Factors Affecting Responsivity of Unilamellar Liposomes to 20 kHz

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Articles Factors Affecting Responsivity of Unilamellar Liposomes to 20 kHz Ultrasound Hung-Yin Lin and James L. Thomas* Department of Chemical Engineering, Columbia University, New York, New York 10027 Received January 14, 2004. In Final Form: May 4, 2004 Ultrasound is commonly used in the preparation of unilamellar liposome dispersions and is often considered for cell membrane disruption for drug delivery or DNA transfection applications. To better understand the physical and chemical properties of lipid membranes that render them susceptible to ultrasonic permeabilization, the roles of temperature, lipid composition (cholesterol and PEG-lipid content), and liposome size have been studied. The results of these studies suggest that lipid packing is very important to ultrasound responsiveness; surprisingly, cohesive energy and tensile strength are not. Taken together, the experimental results implicate a defect-mediated permeabilization mechanism, rather than pore formation or membrane tearing. The implications of this work for drug release from liposomes and ultrasoundmediated DNA transfection are discussed.

Introduction Since the early years of liposome research, ultrasound has been used to disrupt large multilamellar vesicular aggregates and force their reorganization into unilamellar structures (which are generally more suitable for biochemical and biophysical studies).1 Recently, there has been increasing interest in using ultrasound to permeabilize cellular membranes, for DNA transfection2-4 and drug delivery.5-12 Despite this long history and high level of current interest, the factors that determine the ultrasound responsivity of various lipid bilayer membranes have not been clearly elucidated. That different bilayer membranes exhibit remarkably different responsivity to ultrasound was shown in an earlier study by the present researchers.13 In that work, it was found that bilayers containing modest amounts of PEGylated lipids, or other surface-active molecules with oligo-ethylene glycol headgroups, show as much as a 10* To whom correspondence should be addressed. Current address: Department of Physics and Astronomy, University of New Mexico, 800 Yale Blvd. NE, Albuquerque, NM 87131. E-mail: [email protected]. (1) Huang, C. Biochemistry 1969, 8, 344-352. (2) Cochran, S. A.; Prausnitz, M. R. Ultrasound Med. Biol. 2001, 27, 841-850. (3) Liu, J.; Lewis, T. N.; Prausnitz, M. R. Pharm. Res. 1998, 15, 918-924. (4) Shohet, R.; Chen, S.; Zhou, Y.-T.; Wang, Z.; Meidell, R.; Unger, R.; Grayburn, P. Circulation 2000, 101, 2554-2556. (5) Unger, E. C.; Hersh, E.; Vannan, M.; Matsunaga, T.; McCreery, T. Prog. Cardiovasc. Dis. 2001, 44, 45-54. (6) Unger, E.; McCreery, T.; Sweitzer, R.; Caldwell, V.; Wu, Y. Q. Invest. Radiol. 1998, 33, 886-892. (7) Rapoport, N. Colloids Surf., B 1999, 16, 93-111. (8) Rapoport, N.; Munshi, N.; Pitina, L.; Pitt, W. G. Polym. Prepr. (Am. Chem. Soc., Div. Polym. Chem.) 1997, 38, 620-621. (9) Frinking, P.; Bouakaz, A.; de Jong, N.; Ten Cate, F. J.; Keating, S. Ultrasonics 1998, 36, 709-712. (10) Wu, J. R.; Chappelow, J.; Yang, J.; Weimann, L. Ultrasound Med. Biol. 1998, 24, 705-710. (11) May, D.; Dayton, P.; Chomas, J.; Allen, J.; Ferrara, K. Proc. IEEE Ultrasonics Symp. 2000, 2, 1429-1432. (12) Ng, K.; Liu, Y. Med. Res. Rev. 2002, 22, 204-223. (13) Lin, H.-Y.; Thomas, J. L. Langmuir 2003, 19, 1098-1105.

fold enhancement of their response to 20 kHz insonation, as compared to egg phosphatidylcholine controls. Moreover, Rapoport and co-workers have found that cancer cells treated with Pluronic block copolymers (PEG-PPOPEG triblocks) show a much enhanced ultrasound sensitivity as compared to normal cells treated in the same way.8,14,15 In both of these cases, the physical or chemical mechanisms that are responsible have not yet been identified. Thus, the goal of the experiments reported herein was to identify physical and chemical factors that may affect the responsivity of lipid bilayer membranes to ultrasound. Such knowledge could be useful in the design of ultrasonically activated lipid-based drug delivery and could also help to understand differences in cellular responses. Leakage of liposomally entrapped hydrophilic compounds (in the absence of ultrasound) has been studied for many years.16 The most straightforward approach, which we also employ here, is to entrap a water-soluble self-quenching fluorescent dye, using the relief of selfquenching as an indicator of liposome leakage.17 A variety of factors have been shown to influence liposome permeability.18 Permeability is enhanced near the phase transition temperature;19,20 it is reduced by the incorporation of cholesterol.21 Detergents and other amphiphiles with large headgroups also increase permeability markedly, at concentrations well below that required for solubiliza(14) Rapoport, N. Y.; Herron, J. N.; Pitt, W. G.; Pitina, L. J. Controlled Release 1999, 58, 153-162. (15) Marin, A.; Muniruzzaman, M.; Rapoport, N. J. Controlled Release 2001, 71, 239-249. (16) Bangham, A. D.; Standish, M. M.; Miller, N. Nature 1965, 208, 1295-1297. (17) Allen, T. M. In Liposome Technology; Gregoriadis, G., Ed.; CRC Press: Boca Raton, FL, 1984; Vol. III, pp 177-182. (18) Exerowa, D.; Kashchiev, D.; Platikanov, D. Adv. Colloid Interface Sci. 1992, 40, 201-256. (19) Marsh, D.; Watts, A.; Knowles, P. F. Biochemistry 1976, 15, 3570-3578. (20) Marcelja, S.; Wolfe, J. Biochim. Biophys. Acta 1979, 557, 2431. (21) Papahadjopoulos, D.; Jacobson, K.; Nir, S.; Isac, T. Biochim. Biophys. Acta 1973, 311, 330-348.

10.1021/la049866z CCC: $27.50 © 2004 American Chemical Society Published on Web 06/15/2004

Ultrasound Responsivity of Unilamellar Liposomes

tion.22-25 In general, high permeability in liposomes is correlated with a low compressibility modulus,26 which is consistent with either a defect- or a pore-mediated permeation process. In the case of ultrasound-mediated liposome release, it has not yet been determined whether the same principles carry over. The experiments presented herein address this issue, and the results indicate some significant differences between ultrasound-driven permeability and passive permeability. Experimental Section Lipids and Chemicals. The lipids egg yolk L-R-phosphatidylcholine (PC), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-Nmethoxy(poly(ethylene glycol))2000 (DPPE-PEG2000), and 1,2dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3benzoxadia zol-4-yl) (Ammonium Salt) (NBD-DPPE) were purchased from Avanti Polar Lipids (Birmingham, AL). Dithionite (sodium hydrosulfite, tech. ca. 85%) was obtained from Acros Organics (NJ). Calcein (fluorexon) and Sephadex G50 (20-80 µm) were from Aldrich Chemical Co. (Milwaukee, WI). Cholesterol, N-[2-hydroxyethyl] piperazine-N′-[2-ethanesulfonic acid] (HEPES), and Triton X-100 (TX-100) were purchased from Sigma (St. Louis, MO). Disodium ethylenediamine tetraacetate (EDTA), sodium azide, and sodium chloride were purchased from Fisher Scientific (Fair Lawn, NJ). Chemicals were used as received. Preparation of Liposomes. Stock lipids in CHCl3 were dried under nitrogen and then under vacuum overnight. For permeability measurements, the lipids were vortex mixed and resuspended in a 50 mM calcein solution, pH 8.0. The lipid suspension was then passed through two stacked polycarbonate filters (Nuclepore filters, Whatman Inc., Clifton, NJ) 19 times27 in a “mini-extruder” (Avanti Polar Lipids, Birmingham, AL). 100 nm polycarbonate filters were generally used; in some experiments to test size dependence, 30, 50, and 200 nm filters were also used. The extrusion temperature was above the lipid main chainmelting transition: DPPC was extruded at 55 °C, and egg PC was extruded at room temperature. Unentrapped calcein was then removed by size exclusion chromatography in a Sephadex G50-packed 10 cm column (0.9 cm diameter), eluted with an isotonic buffer (5 mM HEPES, 0.6% NaCl, 1 mM EDTA, 0.02% NaN3, pH ) 7.6) at room temperature (ca. 20 °C). Ultrasound Apparatus and Insonation. The 20 kHz Ultrasonic processor (model VC130PB, Sonics & Materials Inc., Newtown, CT) was used.28,29 The probe of the sonicator was immersed into a polystyrene cuvette (1 mL path) through a clearance hole in a Teflon cap, while the cuvette was held in the fluorimeter, an SLM Aminco 8000. The probe was immersed approximately 1 cm into a 3 mL sample, initially containing only buffer (5 mM HEPES buffer, 0.6% NaCl, 1 mM EDTA, pH ) 7.6) and a stir bar. Next, 60 µL of liposome stock solution (ca. 0.06 µmol of lipid) was added through an injection port while stirring. All solutions were degassed by applying a “house” vacuum (ca. 0.1 atm) for ca. 30 min prior to experiments. The fluorimeter holds the cuvette in a copper heat transfer block, the temperature of which is controlled by a water circulator (Isotemp 3016, Fisher Scientific Co., Morris Plains, NJ). Because ultrasonic energy causes substantial sample heating, we measured the temperature of the sample by direct immersion of a thermocouple probe (model HH21, Omega Engineering Inc., Stamford, CT) and kept the (22) Helenius, A.; Simons, K. Biochim. Biophys. Acta 1975, 415, 2979. (23) Silvander, M.; Johnsson, M.; Edwards, K. Chem. Phys. Lipids 1998, 97, 15-26. (24) Nikolova, A. N.; Jones, M. N. Biochim. Biophys. Acta 1996, 1304, 120-128. (25) Puech, P.-H.; Borghi, N.; Karatekin, E.; Brochard-Wyart, F. Phys. Rev. Lett. 2003, 90, 128304. (26) Nagle, J. F.; Scott, J. H. L. Biochim. Biophys. Acta 1978, 513, 236-243. (27) Hope, M. J.; Bally, M. B.; Webb, G.; Cullis, P. R. Biochim. Biophys. Acta 1985, 812, 55-65. (28) Husseini, G. A.; Myrup, G. D.; Pitt, W. G.; Christensen, D. A.; Rapoport, N. Y. J. Controlled Release 2000, 69, 43-52. (29) Husseini, G. A.; Rapoport, N. Y.; Christensen, D. A.; Pruitt, J. D.; Pitt, W. G. Colloids Surf., B 2002, 24, 253-264.

Langmuir, Vol. 20, No. 15, 2004 6101 sample within (1 °C of the target temperature for all experiments. The water circulator system was controlled via a PC RS232 serial port with Labview Software V5.0 (National Instruments Corp., Austin, TX). The duration and power of sonication was also computer controlled with VibraCell PC Control (Sonics & Materials Inc., Newtown, CT). Experiments were run at 25% duty cycle, using “20%” of full power (ca. 1 W/cm2), unless otherwise noted. Fluorescence Monitoring of Calcein Release from Liposomes. Excitation and emission wavelengths were set at 488 and 520 nm, respectively. After 60 µL of liposome stock solution was added, the fluorescence signal (from residual unentrapped dye, and incomplete self-quenching of internal dye) was allowed to stabilize for ca. 100 s. Ultrasound was then applied for 5 min, during which time the fluorescence was continuously monitored. At the conclusion of each experiment, the detergent Triton X-100 (10 µL of 10 wt %) was then added to rupture liposomes completely and to achieve complete calcein release. The fluorescence increase was normalized as ∆I(t) ) [I(t) - I0]/[Imax - I0], where I is the measured fluorescence intensity, I0 is the fluorescence intensity before liposome addition, and Imax is the maximum fluorescence intensity after Triton X-100 addition.30 In control experiments on buffer solutions, no direct signal from the insonation (i.e., sonoluminescence) was observed under our observation conditions. Osmolarity/Ionic Strength of Buffers. 5 mM HEPES buffer solutions were prepared with different NaCl concentrations (also containing 1 mM EDTA and 0.02% sodium azide at pH 7.4), and the osmotic strength of each buffer was measured with an Advanced Instruments (Norwood, MA) 3D3 freezing point osmometer. The buffers were then preheated to 37 °C in a water circulator (Isotemp 3016, Fisher Scientific Co., Morris Plains, NJ) for permeability measurements. Unilamellarity of Liposomes. Dithionite (S2O4-) and the spontaneously produced SO2- radical rapidly and irreversibly quench the fluorescent lipid NBD-PE in the outer leaflet of liposomes.31,32 The fluorescence of unilamellar liposomes containing NBD-PE is thus reduced by at least 50% on adding dithionite; smaller unilamellar liposomes are subject to even greater quenching, because of the significantly larger area of the outer leaflet. Light Scattering by Liposomes. Quasielastic light scattering (QELS) of vesicle suspensions after insonation was studied with a Coulter N4 Plus (Beckman Coulter Inc., Miami, FL), equipped with a 632.8 nm 10 mW helium-neon laser light source. The most reproducible scattering signals were obtained at 30.2° and 62.6°; measurements at these angles were averaged after fitting. Particle size distributions were calculated from autocorrelation data analysis by the Size Distribution Processor (SDP) (an implementation of the Contin algorithm).33 For fitting, 31 bins distributed logarithmically between 1 and 1000 nm were chosen. Samples were filtered with 0.45 µm syringe filters (GHP Acrodisc, Gelman Sciences, Ann Arbor, MI) just prior to measurement. The collection times for the autocorrelation function were 2-4 min.

Results and Discussion Temperature Dependence. In a previous paper,13 it was shown that liposomes containing PEG-lipids are more responsive to 20 kHz ultrasound than liposomes without this polymeric component. To better study the effects of temperature and lipid phase on liposome response, dipalmitoyl phosphatidylcholine (DPPC) was chosen as the chief liposome constituent (rather than egg PC, which contains a mixture of lipids with no clear acyl chain-melt transition). The 100 nm liposomes containing the selfquenching fluorophore calcein were prepared by the (30) Duzgunes, N.; Straubinger, R. M.; Baldwin, P. A.; Friend, D. S.; Papahadjopoulos, D. Biochemistry 1985, 24, 3091-3098. (31) McIntyre, J. C.; Sleight, R. G. Biochemistry 1991, 30, 1181911827. (32) Sofou, S.; Thomas, J. L. Biosens. Bioelectron. 2003, 18, 445455. (33) Provencher, S. W. Comput. Phys. Commun. 1982, 27, 213-227.

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Figure 1. Fluorescence increases from the relief of selfquenching of calcein dye entrapped in liposomes. Liposomes composed of DPPC:DPPE-PEG2000 (100:8) were prepared with 50 mM entrapped calcein. Liposomes were loaded after 100 s and allowed to stabilize at the set temperature, (A) at 28, 37, 39, 43, 46, and 51 °C, respectively. Insonation at 20 kHz (20% full power and 25% duty cycle, approximately 0.5 W/cm2) caused rapid fluorescence increase while on (from 200 to 500 s), with no persistent permeabilization when switched off (ca. from 500 to 600 s). In the absence of insonation, fluorescence was stable, except near the transition temperature where slow leakage was evident. Triton X-100 (TX-100) was added at the conclusion of each measurement to determine the maximal fluorescence from complete dye release. (B) Effect of ultrasound on DPPC: DPPE-PEG2000 (100:8) (solid line) and pure DPPC (dashed line) liposomes at 41 °C. Sonication procedures are the same as those used in Figure 1A. At a temperature so close to the phase transition, the ultrasound causes a sigmoidal response that is likely due to local heating.

extrusion method, from DPPC alone, and with 8 mol % PEG2000-DPPE. Results on PEG-containing liposomes are shown in Figure 1. In these measurements, a small amount (60 µL) of a liposome stock solution was added to a cuvette containing 3 mL of buffer at the temperature indicated; after stabilization of the fluorescence signal (ca. 100 s), ultrasound was applied to the sample via a probe sonicator. Both PEG-containing and PEG-free liposomes showed temperature-dependent prompt leakage of dye (as judged by the fluorescence increase) in the absence of ultrasound. Greatly enhanced liposome permeability in the vicinity of the phase transition is well established (and is a viable mechanism for engineering temperature-responsive liposomes).34 The prompt leakage observed in our liposomes (both PEGylated and pure DPPC) appears to be caused by this phenomenon; a temperature jump to just above the phase transition (34) Anyarambhatla, G. R.; Needham, D. J. Liposome Res. 1999, 9, 491-506.

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temperature results in much greater prompt leakage than a jump to a higher temperature, for which the time spent near the phase transition would be expected to be shorter. The increasing rate of leakage at the highest temperature (51 °C) (as opposed to the prompt leakage) suggests that the inherent responsivity to ultrasound increases with temperature and led us to refine our analysis to distinguish between steady and prompt leakage, as described below. The total ultrasonic power delivered to the sample was estimated to be 0.07 W (20% of full power with 25% duty cycle), determined by temperature increases in a thermally insulated cuvette. As shown in previous work, liposomes show little response to ultrasound below the cavitation threshold;35 the intensity of 20 kHz ultrasound used in this study was sufficient to cause cavitation in the solution in the vicinity of the probe tip.36 It should be recognized that higher ultrasound frequencies are less capable of causing solution cavitation and would be expected to require higher powers to effect liposome lysis. Because of the periodic deposition of heat into the sample from the ultrasound probe, exact temperature control could not be maintained, even with a heating/cooling bath. By using an immersed thermocouple, we estimate that the bulk sample temperatures never deviated by more than (1 °C from the desired temperature. Nonetheless, in the vicinity of the phase transition, the combination of ultrasound-induced permeabilization and phase transition effects is difficult to disentangle, as shown in Figure 1B. In this experiment, DPPC liposomes with (solid line) or without (dashed line) PEG-lipid were added to buffer at 41 °C. Shortly after the initiation of insonation, there was a dramatic, exponential increase in fluorescence, followed by a slower increase that followed first-order kinetics. Thus, the temperatures reported in this figure and subsequent figures must be taken as “nominal” temperatures with significant ((1 °C) uncertainty. Because the “starting” fluorescence (after stabilization) for ultrasound experiments at different temperatures varies greatly, it is important to properly normalize the resulting ultrasound-induced fluorescence increases. For further analysis of the data in Figure 1A, the rates of fluorescence change on applying ultrasound were normalized to the amount of fluorescence remaining entrapped, determined by complete liposome lysis with the detergent TX-100 at the conclusion of each run. The prompt leakage of calcein (as quantified by the fluorescence increase on adding to buffer at various temperatures) is shown in Figure 2, for both pure DPPC 100 nm liposomes and PEG-containing DPPC liposomes. For both compositions, a maximum in the prompt leakage occurs near the main chain-melting transition temperature Tm, that is, 41.4 ( 0.1 °C.37,38 The presence of PEGlipids in the membrane did not detectably modify the temperature of maximum prompt leakage. This is expected, because the PEG lipids also carry dipalmitoyl tails and do not appreciably alter the phase behavior.39,40 The general aspect of the prompt leakage curves in Figure 2 (i.e., a peak at the transition temperature with somewhat (35) Lin, H.-Y.; Thomas, J. L. Langmuir 2003, 19, 1098-1105. (36) Mason, T. J.; Lorimer, J. P. Sonochemistry: theory, applications and uses of ultrasound in chemistry; Ellis Horwood Ltd.: Chichester, 1988. (37) Biltonen, R. L.; Lichtenberg, D. Chem. Phys. Lipids 1993, 64, 129-142. (38) Koynova, R.; Caffrey, M. Biochim. Biophys. Acta 1998, 1376, 91-145. (39) Vermehren, C.; Kiebler, T.; Hylander, I.; Callisen, T. H.; Jorgensen, K. Biochim. Biophys. Acta 1998, 1373, 27-36. (40) Hashizaki, K.; Itoh, C.; Sakai, H.; Yokoyama, S.; Taguchi, H.; Saito, Y.; Ogawa, N.; Abe, M. Colloids Surf., B 2000, 17, 275-282.

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Figure 2. Prompt leakage of DPPC (O) and DPPC:DPPEPEG2000 (100:8) (4) liposomes at temperatures from 27 to 50 °C in the absence of ultrasound. Maximum prompt leakage occurred at 43 °C for both liposome compositions.

higher leakage above Tm than below) is reminiscent of previous studies of the temperature dependence of liposome permeabilization.41 However, the prompt leakage is certainly not a steady-state measurement of permeability. To reach temperatures above Tm, liposomes were necessarily driven through Tm in the experimental protocol; thus, the leakage above Tm occurs partly while the temperature is crossing Tm. The application of 20 kHz ultrasound to the liposome suspensions resulted in increased rates of calcein permeation. Calcein permeation rates were characterized in two ways: by initial (normalized) leakage rates (Figure 3A), obtained from the slope of the fluorescence increase immediately after beginning insonation; and by maximum (normalized) leakage rates (Figure 3B). The open circles represent unsonicated controls. The maximum leakage rates have a large peak at the phase transition temperature, while the initial rates have a small or nonexistent peak. Because the application of ultrasound causes heating, we believe that the maximum leakage rates reflect thermal as well as ultrasonic stimulation of liposome leakage: in many instances near Tm, the maximum leakage rate was obtained after a brief period of insonation (as in Figure 1B). The results presented in Figure 3A show several important features of the ultrasound response of liposomes. The ultrasound-induced leakage from DPPC or PEG/ DPPC liposomes is temperature-dependent, but the PEGcontaining liposomes are especially so, with a 7-fold higher responsivity at 51 °C than at 28 °C. The basal leakage rate does not increase nearly so much over this temperature range. It is of course possible that some of the temperature dependence reflects changing properties of the solutionsfor example, it may be somewhat easier to nucleate cavitation at elevated temperatures, because of the reduced air solubility in the buffer solution. However, the PEG-containing liposomes differ markedly from the pure DPPC, implying that some of the temperature dependence is caused by temperature-dependent lipid properties. It is also noteworthy that, at least within the uncertainty of our experiment, we do not find a clear and unambiguous “step change” in the ultrasound responsivity at Tm. Power Dependence. As observed previously,13 there is a threshold intensity for ultrasound-induced lipid membrane disruption that appears to correspond to the onset of solution cavitation, Figure 4. The Sonics & (41) Thomas, J. L.; Devlin, B. P.; Tirrell, D. A. Biochim. Biophys. Acta 1996, 1278, 73-78.

Figure 3. Initial (A) and maximum (B) ultrasound-induced leakage rates for DPPC (b) and DPPC:DPPE-PEG2000 (100:8) (2) liposomes. Rates were normalized to the amount of fluorescence remaining entrapped. The initial rate was measured at the beginning of the insonation period, while the maximal rate was enhanced near the phase transition. Open symbols are unsonicated controls measured over the same time periods and at the same temperatures.

Figure 4. Initial leakage rate for PEG-containing liposomes at different temperatures under 20 kHz insonation. All temperatures showed a threshold at about 10% of maximum power, ca. 1.5 W/cm2, near the onset of cavitation. There is a general increase in responsivity with temperature, with a strong enhancement near the phase transition (41 °C).

Materials probe can be run at different “percentage” power settings; only at power settings at or above 13% do PEGcontaining liposomes show significantly enhanced leakage of the fluorescent probe. Although the “percentage” power setting is not a precise, linear measure of the actual output power, a measurement of sample heating shows linearity up to ca. 20% power. Thus, the lack of a response at “10%” power was not caused by an absence of effective insonation.

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Table 1. Initial Leakage Rates, Immediately after Beginning Insonation with 20 kHz Ultrasounda lipid conc (mg/L)

initial leakage rate, % per s

0.25 1.25 2.5 15 25 125

0.55 ( 0.14 0.38 ( 0.08 0.37 ( 0.08 0.35 ( 0.06 0.24 0.39

a EYPC/8 mol % PEG2000-DPPE 100 nm liposomes were studied. The leakage rate was normalized to the total fluorescence increase upon liposome disruption with detergent. As can be seen from the measurements, there is no clear trend with increasing liposome concentration, strongly suggesting that liposome-liposome interactions are not important to US-induced leakage.

Concentration Dependence. Experiments were performed to explore the possible effect of liposome concentration on ultrasound responsivity. In principle, ultrasonic response could involve liposome-liposome interactions, which would result in faster disruption at higher concentrations. As shown in Table 1, lipid concentrations were varied by a factor of 500, with no significant change in the rate of ultrasound-induced leakage. Size Dependence. Although lipid bilayers have a low bending modulus, curvature strain is still present in the smallest liposomes, where it is thought to cause instability to fusion.42 To explore the role of liposome curvature on ultrasound responsivity, liposomes were prepared by extrusion through polycarbonate membranes with different pore sizes: the size of liposomes produced by extrusion has been shown to be closely correlated to the extrusion pore size.43 The results for egg phosphatidylcholine liposomes are shown in Figure 5A. All data have been normalized to the maximum total fluorescence increase, obtained by addition of detergent at the end of each run. Insonation of the liposomes was initiated after signal stabilization, at about 220 s on the time axis. For egg PC liposomes, there is a strong size dependence of the responsivity to ultrasound, with the smallest liposomes responding most dramatically. Leakage rate measurements were also performed for egg PC liposomes containing PEG lipid. The results for both liposome compositions are shown in Figure 5B, with the initial leakage rate plotted against the pore size of the extrusion filters. (Pores larger than 200 nm were not used, because larger pores do not produce exclusively unilamellar liposomes.43 Unilamellarity of the extruded liposomes used in these experiments was tested by the dithionite assay, Table 2.31,32) Interestingly, while the egg PC liposomes show a strong size dependence of the ultrasound-induced dye leakage, the PEG-containing formulations do not. For the larger liposomes, PEG significantly enhances the leakage rates, but for small liposomes it depresses them; the overall result is a weak dependence on size, with a slight trend to greater responsivity for the larger sizes. The curvature stress in small, extruded liposomes results from monolayer curvature, not from leaflet area differencessduring extrusion, lipids can partition themselves between the inner and outer leaflets to equilibrate the area per lipid in each leaflet. The fact that they do so is supported by the dithionite measurements, which show an increasing proportion of outer leaflet lipids in smaller liposomes, as well as by NMR experiments using mem(42) Magotoshi, M.; Abu-Zaid, S. S.; Noriaki, T. Int. J. Pharm. 1983, 17, 215-224. (43) Mayer, L. D.; Hope, M. J.; Cullis, P. R. Biochim. Biophys. Acta 1986, 858, 161-168.

Figure 5. (A) Egg yolk PC liposomes prepared by extrusion through smaller pores show increased responsivity to ultrasound. Liposomes were prepared by extrusion through polycarbonate filters with pore sizes of 30 nm (solid line), 50 nm (dotted line), or 100 nm (dashed line). The experiment temperature was 30 °C. (B) Initial leakage rates for liposomes of different compositions (EYPC, b; EYPC with 8 mol % PEG2000DPPE, [) versus extrusion pore size. Empty symbols show unsonicated controls. The PEG-containing liposomes do not show increased response at small sizes. Table 2. Unilamellarity of Liposomes (1 mol % NBD-DPPE in EYPC) Was Measured by Using Membrane-Impermeant Dithionite, Which Quenches NBD-DPPE Lipids in the Outermost Leaflet of Liposomesa polycarbonate filter pore size (nm) reduction in fluorescence, % 30 50 100 200

72.15 ( 2.188 59.09 ( 1.185 61.12 ( 3.440 63.17 ( 0.785

a The reduction in fluorescence indicates the proportion of accessible lipids; for unilamellar liposomes, slightly more than onehalf the lipids are in the outer leaflet, because of its larger radius as compared to that of the inner leaflet. Multilamellar liposomes would have a smaller reduction in fluorescence (i.e.,