Spin Labeling. Theory and Applications; Berliner, L. J., Reuben, J., Eds.; Plenum Press: New York, 1989; Vol. 8, p 1 and references therein. (30) Budil; D. E.; Lee, S.; Saxena, S.; Freed, J. H. J. Magn. Reson. 1996, 120, 155. (31) Meirovitch, E.; Nayeem, A.; Freed, J. H. J. Phys. Chem. 1984, 48, 3454. (32) Ge, M.; Freed, J. H. Biophys. J. 1998, 74, 910. (33) Butterfield, D. A. Biological Magnetic Resonance; Berliner, L. J., Reuben, J., Eds.; Plenum Press: New York, 1982; Vol. 4, p 1. (34) Hemminga, M. Chem. Phys. Lipids 1983, 32, 323.

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Figure 1. Turbidity as a function of the LCOB concentration at different times after the preparation of EPC liposome dispersions: (9) 10 min; (4) 48 h; (2) 95 h; (b) 167 h. The EPC concentration in all samples was 10-3 mol/dm3. The LCOB molar fractions are indicated in the figure. directors, was taken into account by averaging spectra over 30 different orientations (NORT)30). Turbidity Measurements. A Hewlett-Packard 8453 UV-visible spectrophotometer connected to a LAUDA RC6 CP thermostat set to 298 K was used for the turbidity runs. Quartz 1 cm length cuvettes were used and turned upside down a couple of times before measurement. The absorbance of each dispersion was measured against a blank of HEPES buffer solution. Cryogenic Transmission Electron Microscopy. Electron microscopy investigations were performed with a Zeiss EM 902A instrument operating at 80 kV. The technique used for cryoTEM examination is described in detail elsewhere35-37 and can be briefly summarized as follows. Thin sample films (10-500 nm) of the mixed systems were prepared at controlled temperature (298 K) and controlled relative humidity (98-99%) within a custom-built environmental chamber. The films were thereafter vitrified by quick freezing in liquid ethane and transferred to a transmission electron microscope for examination. Temperature was kept below 108 K during both transfer and examination. A large number of areas were examined for each sample, and the micrographs shown were chosen to give a representative picture of the sample structure.

Results and Discussion Turbidity Measurements. To get a first indication on the effects caused by LCOB on EPC liposomes, we carried out a series of systematic turbidity measurements. Figure 1 shows the absorbance recorded at 350 nm (at this wavelength the lactosyl carborane did not absorb significantly up to 10-2 M) for extruded samples containing 10-3 M EPC and increasing amounts of LCOB. The measurements showed that the turbidity initially increased with increasing LCOB concentration to reach a maximum value at [LCOB] ) 1.7 × 10-3 M, corresponding to xLCOB ) 0.63. At higher LCOB concentrations, we observed instead a pronounced and concentration-dependent decrease in the turbidity. Upon prolonged aging (up to 7 days from sample preparation), a general increase in the turbidity was detected. The increase was particularly dramatic for the (35) Almgren, M.; Edwards, K.; Karlsson, G. Colloids Surf., A 2000, 174, 3. (36) Bellare, J. R.; Davis, H. T.; Scriven, L. E.; Talmon, Y. J. Electron Microsc. Technol. 1988, 10, 87. (37) Dubochet, J.; Adrian, M.; Chang, J.; Homo, J.; Lepault, J.; McDowall, A. W.; Schultz, P. Q. Rev. Biophys. 1988, 21, 129.

Figure 2. cryo-TEM micrograph of a 5 × 10-3 M LCOB solution in HEPES buffer. The inset shows a cluster of small particles close to the edge of the polymer film. T ) 298 K. Bar ) 100 nm.

sample with the highest LCOB molar fraction, i.e., xLCOB ) 0.82, and 48 h after sample preparation visual inspection revealed in this case a macroscopic phase separation. The increase in turbidity observed for xLCOB ) 0.63, and below, suggested that the presence of LCOB induced either growth or aggregation of the liposomes. The drop in turbidity at higher LCOB concentrations was, on the other hand, taken as an indication of micelle formation. To further characterize the aggregate structure and phase behavior in the EPC/LCOB mixed system, we carried out a series of cryo-TEM and ESR measurements. cryo-TEM and ESR Investigations of the Pure LCOB/Water System. Figure 2 shows the microstructure found in buffer solutions containing 5 × 10-3 M LCOB. The micrographs were dominated by electron dense, roughly spherical, objects with a size in the range of 1015 nm. In addition to these structures, the sample also contained a proportion of more complex aggregates and clusters (see enlarged inset). The occurrence of aggregates and clusters was rather common. From their appearance in the micrograph it is tempting to identify the small spherical particles in Figure 2 as large globular micelles. It is difficult to see, however, how the geometry and the small volume of the ortho-carborane cage (the HYPERCHEM calculus program suggests a van der Waals volume of about 12 Å3) could be compatible with packing into micelles with diameters of 10 nm, or more. Further, from the chemical structure of the carborane moiety it is clear that the interior of the LCOB particles cannot possess the same liquidlike properties that are typically found in the hydrophobic core of conventional micelles. The ESR measurements reported below show, however, that the LCOB structures are capable of dissolving fatty acid derivatives, and this fact indicates that the packing of the carborane units is characterized by a certain amount of disorder. The exact nature of the LCOB aggregates cannot be determined based on the present investigations but will have to await further analysis.

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Figure 3. ESR spectra of 5-DSA (2 × 10-5 M) as a function of LCOB content in HEPES buffer solutions: (a) 8 × 10-4 M; (b) 10-3 M; (c) 3 × 10-3 M; (d) 10-2 M. Asterisks in spectrum b indicate the absorption component corresponding to 5-DSA in aqueous monomeric form. T ) 298 K.

ESR analysis of solutions containing different concentrations of LCOB in HEPES allowed us to obtain an estimate of the critical aggregation concentration. Figure 3 shows the 298 K ESR spectra of 5-DSA (2 × 10-5 M) in LCOB solutions at different concentrations. The probe is preferentially soluble in hydrophobic milieus, such as the interior of micelles and related self-aggregated structures, but spectra recorded in HEPES buffer in the presence of LCOB e 8 × 10-4 M (Figure 3a) exhibited the usual threeline pattern observed for aqueous solutions where the probe is free to move unrestricted. This indicated that all the LCOB molecules were dissolved as monomers at this concentration. When the LCOB concentration was increased to 10-3 M or more, the ESR spectra became complicated, however, by the superposition of signals arising from 5-DSA probes located (1) in an aqueous environment and (2) in an environment offering a more restricted mobility (Figure 3b-d). The latter signals arose presumably from probe molecules situated in multimolecular aggregates formed by LCOB, and thus it was reasonable to assume that LCOB started to self-aggregate at a concentration between 8 ×10-4 and 10-3 M. cryo-TEM of LCOB Containing Liposomes. Micrographs obtained from pure EPC reference samples were dominated by unilamellar liposomes with diameters ranging from 80 to 120 nm (Figure 4a). The liposomes often appeared invaginated, a phenomenon frequently observed for EPC liposomes in saline medium and commonly attributed to osmotic shrinkage caused by a small amount of water evaporation during the sample preparation.38,39 Addition of low concentrations of the (38) Vinson, P. K.; Talmon Y.; Walter A. Biophys. J. 1989, 56, 669.

Figure 4. cryo-TEM micrographs of samples containing EPC and LCOB in molar fractions of (a) 0 and (b and c) 0.63. Black arrows in (a) denote ice crystals deposited on the sample surface after vitrification, and the white arrow indicates a multilamellar liposome showing signs of radiation damage. Note the extremely small liposomes with diameters of between 10 and 30 nm in (b) and the large cluster consisting of a vast number of small size liposomes in (c). Bar ) 100 nm.

lactosyl-carborane did not appear to affect the liposomal size or structure: cryo-TEM pictures of EPC liposomes containing LCOB in concentrations corresponding to xLCOB ) 0.25 revealed a morphology similar to that shown in Figure 4a (data not shown). A significant change in the size distribution was observed, however, when the LCOB molar fraction was increased to 0.44. At this concentration the lactosyl-carborane induced the formation of a popula(39) Baillie, A. J.; Florence, A. T.; Hume, L. R.; Muirhead, G. T.; Rogerson, A. J. Pharm. Pharmacol. 1985, 37, 863.

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tion of liposomes with diameters ranging from 10 to 40 nm. As the LCOB concentration was further increased, the number of small liposomes increased. At xLCOB ) 0.63 (Figure 4b,c) extremely small liposomes with diameters ranging from 10 to 30 nm were frequently observed in coexistence with liposomes in the size range of 80-100 nm. A similar behavior has previously been detected by cryo-TEM in liposomal dispersions of phospholipids and nonionic surfactants, such as octyl-glucoside and octa(ethylene glycol) n-dodecyl monoether (C12E8).40 Small liposomes with diameters of about 20 nm have, furthermore, been reported to exist in certain complex systems41-44 as for instance in the water/1-octanol/Triton X-100/ cetylpyridinium chloride system.42 The micrographs obtained at xLCOB ) 0.63 exposed a second interesting effect caused by the incorporation of LCOB: both populations of the liposomes developed a tendency to aggregate and form clusters, a phenomenon which coincided with the maximum in the turbidity profile shown in Figure 1. The above-mentioned tendency for aggregation disappeared when the molar fraction of LCOB was increased to 0.73. As seen in Figure 5a the liposomes now appeared well separated in the micrographs. This was presumably due to the high density of lactosyl polar headgroups at the surface of the liposomes, which strengthened the repulsive hydration forces between the particles. Both size distributions (small and large liposomes) were, however, still present, and the bilayer integrity of the liposomes remained intact. The aggregate structure remained essentially the same when the LCOB concentration was increased to xLCOB ) 0.79 (Figure 5b). However, the number of small liposomes had increased at the expense of the larger ones, and thus the average size of the liposomes decreased. This might explain the lower turbidity recorded at xLCOB ) 0.79 compared to xLCOB ) 0.73 (Figure 1). It is noteworthy that the large liposomes in panels a and b of Figure 5 displayed irregular bilayer profiles, as if the liposomes were delimitated by a rippling bilayer. The deviation from the smooth bilayer profile exhibited by pure EPC liposomes (Figure 4) suggests the existence of domains of segregated LCOB molecules that cause a locally different curvature. Similar irregular liposomes have been observed in other systems, such as in mixtures of monoolein, sodium oleate, and oleic acid.45 Figure 5c shows that further increase in the LCOB concentration resulted in the onset of liposome solubilization. At xLCOB ) 0.82 the sample contained only a limited number of liposomes and most of them displayed bilayer openings from which seemed to originate short threadlike micelles, a few tens of nanometers in length. To check the stability of the EPC/LCOB dispersions, we repeated the cryo-TEM investigations at regular times during the first week after sample preparation. No significant morphological changes were noted in the LCOB/EPC mixed systems having LCOB concentrations corresponding to xLCOB ) 0.44, or less. However, in samples containing high concentrations of the lactosyl-carborane, we observed some interesting effects of aging. Figure 6a (40) Johnsson, M.; Edwards, K. Langmuir 2000, 16, 8632. (41) Bernheim-Groswasser, A.; Zana, R.; Talmon, Y. J. Phys. Chem. B 2000, 104, 12192. (42) Oberdisse, J.; Couve, C.; Appel, J.; Berret, J. F.; Ligoure, C.; Porte, G. Langmuir 1996, 12, 1212. (43) Oberdisse, J.; Regev, O.; Porte, G. J. Phys. Chem. B 1998, 102, 1102. (44) Oberdisse, J.; Porte, G. Phys. Rev. E 1997, 56, 1965. (45) Borne´, J.; Nylander, T.; Khan, A. J. Phys. Chem. B 2002, 106, 10492.

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Figure 5. cryo-TEM micrographs of samples containing EPC and LCOB in molar fraction of (a) 0.73, (b) 0.79, (c) 0.82. The arrow in (a) denotes a multilamellar liposome. Note the irregularity of the bilayer surface in (a) and (b). Note also that the liposomes did not aggregate but appeared well separated. Bar ) 100 nm.

shows a typical micrograph of the xLCOB ) 0.63 dispersion vitrified 7 days after preparation. The sample was still characterized by intact and predominantly aggregated liposomes but displayed a strong tendency to form elongated or even tube-shaped liposomes. The tubules exhibited swollen ends, and their length could reach several hundred nanometers. For samples containing xLCOB ) 0.73 the structural changes caused by aging were even more dramatic. cryo-TEM images obtained 1 week after sample preparation showed that most of the closed

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Figure 6. cryo-TEM micrographs of EPC/LCOB liposome dispersion with (a) xLCOB ) 0.63 and (b) xLCOB ) 0.73 vitrified 7 days after sample preparation. Note the long tubular liposomes in (a) and the open liposomes and short threadlike micelles in (b). Bar ) 100 nm.

liposomes (compare Figure 5a) had been broken down into open structures and short threadlike micelles (Figure 6b). The reason behind the structural changes observed in the aged samples remains to be thoroughly investigated, but part of the explanation may be attributed to the extremely hydrophobic nature of the carborane part of the LCOB molecule. It would be very interesting to speculate on typical flipping times of LCOB molecules in the vesicles. However the poor characterization of the thermodynamical features of the LCOB molecule itself prevented this approach. The hydrophobic nature of LCOB can be expected to slow the redistribution of the LCOB between the aqueous solution and the liposomes and thus comparably long times may be needed for the solubilization of the liposome bilayers and the equilibration of mixed micelles. On the basis of this line of argument, the structures displayed in Figure 6 probably represent a stage closer to the true equilibrium than the closed, near spherical, liposomes found in fresh samples. However, it cannot be excluded that the accumulation of hydrolysis products, such as lysolipids and free fatty acids, in the aged sample might contribute to the observed structural changes. In particular, micelles have been reported to form in pure EPC samples upon incubation for 3 weeks at room temperature.46 Since the EPC liposomes in the present

Figure 7. cryo-TEM micrographs of phase-separated EPC/ LCOB liposome dispersion with xLCOB ) 0.82 vitrified 7 days after sample preparation: (a) branched and entangled threadlike micelles found in the upper phase (bar ) 100 nm); (b) polydisperse multilamellar liposomes originating from the lower phase (bar ) 500 nm).

study were supplemented with a micelle-promoting component, i.e., the LCOB, it is plausible that a smaller amount of hydrolysis products, and thus a shorter time, was required to induce breakdown of the liposome structure. As mentioned earlier, samples with LCOB concentrations corresponding to xLCOB ) 0.82 started to phase separate within 48 h after sample preparation. cryo-TEM images obtained from the upper, semitransparent phase 7 days after sample preparation revealed toroidal, branched, and entangled threadlike micelles (Figure 7a). To capture the structures present in the lower phase, which consisted of a white precipitate, we prepared TEM specimens from samples that had been vigorously shaken. Micrographs obtained after this treatment disclosed the presence of a polydisperse population of uni- and multi(46) Bergstrand, N.; Edwards, K. Langmuir 2001, 17, 3245.

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lamellar liposomes (Figure 7b). It should be pointed out that multilamellar liposomes in the size range of 0.5-1 µm, which represents the upper size limit for cryo-TEM, were frequently observed, and therefore it is possible that the bulk sample also contained some lamellar structures that were too large to be visualized by the present technique. Anyhow, the results presented in Figure 7 suggested that the xLCOB ) 0.82 dispersions with time separated into two distinct phases containing threadlike micelles and multilamellar liposomes, respectively. A similar macroscopic phase separation has previously been detected at high surfactant concentrations in several EPC/ nonionic surfactant systems.46,47 Aggregate Structure after External Addition of LCOB. In the previous paragraph we have discussed the aggregate structure in dispersions that were prepared by mixing LCOB and EPC before hydration and extrusion of the samples. For comparison we also investigated the structural effects brought about by external addition of LCOB to premade EPC liposomes. In this case the initial distribution of the lactosyl carborane between the inner and outer leaflet of the lipid bilayer becomes asymmetric. Our investigations showed that this fact primarily shifted the concentrations of LCOB needed for the inducement of liposome solubilization and mixed micelle formation to lower values. The type of aggregates formed, as well as their sequence of appearance, seemed to remain unaltered. Again we noted that the aggregate structure changed with time, and the samples were therefore followed over several days. The external addition of LCOB in concentrations corresponding to xLCOB ) 0.44 resulted in the formation of a population of small liposomes having diameters ranging from 20 to 40 nm (results not shown). In fresh samples these small liposomes coexisted with closed 80100 nm liposomes and the overall sample structure was thus similar to that observed after symmetric distribution of somewhat higher LCOB concentrations (compare panels b and c of Figure 4). However, when the sample was reexamined 4 days later, liposomes with holey, or perforated, bilayers were detected in coexistence with bilayer fragments and cylindrical micelles (results not shown). Figure 8 shows the aggregate structure captured in samples with LCOB/EPC molar ratios between 0.63 and 0.79. In all cases addition of LCOB promoted the formation of small liposomes and tubular structures. The latter often displayed a waistlike narrowing (pointed out by black arrows in panels a and c of Figure 8)sas if the tubes were in the process of budding off small liposomes. Threadlike cylindrical micelles were, furthermore, detected in all samples. At the higher LCOB concentrations the EPC/ LCOB mixed micelles formed immediately after addition of the lactosyl-carborane, whereas at lower LCOB concentrations a few days of incubation was needed before the micelles appeared. As exemplified in Figure 8b the micelles exhibited a strong tendency to form hook- or loopshaped ends. The micrographs in panels d and e of Figure 8 show how the gradual breakdown of individual liposomes eventually resulted in the formation of tight balls of entangled threadlike micelles. It is important to note that after 7 days of incubation at 298 K all the samples shown in Figure 8 still contained an appreciable amount of lamellar structures in the form of liposomes and bilayer fragments. A small population of liposomes was detected also when the EPC samples were supplemented with LCOB in molar fractions corresponding to 0.82. In agreement with our (47) Almgren, M. Biochim. Biophys. Acta 2000, 1508, 146.

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earlier observations, the sample displayed a phase separation and 4 days after LCOB addition a white precipitate and a semitransparent upper phase could be clearly distinguished. The aggregate structure in the two phases was similar to that shown in Figure 7, and no significant morphological changes were observed in samples that had been incubated at 298 K for 7 days. ESR Measurements. To explore the effects caused by LCOB on the bilayer fluidity, we carried out a series of systematic ESR measurements using n-doxyl stearic acid. In particular, 5-DSA and 16-DSA (2) were used to monitor local dynamic properties of lipid molecules in the bilayers of carborane-free and carborane-loaded liposomes. 5-DSA reported on the mobility near the polar headgroup region, whereas 16-DSA sensed dynamic properties toward the center of the bilayer. The final n-DSA:EPC molar ratio was kept below 1:100 in order to avoid line broadening effects in the ESR spectra. All measurements were, furthermore, carried out on freshly prepared liposome samples, and when present, LCOB was mixed with EPC before the hydration and extrusion procedure. Figure 9a compares the ESR spectrum of 5-DSA inserted into pure EPC liposomes (full line) with the spectrum of the same probe inserted into liposomes containing LCOB at concentrations corresponding to xLCOB 0.63 (dotted line). As commonly accepted for 5-DSA in ordered assemblies, we assumed that the paramagnetic moiety, i.e., the nitroxide group, was localized in the proximity of the water/ hydrocarbon interphase. A very small fraction of the overall adsorption (indicated by the arrows), which did not affect the original line shape, was due to probe in aqueous monomeric form. As seen in Figure 9a the ESR spectrum recorded in pure EPC liposomes displayed the typical anisotropic absorption behavior usually found for 5-DSA in hindered domains. Both line shape and partial resolution of the hyperfine tensor components were satisfactory reproduced by using the procedure given by Freed and co-workers.29,30 Figure 9b compares the experimental spectrum with the shape computed with the parameter reported in Tables 1 and 2. The structural and dynamic parameters obtained (Table 1) reveal that the mean correlation time of the probe was 1.2 × 10-9 s with a significant motional anisotropy (the anisotropy parameter n ) τ⊥/τ|| used for the best-fit calculation was 10), which suggested a partial immobilization inside the phospholipid bilayer. The order parameter S20 ) 0.51 was indicative of relevant order near the water/membrane interface. Only very slight differences in the line shape and in the motional parameters were observed when the liposomes contained LCOB in molar fraction up to 0.25 (Table 2). This finding showed that the presence of relatively small amounts of the lactosyl-carborane did not appreciably modify the environment in which the nitroxide was embedded. The extreme peaks separation 2A′|| was taken as a valid parameter to reflect changes in the probe mobility, and the following shows how 2A′|| changed with xLCOB: xLCOB ) 0, 2A′zz ) 5.14 ( 0.02 mT; xLCOB ) 0.25, 2A′zz ) 5.19 ( 0.02 mT; xLCOB ) 0.63, 2A′zz ) 5.33 ( 0.02 mT. More rigorously, the calculated motional and order parameters reported in Table 2 provide clear evidence that the mobility decreased with respect to LCOB-free liposomes. In particular the order parameter S20, which gives information about the angular amplitude of motion of the labeled chain segment, increased steadily in the entire range of LCOB molar fractions. The same behavior was obtained for the probe rotational mobility, represented by τc, which increased from 1.2 × 10-9 s at xLCOB ) 0.25

Liposomes Loaded with a Carboranyl Compound

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Figure 8. cryo-TEM micrographs obtained after external addition of LCOB to premade EPC liposomes: (a) xLCOB ) 0.63 after 10 min; (b) xLCOB ) 0.63 after 48 h; (c) xLCOB ) 0.73 after 10 min; (d) xLCOB ) 0.73 after 48 h; (e) xLCOB ) 0.79 after 10 min. Black arrows in (a) and (c) indicate waistlike structures displayed by elongated liposomes. Arrows marked P and Q in (b) indicate loop-shaped ends of threadlike micelles and superimposed tubular liposomes, respectively. White arrows in (c) point out some threadlike micelles. Arrow marked F in (d) shows a radiation-damaged multilamller liposome, whereas the white arrows point out ice crystals and the black arrow indicates a liposome captured in the intermediate stage of the solubilization process. See text for further details. Bar ) 100 nm.

to 2.4 × 10-9 s at xLCOB ) 0.79. Table 2 includes magnetic parameters retrieved also for xLCOB ) 0.82. Since the results at this concentration are complicated by the presence of open liposomes and mixed micelles, we refrain, however, from discussing the reported data. As in several other cases, the correlation times reported in this work could not be assumed to exactly reflect the

motion of the entire spin probe. Typically τc is identified with the Debye-Stokes-Einstein reorientational time

τc ) 4πa3η/3kBT where a is the hydrodynamic radius of the reorienting species, η is the viscosity, kB the Boltzmann constant, and

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Rossi et al.

Table 1. Best-Fit Magnetic Parameters Used To Reproduce the 298 K ESR Spectra of n-DSA in Buffer Solution and in EPC Liposome Dispersionsa gxx

gyy

gzz

〈g〉

Axx (mT)

Ayy (mT)

5-DSA 16-DSA

2.0080 2.0080

2.0062 2.0060

2.0029 2.0029

2.0057 2.0056

HEPES buffer 0.62 0.58 0.62 0.58

5-DSA 16-DSA

2.0083 2.0083

2.0070 2.0070

2.0029 2.0029

2.0061 2.0061

EPC liposomes 0.55 0.55 0.55 0.55

a

Azz (mT)

〈A〉 (mT)

τc (ns)

n

3.54 3.54

1.58 1.58

0.08 0.05

1 1

3.28 3.28

1.46 1.46

1.2 0.59

10 1

S20

0.51 0.06

Accuracy: gii ) (0.003; Aii ) (0.02 mT; τc ) 10%; S20 ) (0.02. Table 2. Best-Fit Magnetic Parameters Which Reproduced 298 K ESR Spectra of 5-DSA in EPC/LCOB Liposomes at Different LCOB Molar Fractionsa xLCOB

n

τ⊥ (ns)

τ| (ns)

τc (ns)

S20

0 0.25 0.42 0.58 0.63 0.73 0.79 0.82

10 10 10 7 5 5 3 3

2.6 2.6 2.9 3.0 3.1 3.3 3.5 3.6

0.26 0.26 0.29 0.43 0.63 0.74 1.16 1.20

1.2 1.2 1.3 1.6 1.8 2.0 2.4 2.5

0.51 0.51 0.52 0.52 0.53 0.54 0.55 0.55

a

Figure 9. (a) Experimental ESR spectra of 5-DSA (10-5 M) in pure EPC liposomes (full line) and in EPC liposomes containing LCOB at xLCOB 0.63 (dotted line). T ) 298 K. The two enlargements indicate the first and third absorption of the overall spectra. (b) Experimental ESR spectra (full line) reported in part a and the corresponding simulated spectra (dotted line).

T the temperature. However, the hydrodynamic radii which are tipically calculated from this relation are well below what may be reasonably expected from the paramagnetic probe sizes. In several cases, adjustable parameters have been inserted in the above relationship for τc which account for this problem (see, for example, the stiffness parameters given by McClung and Kivelson48). Recently Morandi et al.49 have studied by ESR and QELS the incorporation properties of 1 in DOTAP/DOPE cationic liposomes and have found ESR results that were in line with those reported in this paper. (48) McClung, R. E. D.; Kivelson, D. J. Chem Phys. 1968, 49, 3380. (49) Morandi, S.; Ristori, S.; Berti, D.; Panza, L.; Becciolini, A.; Martini, G. J. Phys. Chem, submitted for publication.

Accuracy as in Table 1.

To probe the dynamic properties at a position located deeper within the hydrophobic region of the bilayer, we complemented our ESR investigations with measurements done with 16-DSA (2). The spectrum of 16-DSA in pure EPC liposomes was reproduced with τc ) 5.9 × 10-10 s, which was indicative of a relatively high mobility of the paramagnetic unit in the hydrophobic domain of the bilayer, and S20 ) 0.06. This meant that practically no order was sensed by the radical. Moreover the anisotropy parameter n ) 1 suggested an almost free, isotropic motion of the chain points in the terminal positions. The low isotropic coupling constant value (1.46 mT), which was the same as that observed for 5-DSA, reflected a hydrophobic environment around the N-O unit. 16-DSA gave invariantly the same spectrum in both pure EPC liposomes and EPC/LCOB mixed system containing xLCOB molar fractions up to 0.79. At xLCOB ) 0.82, where threadlike micelles and open liposomes were detected, 16-DSA gave the same results as the xLCOB e 0.79. This radical was therefore unaffected by the presence of LCOB. This finding came as no surprise since the average length of the ortho-carborane cage is merely 3.5 Å (calculated from HYPERCHEM program) and the distance from the headgroup to the doxyl group in a fully stretched 16-DSA molecule is estimated to be ∼22 Å.50 Taken together our ESR data suggest that inclusion of LCOB in the lipid membrane decreased the fluidity in the interface region of the bilayer whereas the liquidlike core of the membrane remained unaffected. Conclusions The results obtained in this study showed that the boronated amphiphilic compound LCOB self-aggregated and formed dispersed micelle-like particles in aqueous solution. Further, our investigations revealed that surprisingly high concentrations of lactosyl-carborane can be incorporated into the bilayers of EPC liposomes. In fresh samples intact liposomes could be prepared from LCOB/EPC mixtures, where the LCOB molar fraction was (50) Romanelli, M.; Ristori, S.; Martini, G.; Kang, Y.-S.; Kevan, L. J. Phys. Chem. 1994, 98, 2125.

Liposomes Loaded with a Carboranyl Compound

as high as 0.79. Lower concentrations were tolerated in aged samplessafter 1 week of incubation at room temperature clear signs of lipid solubilization were detected in samples having a LCOB molar fraction of 0.73 or higher. The above results are encouraging and suggest that phospholipid liposomes may indeed constitute suitable carriers for iv administration of LCOB and related carboranyl glycosides. Our investigations showed, however, that careful optimization of the LCOB content is needed before liposomal formulations are considered for in vivo applications. In particular, the propensity of LCOB to induce liposome aggregation may cause blocking of veins and other unwanted side effects. Above the saturation limit the carborane induced essentially the same sequence of structural changes as observed with regular, hydrocarbon based, nonionic surfactants. Despite its unusual chemical structure, LCOB was thus capable of both stabilizing liposome openings

Langmuir, Vol. 19, No. 14, 2003 5617

and solubilizing the EPC component into threadlike mixed micelles. Acknowledgment. The authors are in debt to the University of Florence and to Consorzio per lo Sviluppo dei Sistemi a Grande Interfase, CSGI, Italy, for financial and instrumental support. In addition The Swedish Research Council and The Swedish Cancer Society are gratefully acknowledged for their economic support. Dr. Luigi Panza is thanked for kindly providing us with the boronated substance. Authors are also in debt to Professor Stefan Sjo¨berg and Mr. Ludvig Eriksson for NMR measurements and to Dr. Jean Pettersson for ICP-MS boron determinations. Simona Rossi is grateful to the “C. M. Lerici” Foundation for financial support to spend some time at the Department of Physical Chemistry, Uppsala University. LA0209841