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Langmuir 2002, 18, 505-511

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Control over PEGylated-Liposome Aggregation by NeutrAvidin-Biotin Interactions Investigated by Photon Correlation Spectroscopy Patrick Vermette,*,†,‡ Sarah Taylor,† Dave Dunstan,§ and Laurence Meagher†,‡ CSIRO Molecular Science, Bag 10, Clayton South, VIC 3169, Australia, Cooperative Research Centre for Eye Research and Technology (CRCERT), Rupert Myers Building, The University of New South Wales, Sydney, NSW 2052, Australia, and CRC for Bioproducts, Department of Chemical Engineering, The University of Melbourne, Melbourne, VIC 3010, Australia Received June 29, 2001. In Final Form: October 22, 2001 Photon correlation spectroscopy has been used to determine the degree and sizes of PEGylated-liposome aggregation. The aggregation of the liposomes has been induced by NeutrAvidin-biotin adsorbed to the surfaces of the vesicles. None of the liposome preparations described herein exhibited aggregation before the introduction of NeutrAvidin. The critical ratios of the NeutrAvidin-biotin required to induce vesicle aggregation have been measured and are reported in this work. The aggregation is found to be dependent upon the ratio of NeutrAvidin to exposed biotinylated lipid and also the exposed biotinylated lipid fraction.

Introduction In the past decade, liposomal drug delivery systems have come of age, with some antifungal and anticancer preparations having received final approval for clinical use in a number of countries.1 Generally, PEGylated unilamellar liposomes are preferred for therapeutic applications.2 However, these single-walled (unilamellar) vesicles do not allow multifunctional drug delivery. The use of multifunctional drug delivery can be appealing for numerous biomedical applications, and the use of aggregated liposomes could make such a strategy possible. But before it can be claimed that liposome aggregates can meet all the requirements of the “real-life” in vivo situation (e.g., opsonization, biological stability, appropriate pharmacokinetics and pharmacodynamics, etc.), the concept must be investigated in more detail. Numerous authors have studied the aggregation reactions of colloidal particles, such as colloidal gold, latex particles, and silica particles.3 Colloidal gold particles, for example, will aggregate in the presence of pyridine. This, however, refers to nonspecific aggregation phenomena. On the other hand, von Schulthess et al.4 were among the first researchers to investigate the aggregation of small latex particles coated with human serum albumin (HSA) by anti-HSA antibody; this is a good example of colloidal aggregation by specific interaction. In a series of studies, Powers et al.5,6 reported methods for the purification of specific biomolecules in which target * To whom correspondence should be addressed. New address: Prof. Patrick Vermette, Department of Chemical Engineering, Intelligent Materials and Systems Institute, Universite´ de Sherbrooke, 2500, boul. Universite´, Sherbrooke, Que´bec, Canada, J1K 2R1. Phone: 819-821-8000, ext. 2826. Fax: 819-821-7955. E-mail: [email protected] or patrick_vermette@ hotmail.com. † CSIRO Molecular Science. ‡ Cooperative Research Centre for Eye Research and Technology. § The University of Melbourne. (1) Allen, T. Curr. Opin. Colloid Interface Sci. 1996, 1, 645-651. (2) Marjan, J.; Allen, T. Biotechnol. Adv. 1996, 14, 151-175. (3) Hunter, R. Foundations of Colloid Science; Oxford Science Publications: New York, 1989; Vol. 1. (4) von Schulthess, G.; Benedek, G.; De Blois, R. Macromolecules 1983, 16, 434-440. (5) Powers, J.; Kilpatrick, P.; Carbonell, R. Biotechnol. Bioeng. 1989, 33, 173-182. (6) Powers, J.; Kilpatrick, P.; Carbonell, R. Biotechnol. Bioeng. 1990, 36, 506-519.

proteins were bound specifically from complex mixtures to small (500-700 Å in diameter) unilamellar vesicles. The authors observed that when the target protein had multiple specific binding sites, such as avidin with four equivalent binding sites for biotin or an IgG antibody with two equivalent binding sites for its antigenic epitope, crosslinking of the lipid vesicles occurred leading to network formation and ultimately precipitation.5,6 In further work from the same research group, the aggregation kinetics of the vesicles was followed using photon correlation spectroscopy (PCS) to measure the mean particle size.7 A model based on Smolukowski kinetics revealed that the aggregation kinetics was primarily determined by the biotin density on the liposome surface, the stoichiometric ratio of avidin molecules to liposomes, and the liposome concentration.7 The aggregation of lipid vesicles has been also studied by the group of Zasadzinski. In fact, Chiruvolu et al.8 reported the association of lipid vesicles by means of sitespecific ligand-receptor (biotin-streptavidin) coupling. Adding the streptavidin to the vesicle suspension changed the suspension from clear and bluish to opaque and cloudywhite within a few seconds.8 Only phenomenological observations were reported, however. In further work from Zasadzinski’s group, Kisak et al.9 reported that liposome aggregation can be made self-limiting by controlling the ratio of reactive groups (ligands such as biotin coupled to phospholipids and incorporated in a vesicle membrane) on the liposome surface to cross-linking agents (multifunctional receptors such as avidin or streptavidin) in solution. Farbman-Yogev et al.10 thereafter reported a statistical thermodynamic model to describe the aggregation of large particles (e.g., biotinylated-lipid vesicles) mediated by small, cross-bridge particles (e.g., avidin). The system exhibited a first-order phase transition from a dilute to a condensed phase of vesicles once the average number of “stickers” per vesicle exceeded a certain threshold value. (7) Lynch, N.; Kilpatrick, P.; Carbonell, R. Biotechnol. Bioeng. 1996, 50, 151-168. (8) Chiruvolu, S.; Walker, S.; Israelachvili, J.; Schmitt, F. J.; Leckband, D.; Zasadzinski, J. A. Science 1994, 264, 1753-1756. (9) Kisak, E.; Kennedy, M.; Trommeshauser, D.; Zasadzinski, J. Langmuir 2000, 16, 2825-2831. (10) Farbman-Yogev, I.; Bohbot-Raviv, Y.; Ben-Shaul, A. J. Phys. Chem. A 1998, 102, 9586-9592.

10.1021/la0109967 CCC: $22.00 © 2002 American Chemical Society Published on Web 12/27/2001

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In an effort to detect and measure pollutants and contaminants in the environment and in food sources, Durst et al.11 have recently developed a sensor based on liposome immuno-aggregation. This assay was reported to be useful to detect polychlorinated biphenyls.11 The main aim of this work was to investigate the aggregation of PEGylated liposomes by NeutrAvidinbiotin interactions, measured using photon correlation spectroscopy and transmission electron microscopy (TEM). Poly(ethylene glycol) (PEG)-stabilized liposomes were selected because they have been successfully employed for drug delivery.1,2,12 Liposome aggregation was induced by adding an aliquot of a NeutrAvidin solution to biotinylated-vesicle suspensions, forming mixtures where the ratio of NeutrAvidin to biotin-lipids was varied systematically. The stability of the liposomes upon aggregation by NeutrAvidin was also monitored. Materials N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES) (99.5% purity)] and t-octylphenoxypolyethoxyethanol (Triton X-100) were purchased from Sigma Chemical Co. (St-Louis, MO). 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC) (purity >99%), cholesterol (CHOL) (purity >99%), N-(w-(biotinoylamino) poly(ethylene glycol) 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG(2000)-biotin) (purity >96%), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(poly(ethylene glycol) 2000) (DSPE-PEG(2000)) (purity >99%) were purchased from Northern Lipids Inc. (Vancouver, BC, Canada). NeutrAvidin (ImmunoPure NeutrAvidin biotin-binding protein) and D-biotin (ImmunoPure d-biotin) were purchased from Pierce (Rockford, IL). NeutrAvidin is a modified avidin with low nonspecific binding properties. The NeutrAvidin protein does not contain carbohydrate, thus eliminating the potential of binding to lectins (information obtained from Pierce, Rockford, IL). NeutrAvidin is also a less expensive alternative to streptavidin. 5-(and-6)-Carboxyfluorescein mixed isomers (purity 99%) were obtained from Molecular Probes, Inc. (Eugene, OR).

Methods Preparation of Buffer. The buffer used to prepare the liposome suspensions contained 10 mM HEPES pH 7.4 and a NaCl concentration which was adjusted to an osmolarity of 290 mOsm. The osmolarity of the buffer solution was adjusted to 290 mOsm using The Advanced Osmometer, model 3D3 (Advanced Instruments Inc., Norwood, MA). Milli-Q gradient water (Millipore Australia Pty Limited, North Ryde, NSW, Australia) with a resistivity of not less than 18.2 MΩ cm was used to prepare the buffer solutions. The buffer solution was filtered using 0.2 µm filters (Minisart, steril Go¨ttingen, Germany) before being used in the preparation of the liposome suspensions. Preparation of Liposomes. Procedures for obtaining extruded liposomes have been previously reported by other workers.12-15 Briefly, unilamellar vesicles (ULVs) were prepared in a round-bottom flask by first mixing DSPC and cholesterol (2:1 mole ratio) with varying amounts of DSPC-PEG(2000)biotin or DSPE-PEG(2000) in HPLC-grade chloroform. Approximately 144 µmol of lipids was deposited from 2 mL of HPLCgrade chloroform in a thin film on the interior of a 50 mL roundbottom flask by rotary evaporation under reduced pressure of approximately 100 mmHg for 3 h. Following addition of HEPES buffer, lipids were hydrated in the dark above 65 °C. This temperature is greater than the main transition temperature of the lipid mixture (54.5 °C).16 Hydration was carried out for 24 (11) Durst, R. A.; Roberts, M. A.; Siebert, S. T. A.; Reeves, S. G. U.S. Patent 6,086,748, 2000. (12) Lasic, D. Liposomes: From Physics to Applications; Elsevier: New York, 1995. (13) Hope, M.; Bally, M.; Mayer, L.; Janoff, A.; Cullis, P. Chem. Phys. Lipids 1986, 40, 89-107. (14) Hope, M.; Wong, K.; Cullis, P. J. Electron Microsc. Technol. 1989, 13, 277-287. (15) Mayer, L.; Hope, M.; Cullis, P. Biochim. Biophys. Acta 1986, 858, 161-168.

Figure 1. Schematic diagram (not to scale) of the aggregated liposomes. NeutrAvidin mixed with biotinylated liposomes binds rapidly to the liposomes. The liposomes diffuse, collide with other liposomes, and form aggregates (ref 7). h, followed by vortexing. The multilamellar vesicle (MLV) suspension produced above was subjected to 10 freeze-thaw cycles, involving quenching in dry ice + acetone, followed by an immersion in a 65 °C water bath. Unilamellar vesicles were finally produced by extrusion through 100, 200, 400, or 600 nm pore polycarbonate Nucleopore track-etch membranes (Avanti Polar Lipids Inc., Alabaster, AL) using the Avanti Mini-Extruder (Avanti Polar Lipids Inc., Alabaster, AL) operated at 65 °C. Liposome Aggregation. Liposome aggregation viaNeutrAvidin-biotin interactions was induced by adding an aliquot of a NeutrAvidin solution to a liposome suspension at a fixed ratio R of receptor (NeutrAvidin) to DSPE-PEG(2000)-biotin (as shown in Figure 1). Samples were gently shaken after addition of the protein solution to ensure thorough mixing. As reported previously,8,9 the samples appeared cloudy within a few seconds after the addition. The samples were then allowed to aggregate for 24 h in the dark. Two control experiments were carried out in order to determine if liposome aggregation was specific or nonspecific. In one control experiment, a 2.5 mg/mL (total lipid concentration) liposome suspension containing no biotin (DSPC/CHOL/DSPE-PEG(2000), 2:1:2 mol % mole ratio) was mixed with a NeutrAvidin solution at a ratio R of 0.5. In another control experiment, a 2.5 mg/mL (total lipid concentration) liposome suspension containing biotin (DSPC/CHOL/DSPE-PEG(2000)-biotin, 2:1:2 mol % mole ratio) was mixed with a preblocked NeutrAvidin solution at a (16) Kenworthy, A.; Simon, S.; McIntosh, T. Biophys. J. 1995, 68, 1903-1920.

PEGylated-Liposome Aggregation ratio R of 0.5. The binding of DSPE-PEG(2000)-biotin to NeutrAvidin molecules can be blocked by pre-exposing NeutrAvidin to an excess of biotin (10 mM d-biotin).17-19 The resulting mixtures were incubated for 24 h before use. Photon Correlation Spectroscopy. Dynamic light scattering measurements were performed using a Malvern 4700 apparatus (Malvern Instruments Ltd., Malvern, Worcs., U.K.) with a 10 mW AR+ ion laser at 488 nm. Analysis was performed at an angle of 90° and a temperature of 25 °C. Suspensions of the original liposomes and aggregated liposomes were diluted to 0.005 mg/mL (total lipid concentration). For each sample, three measurements were performed to obtain meaningful information. The dilute particle concentration in the samples ensures that multiple scattering and particle-particle interactions were negligible in this system. The time autocorrelation functions were analyzed by the inverse Laplace transform algorithm, CONTIN. For clarity and comparison purposes, the volume-weighted size distributions and the calculated volume-weighted mean diameters of the liposome aggregates were used. Volume-weighted size distributions also give a more representative scattered intensity; the intensity-weighted size distributions are biased more toward larger particles, while the number-weighted size distributions are biased more toward smaller particles.20 Transmission Electron Microscopy. Suspensions of the original and aggregated liposomes were first diluted (multilamellar 1/20 (v/v), 600 nm 1/75 (v/v), 400 nm 1/75 (v/v), 200 nm 1/75 (v/v), 100 nm 1/75 (v/v), aggregated liposomes 1/20 (v/v)) in Milli-Q water. Next, 3 µL of the diluted liposome suspensions were mixed with 3 µL of 2% ammonium molybdate (pH 7) and placed on Formvar-coated grids containing a layer of carbon for a 25 min period. The excess of solution was removed by placing filter paper against the side of the grid with the remainder left to dry on the grid at room temperature. These negatively stained samples were viewed under a Philips CM100 transmission electron microscope. For each sample, three micrographs were taken to obtain meaningful information. Liposome Stability. Measurements of liposome stability were performed using the fluorescence self-quenching (FSQ) method.21,22 As demonstrated previously,21,22 carboxyfluorescein (CF), a water-soluble derivative of fluorescein, trapped at high concentration inside liposomes emits only a few percent of the fluorescence that would result if the molecules are released and diluted into the surrounding medium. For these stability experiments, liposomes were produced using the same technique described in Preparation of Liposomes, except that the lipids were hydrated in the dark using a solution containing 85 mM CF and 10 mM HEPES at pH 7.4. As before, the osmolarity of this CF solution was determined to be approximately 290 mOsm. Liposomes were thereafter produced as described above. Separation of the dye-containing vesicles from nonentrapped material was achieved by gel chromatography, which involved passage through a 2.5 × 25 cm column of Sephadex G-50 Fine (Amersham Pharmacia Biotech., Castle Hill, NSW, Australia). The column was eluted at room temperature with the HEPES buffer described in Preparation of Buffer at a flow rate of approximately 20 mL/ min, which was adjusted using a peristaltic pump connected to the column inlet. The total lipid concentration was determined using a phosphorus assay by the Australian Government Analytical Laboratories (AGAL, South Melbourne, VIC, Aus(17) Chaiet, L.; Wolf, F. Arch. Biochem. Biophys. 1964, 106, 1-5. (18) Green, N.; Konieczny, L.; Toms, E.; Valentine, R. Biochem. J. 1971, 125, 781-791. (19) Green, N. Avidin and Streptavidin. In Avidin-Biotin Technology; Wilchek, M., Bayer, E., Eds.; Methods in Enzymology, Vol. 184; Academic Press: New York, 1990; pp 51-67. (20) Berne, B.; Pecora, R. Dynamic Light Scattering; John Wiley & Sons: New York, 1976. (21) Lelkes, P. Methodological aspects dealing with stability measurements of liposomes in vitro using the carboxyfluorescein-assay. In Liposome Technology. Volume III: Targeted Drug Delivery and Biological Interaction; Gregoriadis, G., Ed.; CRC Press: Boca Raton, FL, 1984; pp 225-246. (22) Weinstein, J.; Ralston, E.; Leserman, L.; Klausner, R.; Dragsten, P.; Henkart, P.; Blumenthal, R. Self-quenching of carboxyfluorescein fluorescence: Uses in studying liposome stability and liposome-cell interactions. In Liposome Technology. Volume III: Targeted Drug Delivery and Biological Interaction; Gregoriadis, G., Ed.; CRC Press: Boca Raton, FL, 1984; pp 183-204.

Langmuir, Vol. 18, No. 2, 2002 507 tralia). Briefly, the samples were digested with nitric acid using microwave digestion, and the solutions were analyzed by inductively coupled plasma atomic emission spectroscopy (ICPAES). The total lipid concentration of the liposome suspension collected at the column outlet was adjusted to the desired concentration using the HEPES buffer described in Preparation of Buffer. Liposomes containing CF were aggregated as mentioned in Liposome Aggregation, and the release of CF from liposomes was monitored using a Perkin-Elmer LS50-B spectrofluorometer (Perkin-Elmer Co., Wellesley, MA). Experiments were typically performed as follows: 0.5 mL of the appropriate medium (either 0.5% (wt/v) Triton X-100 solution made with Milli-Q water or HEPES buffer) was added to a 1 × 1 cm quartz fluorometer cuvette (1 cm path length). At low concentration and neutral pH, Triton X-100 has been shown to instantaneously disrupt the vesicles and liberate their contents without significant interference with the intrinsic fluorescence of the CF dye.21,22 Pure CF has its absorption maximum (at pH 7.5) at 487 nm, which is not changed by the addition of 0.5% (wt/v) Triton X-100.21,22 Then, 0.5 mL of vesicle suspension was added and mixed with the medium by covering the cuvette with a Teflon cap and gently inverting it. The fluorescence was monitored 60 min after the vesicle suspension was added. The fluorescence signal was monitored at 520 nm. Each experiment was triplicate. The dependence of CF release on aggregation and time was calculated by applying eq 4:21,22

fraction of CF remaining in vesicles ) 1 - F/FT

(4)

where F is the fluorescence at 520 nm measured at any time during the experiment and FT is the total CF fluorescence at 520 nm determined after disruption of the vesicles with Triton X-100.

Results Liposomes produced by extrusion have often been described as monodisperse. In the present study, however, liposomes produced by extrusion were found to be not so monodispersed (as shown in Figure 2). In fact, lipid vesicles produced using 100 and 200 nm pore filters (parts A and B of Figure 2, respectively) have a broad size distribution around the size of the pore membranes. Our findings are in good agreement with those of other workers.23-29 The size distributions presented in Figure 2A,B are not monodispersed but rather polydispersed. It has been found in this work that liposomes produced by extrusion were not only polydispersed, but some were also polymodal (as shown in Figure 2C,D). It has also been reported by Ertel et al.30 that lipid vesicles prepared by extrusion through 400 or 600 nm pores were variable and polymodal in size distribution and/or multilamellar. It is well accepted that the blood clearance is strongly influenced by liposome size.1,2,12 For example, the circulation time for large liposomes (either “plain” or PEGylated) is shorter than for small liposomes. It appears therefore mandatory to analyze liposome size distributions and not assume a monodisperse population unless verified. In addition, liposomes produced using 100 and 200 nm pore filters were found to be unilamellar (parts E and D of Figure 3, respectively), while those produced using 400 and 600 nm pore membranes appeared to be more multilamellar (parts C and B of Figure 3, respectively). (23) Egelhaaf, S.; Wehrli, E.; Muller, M.; Adrian, M.; Schurtenberger, P. J. Microsc. 1996, 184, 214-228. (24) Goll, J.; Carlson, F.; Barenholz, Y.; Litman, B.; Thompson, T. Biophys. J. 1982, 38, 7-13. (25) Haskell, R. J. Pharm. Sci. 1998, 87, 125-129. (26) Kojro, Z.; Lin, S.; Grell, E.; Ruf, H. Biochim. Biophys. Acta 1989, 985, 1-8. (27) Lesieur, S.; Grabielle-Madelmont, C.; Paternostre, M.; Ollivon, M. Anal. Biochem. 1991, 192, 334-343. (28) McCracken, M.; Sammons, M. J. Pharm. Sci. 1987, 76, 56-59. (29) Ostrowsky, N. Chem. Phys. Lipids 1993, 64, 45-56. (30) Ertel, A.; Marangoni, A.; Marsh, J.; Hallett, F.; Wood, J. Biophys. J. 1993, 64, 426-434.

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Figure 2. Volume-weighted size distributions of liposomes (DSPC/CHOL/DSPE-PEG-biotin, 2:1:2 mol %) produced using (A) 100 nm, (B) 200 nm, (C) 400 nm, and (D) 600 nm pore polycarbonate filters as obtained by PCS.

Control of the size of liposome aggregates can be achieved by using specific ligand-receptor (biotin-NeutrAvidin) interactions to link the particles, as demonstrated by TEM micrographs (Figure 3F) and PCS (Figure 4). Two control experiments confirmed that liposome aggregation was specific. Indeed, none of the liposome preparations described herein exhibited aggregation before the introduction of NeutrAvidin. Aggregation was only observed when “active” NeutrAvidin molecules (as opposed to “deactivated” preblocked proteins by “free” biotin) and biotinylated lipids were present. The liposome aggregates were found to be polydispersed and (sometimes) polymodal (Figure 4). The size distribution (e.g., polydispersity) of the liposome aggregates presented in Figure 4 was representative of vesicle aggregates produced under other experimental conditions. When the mean of a polydisperse and/or polymodal distribution is taken, it should be noted that the mean should not be used as an absolute value. Rather, the aggregate size obtained by PCS should be used only to facilitate the comparison between the different experimental conditions. At low NeutrAvidin to exposed DSPE-PEG(2000)biotin ratios R (i.e., for 0.1 < R < 0.6), aggregation was extensive and resulted in sedimentation of the vesicles with apparent diameters in excess of a few micrometers (as shown in Figure 5). The measured sizes of the larger aggregates are therefore a minimum estimate. It must be pointed out that the particle size of aggregated liposomes found by PCS should be used with caution as the value obtained under some conditions (e.g., R ) 0.5) is close to the onset of sedimentation. Sedimentation of very large particles can change the nature of the motion of other smaller particles by adding a drift velocity to the measurements.20 For larger values of R, aggregation was limited with the result that the average particle size was greatly reduced (Figure 5). Our findings are in good agreement with those of others.9 At these higher ratios, the aggregation can be considered as self-limiting, resulting in a stable distribution of small aggregates.9 Increasing R further did not change the aggregate size. However, the aggregates were always larger than individual vesicles. By using the fluorescence self-quenching method, we observed that the liposomes released less than 10% of

their content (CF solution) upon aggregation and that this value remained stable over time (up to 2 months at 4 °C). Liposome aggregation also appeared to depend on the total lipid concentration (Figure 5). No appreciable differences were observed over the control of liposome aggregation as a function of the initial liposome size, as shown in Figure 6. Discussion None of the liposome preparations described herein exhibited aggregation before the introduction of NeutrAvidin, thus suggesting that only specific interactions were involved in the aggregation. The receptor-ligand interactions are the only forces that can be considered to induce specific aggregation since they only act at specific points on the liposome surface and only when the interacting molecules are present. It was reported by Chiruvolu et al.8 that this form of aggregation (i.e., specific) produces little deformation or stress on bound liposomes in contrast to the other more nonspecific forces, which can be often associated with increased fragility, lysis, and fusion. Liposome preparations (not-aggregated and aggregated) were observed for 2 months (at 4 °C) and showed no appreciable increase in the mean liposome diameter, indicating that nonspecific aggregation forces do not interfere with the aggregation modulated by NeutrAvidin. In addition, the aggregation behavior with R followed that expected, as depicted in Figure 5. On average, an R value of 1 corresponds to one exposed biotin-lipid per NeutrAvidin molecule (as shown in Figure 1); therefore no appreciable aggregation should occur. At R values of 0.5 (corresponding to two exposed biotin-lipids per NeutrAvidin molecule), 0.33 (corresponding to three exposed biotin-lipids per NeutrAvidin molecule), and 0.25 (corresponding to four exposed biotin-lipid per NeutrAvidin molecule), liposome aggregation should occur. This is in good agreement with the results presented in Figure 5. Due to steric hindrance, it is unlikely that three or four biotin-lipids would bind to one single NeutrAvidin molecule. By varying the DSPE-PEG(2000)-biotin fraction, it was possible to control the aggregate size (as shown in Figure 7). Increasing the biotin-lipid ratio decreased the critical ratio of NeutrAvidin to exposed DSPE-PEG-

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Figure 4. Volume-weighted size distribution of liposomes (DSPC/CHOL/DSPE-PEG-biotin, 2:1:2 mol %) produced using 100 nm pore polycarbonate filters as obtained by PCS (solid line). Volume-weighted size distribution of stable vesicle aggregates obtained from 100 nm liposomes (DSPC/CHOL/DSPE-PEGbiotin, 2:1:2 mol %) for R ) 0.2 and 1 mg/mL total lipid concentration as measured by PCS (dashed line).

Figure 5. Mean volume-weighted vesicle aggregate (DSPC/ CHOL/DSPE-PEG-biotin, 2:1:0.2 mol %) diameter as a function of the NeutrAvidin/exposed DSPE-PEG-biotin ratio for 1, 2.5, and 5 mg/mL total lipid concentrations as measured by PCS. Figure 3. (A) Transmission electron micrograph of vesicles (DSPC/CHOL/DSPE-PEG-biotin, 2:1:0.2 mol %) produced prior to extrusion. Transmission electron micrographs of vesicles (DSPC/CHOL/DSPE-PEG-biotin, 2:1:0.2 mol %) produced using (B) 600 nm pore filters, (C) 400 nm pore filters, (D) 200 nm pore filters, and (E) 100 nm pore filters. (F) Transmission electron micrograph of stable vesicle aggregates obtained from 100 nm liposomes (DSPC/CHOL/DSPE-PEG-biotin, 2:1:0.2 mol %) for R ) 0.2 and 1 mg/mL total lipid concentration.

(2000)-biotin at which the transition from complete to limited aggregation occurred. In other words, at a small biotin-lipid fraction, it would take more NeutrAvidin molecules in solution to allow interaggregate bonds. In fact, there is a much higher probability that a biotinlipid finds a NeutrAvidin molecule at higher biotin-lipid concentrations, therefore decreasing the critical ratio. The diffusion of biotin-lipids is limited by the size of the liposomes; that is, it is extremely slow compared to the size of the biotin molecule itself. In additon, the more lipid-biotin present on the surface of liposomes, the more likelihood that at any time a biotin-lipid can “meet” a NeutrAvidin molecule and form a specific bond. At any time, some of the biotin molecules, because they are at the end of the PEG spacer, which has a random coil configuration at low concentrations, would be buried within the PEG chains (and also possibly in or at the

Figure 6. Mean volume-weighted vesicle aggregates (DSPC/ CHOL/DSPE-PEG-biotin, 2:1:2 mol %) obtained from liposomes made with 100, 200, 400, and 600 nm pore filters for different NeutrAvidin/exposed DSPE-PEG-biotin ratios (measured by PCS).

surface of the lipid bilayer) and not accessible to NeutrAvidin in solution. Quite a number of studies have concentrated on measuring and interpreting the forces and energies involved in avidin-biotin binding and cross-bridging and their dependence on factors such as membrane fluidity

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Figure 7. Mean volume-weighted vesicle aggregate diameter obtained from liposomes with different DSPE-PEG-biotin fractions as a function of the NeutrAvidin/exposed DSPE-PEGbiotin ratio (measured by PCS).

and elasticity.31-33 Among the conclusions of these studies was that the noncovalent avidin-biotin bond was much stronger than the hydrophobic interaction between the hydrocarbon tail of the biotinylated lipid and the vesicle membrane. Thus, it is essential to understand that once avidin is bound to a biotinylated membrane lipid, pulling it off the vesicle would result in detachment of the hydrocarbon tail from the membrane rather than in dissociation of the avidin-biotin bond.31-34 The size and shape of the lipid vesicle aggregates formed upon adding NeutrAvidin to a solution containing biotinylated-lipid vesicles most likely depend on the kinetics associated with the efficient and nearly irreversible binding of the protein to the biotinylated lipids.10 However, it is possible that the long-time behavior of aggregated liposomes may differ from what is observed in this study since cross-bridged vesicles can still dissociate by breaking the weaker bonds between the biotinylated lipid (DSPEPEG(2000)-biotin) and the vesicle membrane.10 This can be amplified by mechanical stress that could be imposed by turbulence in the blood stream, for example. In other words, the equilibrium state of the system would be governed by the formation and dissociation of these biotinylated lipid-vesicle bonds and not the NeutrAvidinbiotin bonds.10 Nevertheless, as reported in our study, the release of the fluorescent dye, CF, was minimal (less than 10%) upon liposome aggregation. This observation indicates that liposomes were able to compensate and remain stable (at least for the period of time investigated in our study, which was 2 months at 4 °C). When interpreting the results presented in this study, it is also very important to bear in mind that the formation (31) Florin, E.-L.; Moy, V. T.; Gaub, H. E. Science 1994, 264, 415417. (32) Leckband, D. E.; Schmitt, F.-J.; Israelachvili, J. N.; Knoll, W. Biochemistry 1994, 33, 4611-4624. (33) Moy, V. T.; Florin, E.-L.; Gaub, H. E. Colloids Surf., A 1994, 93, 343-348. (34) Arora, A.; Marsh, D. Biophys. J. 1998, 75, 2915-2922.

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of lipid vesicle aggregates leads to polydisperse (sometimes polymodal) populations. Control over liposome aggregation is therefore possible, but only to a certain degree. Unfortunately, it is not possible to compare the polydispersity of aggregated liposome suspensions to literature data7-9 because only the mean values were given; how do the means compare? Among the differences between our study and those of others7-9 is the spacer arm between the lipid headgroup and the biotin molecule. In our study DSPE-PEG(2000)biotin was used, whereas DPPE-biotin,8 DHPE-Xbiotin,9 and DMPE-LC-biotin7 were used by others. It may then be argued that this difference will, to some extent, affect the formation (e.g., kinetics, size, polydispersity, etc.) of liposome aggregates. To illustrate the importance of the spacer arm between the lipid headgroup and the biotin molecule, the reader’s attention is directed to a study demonstrating the importance of having a spacer arm that projects far enough from the biotinylated compound in order to obtain a “full” irreversible avidin-biotin bond.18 There is no doubt that the spacer arm will determine if the intermolecular bonding is possible. In addition, the spacer arms X (X: (CH2)5NHCO(CH2)4) (Molecular Probes, http://www.probes.com) and LC (LC: (CH2)5NHCO(CH2)4) (Pierce, Rockford, IL) used in the molecules DHPE-X-biotin and DMPE-LC-biotin, respectively, are more hydrophobic than the PEG(2000) used as a spacer arm in the DSPEPEG(2000)-biotin molecule (used in this study). Thus, the length and hydrophobicity of the spacer arms used in these other studies could have a direct influence on the formation of liposome aggregates. Despite that, by comparing the results of our study and those of Kisak et al.,9 it appears that the specific binding dominates the aggregation, and the detailed nature of the ligand and the receptor do not really matter too much. This point is, however, in need of further study. In addition, it may be speculated that the lipid chains with different carbon atoms might be immiscible. In fact, studies of mixtures of phosphatidylcholine with different hydrocarbon chains by differential scanning calorimetry,35,36 electron paramagnetic resonance spectroscopy,37 and fluorescent probe methods38 have shown that when the chain length differs by four or more carbon atoms there is solid-state immiscibility. Continuous mixtures were only observed if the components differ by no more than two carbon atoms. This issue has not always been appreciated by others. It is also important to appreciate that experimentally determined size distributions are weighted according to the measurement technique applied, and hence the obtained size distribution depends strongly on the method used.23-29 For example, Chiruvolu et al.8 claimed, based on observations with cryo-TEM, that individual lipid vesicles were still spheroidal and minimally stressed upon specific aggregation caused by biotin-streptavidin binding. Cryo-TEM is often considered the technique of choice to provide a “true”, undistorted direct imaging of the particle size. However, this is only true if the liquid film across the holes contains a representative population of liposomes as present in the bulk. In fact, Egelhaaf et al.23 (35) Lentz, B.; Barenholz, Y.; Thompson, T. Biochemistry 1976, 15, 4529-4537. (36) Mabrey, S.; Sturtevant, J. Proc. Natl. Acad. Sci. U.S.A. 1976, 73, 3862-3866. (37) Luna, E.; McConnell, H. Biochim. Biophys. Acta 1978, 509, 462473. (38) Shimshick, E.; McConnell, H. Biochemistry 1973, 12, 23512360.

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demonstrated the effect of demixing on liposome size, whereby the larger liposomes accumulated close to the edge of the grid. In addition, contrast can be a limiting factor in cryo-TEM.39 It derives only from the difference in electron density between the atoms of the amphiphiles and the surrounding water.39 This can be a major limitation when observing small particles (less than 5 nm). As reported in an excellent review article by Almgren et al.,39 it can be difficult to obtain information about the inner architecture of the lipid bilayer, which has a width of approximately 4 nm and is therefore just at the resolution limit of the cryo-TEM technique. In our opinion, freeze-fracture electron microscopy would appear to be more appropriate to detect stress in lipid bilayers.13,14 Although the preparation technique used in cryo-TEM preserves the “natural” state of the liposomes, one therefore still has to pay attention to artifacts, which may arise during the preparation procedure. For qualitative comparison purposes, TEM can be useful to obtain images. In our study, for example, we have been able to make a qualitative assessment as to whether the liposomes were aggregated. TEM micrographs were used to complement the PCS analysis. It should be borne in mind, however, that negative staining can be subject to artifacts that arise as vesicle dispersions are dried on grids.13,14,39 Nonetheless, we were able to determine if liposome aggregation occurred and to assess, with the limitations of the technique, the lamellarity of the liposome populations. PCS is a very powerful technique and can be used for analyzing samples where no other technique is suitable. However, there is the danger that too much is expected of the method, and care must be taken in order to not read too much into the results obtained. For example, its use is more problematic when the size distribution of the sample is broad and the sample material is nonspherical.20,29 This is clearly the case in the present study. The basis of the PCS technique for particle sizing has been described in detail.20,29 Small particles are only very weakly weighted in PCS experiments.20,29 By contrast, large particles are easily detected in PCS and even a very small number of large particles leads to considerable change in the obtained size distribution. This has to be borne in mind when interpreting data from our study and others.7-9 In fact, as reported by McCracken and Sammons,28 one limitation we have noted was the precision obtainable for size information on samples which contain broad size distributions. This in part may be due to the fact that PCS is very sensitive to small amounts of large particles which was clearly the case for aggregated liposomes. The reproducibility of size information on samples with narrow distributions, such as latex spheres used as standards for this work, was less than 5% relative standard deviation. For samples with broad distributions, the limited information inherent in the autocorrelation function can cause large variability in the mean size results for repetitive analysis of the same samples.28 In fact, the reproducibility of lipid vesicle samples showing broad distributions was reported to be in the range of 10-20%.28 To control and to study liposome aggregation, improved precision and models for describing the data may be required. However, amphiphilic structures can be hard and solid-like, but they are more often soft or fluid-like, with the molecules in constant thermal motion within each aggregates twisting, turning, and moving normally relative to the surface. Thus, unlike monodisperse colloidal particles, (39) Almgren, M.; Edwards, K.; Karlsson, G. Colloids Surf., A 2000, 174, 3-21.

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these structures have no definite size or shape but rather a distribution about some mean value.40 For the commercial instrument used for these measurements, conversions from the intensity data to the number distributions were made assuming a solid sphere for the volume factor. A vesicle is not a solid sphere, however, but rather a hollow sphere with an aqueous compartment surrounded by a lipid bilayer. It was found by McCracken and Sammons28 that when the intensity data for lipid vesicles made of lecithin was converted using a hollow-sphere model that the mean number distribution diameter, for example, was approximately 10% larger than that reported by the instrument using the solid-sphere model.28 It may be relevant to perform data collection at a few scattering angles in the range of 30-150° and analyze these by the exponential sampling method.28 This could help to reduce the observed polydispersity of the liposome populations. However, the same general trends would be observed. Our objectives were therefore not to develop a model for liposome aggregation as reported by others7,9 but rather to obtain a qualitative assessment. A possible way to control the size of an aggregated liposome population would be to fractionate the polydisperse vesicle preparation by size-exclusion chromatography (SEC). In this technique, porous packing material (usually Sephadex) is used to separate particles according to their size. The larger particles elute first from the column, followed by smaller particles. In fact, it was shown by others that fractions of lipid vesicles from SEC runs were analyzed by PCS to successfully distinguish small differences in vesicle sizes.28 This will be the subject of a further investigation. Conclusions Lipid vesicles produced by extrusion were polydispersed (sometimes polymodal) around the pore filters. Liposome aggregation was induced by adding an aliquot of a NeutrAvidin solution to biotinylated-vesicle suspensions, forming mixtures where the ratio of NeutrAvidin to biotin-lipids was varied systematically. Control over liposome aggregation was achieved to some degree by varying the mole fraction of biotin-lipids and the R value. In addition, the liposome aggregates were observed to be polydispersed (and sometimes polymodal). Liposome aggregation depends on the ratio of NeutrAvidin molecules to exposed biotin-lipids and on the biotin-lipid fraction. It was also found that liposomes remained stable upon aggregation. In principle, control over liposome aggregation can be useful to produce multicompartmental aggregates of tethered vesicles encapsulated within a larger vesicle.41 These compartmentalized vesicles (called vesosomes) could provide vehicles for multicomponent or multifunctional drug delivery.41 However, clinical and pharmaceutical applications of these vesosomes have not been demonstrated yet. Acknowledgment. We thank Hans J. Griesser for his helpful guidance and collaboration. We are also deeply indebted to Mark Bown for his assistance. This project was kindly supported by grants from the CRC for Eye Research and Technology, CSIRO Molecular Science, and the FCAR-Que´bec (P.V.). LA0109967 (40) Israelachvili, J. N. Intermolecular and Surface Forces, 2nd ed.; Academic Press: New York, 1992. (41) Walker, S.; Kennedy, M.; Zasadzinski, J. Nature 1997, 387, 6164.