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Lateral Phase Separation in Lipid-Coated Microbubbles Mark A. Borden,*,†,⊥ Gary V. Martinez,⊥ Josette Ricker,‡ Nelly Tsvetkova,§,# Marjorie Longo,| Robert J. Gillies,⊥ Paul A. Dayton,† and Katherine W. Ferrara† Department of Biomedical Engineering, Department of Anatomy, Physiology and Cell Biology, Department of Molecular and Cellular Biology, and Department of Chemical Engineering, UniVersity of California, DaVis, California 95616, and Arizona Cancer Center, UniVersity of Arizona, Tucson, Arizona 85724 ReceiVed October 21, 2005. In Final Form: February 3, 2006 In the design of lipid-coated microbubble ultrasound contrast agents for molecular imaging and targeted drug delivery, the surface distribution of the shell species is important because it dictates such properties as ligand location, brush coverage, and amount of drug loading. We used a combination of spectroscopy and microscopy techniques to test the prevailing notion that the main phosphatidyl choline (PC) and lipopolymer species are completely miscible within the monolayer shell. NMR spectroscopy showed that the shell composition is roughly equivalent to the bulk lipid ratio. FTIR spectroscopy showed a sharp melting peak corresponding to the main phase-transition temperature of the main PC species, with no observed pretransitions while scanning from room temperature, indicating a single PC-rich ordered phase. Electron and fluorescence microscopy showed a heterogeneous microstructure with dark (ordered) domains and bright (disordered) regions. Domain formation was thermotropic and reversible. Fluorescent labeling of the lipopolymer following shell formation showed that it partitions preferentially into the disordered interdomain regions. The ordered domains, therefore, are composed primarily of PC, and the disordered interdomain regions are enriched in lipopolymer. Phase heterogeneity was observed at all lipopolymer concentrations (0.5 to 20 mol %), and the degree of phase separation increased with lipopolymer content. The composition and temperature dependence of the microstructure indicates that phase separation is driven thermodynamically rather than being a kinetically trapped relic of the shell-formation process. The overall high variation in microstructure, including the existence of anomalous three-phase coexistence, highlights the nonequilibrium (history-dependent) nature of the monolayer shell.
1. Introduction Lipid-monolayer-coated microbubbles currently are being used as ultrasound contrast agents for echocardiography and show great promise for molecular imaging1,2 and targeted drug delivery.2-4 Proper characterization of the lipid shell is crucial for these applications because it is the shell that imparts many of the functional properties, such as stabilization in storage, sustained persistence in circulation, specific adhesion to vascular targets, compliance to ultrasound-induced oscillations of the gas core, and loading and release of drugs and genes. Despite the monolayer shell’s importance, little is known currently about the composition and phase behavior, and it is generally implied that the constituents fully mix at their bulk concentration.3,5 Previous studies have revealed interesting characteristic properties of the monolayer shell. Kim et al. demonstrated the remarkable mechanical rigidity of the lipid shell, which is quite * Corresponding author. E-mail:
[email protected]. † Department of Biomedical Engineering, University of California, Davis. ‡ Department of Anatomy, Physiology and Cell Biology, University of California. § Department of Molecular and Cellular Biology, University of California. | Department of Chemical Engineering, University of California. ⊥ Department of Radiology, University of Arizona. # Current address: Formulation Department, Bayer Corporation, Berkeley, California 94710. (1) Ferrara, K. W.; Merritt, C. R. B.; Burns, P. N.; Foster, F. S.; Mattrey, R. F.; Wickline, S. A. Acad. Radiol. 2000, 7, 824-839. Dayton, P. A.; Ferrara, K. W. J. Magn. Reson. Imag. 2002, 16, 362-377. (2) Bloch, S. H.; Dayton, P. A.; Ferrara, K. W. IEEE Eng. Med. Biol. Magn. 2004, 23, 18-29. Lindner, J. R. Nat. ReV. Drug DiscoVery 2004, 3, 527-532. (3) Unger, E. C.; Porter, T.; Culp, W.; Labell, R.; Matsunaga, T.; Zutshi, R. AdV. Drug DeliVery ReV. 2004, 56, 1291-1314. (4) Lindner, J. R.; Kaul, S. Echocardiography J. CardioVasc. Ultrasound Allied Tech. 2001, 18, 329-337. (5) Klibanov, A. L. Bioconjugate Chem. 2005, 16, 9-17.
viscous and requires a minimum applied stress (shear yield) to induce deformation.6 Duncan et al. showed that the high compression state of the shell effectively diminishes tension in the surface to zero, thus removing the Laplace overpressure that would otherwise drive the dissolution of the gas core.7 Borden et al. showed that the shell yields a significant barrier to gas transport and hinders the dissolution rate in degassed media.8,9 More recently, Marmottant et al. showed how the monolayer dilatational properties affect the response to large-amplitude ultrasonification, leading to such interesting phenomena as compression-only oscillation.10 These material properties, which are more characteristic of a solid film than a fluid interface, depend strongly on shell composition and microstructure. Although experimental complexity has precluded direct evidence that the shell is indeed a monolayer, a wealth of indirect evidence supports this view.5,6,8,10-14 Typically, two shell components are necessary to form stable microbubbles.12 The main component is saturated diacyl PC with a main phasetransition temperature (Tm) above the working temperature. The (6) Kim, D. H.; Costello, M. J.; Duncan, P. B.; Needham, D. Langmuir 2003, 19, 8455-8466. (7) Duncan, P. B.; Needham, D. Langmuir 2004, 20, 2567-2578. (8) Borden, M. A.; Longo, M. L. Langmuir 2002, 18, 9225-9233. Borden, M. A.; Longo, M. L. J. Phys. Chem. B 2004, 108, 6009-6016. (9) Pu, G.; Longo, M. L.; Borden, M. A. J. Am. Chem. Soc. 2005, 127, 65246525. (10) Marmottant, P.; van der Meer, S.; Emmer, M.; Versluis, M.; de Jong, N.; Hilgenfeldt, S.; Lohse, D. J. Acoust. Soc. Am. 2005, 118, 3499-3505. (11) Klibanov, A.; Rychak, J. J.; Hughes, M. S.; Cantrell, G. L.; Wible, J. H. Biophys. J. 2005, 88, 207A. (12) Klibanov, A. L. In Topics in Current Chemistry; Krause, W., Ed.; SpringerVerlag: New York, 2002; Vol. 222, p 73. (13) Kim, D. H.; Klibanov, A. L.; Needham, D. Langmuir 2000, 16, 28082817. (14) Borden, M. A.; Pu, G.; Runner, G. J.; Longo, M. L. Colloids Surf., B 2004, 35, 209-223.
10.1021/la052841v CCC: $33.50 © 2006 American Chemical Society Published on Web 03/23/2006
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heterogeneity and previous assertions of uniformity in order to reveal the true nature of the microbubble shell. Our goal was to test whether the main phospholipid component, DSPC, and the lipopolymer, DSPE-PEG2000, mix uniformly (Figure 1a) or phase separate (Figure 1b) within the highly condensed state of the microbubble shell. Herein, we present results of a systematic investigation of the phase behavior and microstructure of perfluorobutane-filled microbubbles coated with a binary mixture of DSPC and DSPEPEG2000. We present results from several spectroscopic and microscopic experiments to characterize the shell under various conditions of composition and temperature. We then argue that thermotropic phase separation results in ordered, PC-rich domains surrounded by a glassy, lipopolymer-rich matrix. 2. Materials and Methods
Figure 1. Schematic representation showing (a) complete miscibility vs (b) phase separation in the monolayer plane of a typical lipidcoated microbubble shell. Shown are the effects of monolayer mixing behavior on the side view (top) and view from above (bottom). Individual components are labeled as shown. Adapted from Baekmark et al.15
second component is an emulsifying agent, which typically comprises a hydrophilic polymer moiety, such as PEG, and a lipid anchor. The purpose of the emulsifier is to promote the self-assembly of the monolayer shell and to form a brush layer that shields the bubble against coalescence and immune cell interrogation. Previous work on DSPC/PEG40S-coated, air-filled microbubbles showed that phase separation between PC and the surfactant emulsifier resulted in a polycrystalline shell.6,14 In this case, the phase separation in the microbubble shell was evident from the obvious squeeze-out plateau observed in the pressure-area behavior on the Langmuir trough.14 Control over microstructure was demonstrated using processing procedures such as heat treatment and area dilation/compression schedules.6,14 In practice, lipopolymers are often used as the emulsifier and/ or ligand-bearing species to coat perfluorocarbon bubbles.3,5,13,16 To our knowledge, no prior systematic study has investigated the phase behavior or microstructure of microbubble shells formed by binary mixtures of PC and lipopolymer. In fact, it has been argued that these species are fully miscible and form a uniform shell (Figure 1a).3,13 Presumably, this assertion stemmed from Langmuir trough work on PC/lipopolymer monolayers in which the collapse surface pressure of the mixture, which was similar in magnitude to that of the pure components, was used as the premise to rule out demixing of the two species.17 Indeed, isotherms of DSPC/DSPE-PEG2000 mixtures do not exhibit any obvious squeeze-out plateaus indicative of phase separation,17 as was observed for DSPC/PEG40S mixtures.14 However, we recently observed heterogeneity on microbubble shells composed of PC and lipopolymer, similar to those observed for the DSPC/ PEG40S system.16 The current study was motivated by the need to address the discrepancy between the recently observed (15) Baekmark, T. R.; Elender, G.; Lasic, D. D.; Sackmann, E. Langmuir 1995, 11, 3975-3987. (16) Borden, M. A.; Kruse, D.; Caskey, C.; Zhao, S.; Dayton, P.; Ferrara, K. IEEE Trans. Ultrason. Ferr. 2005, 52, 1992-2002. (17) Kuhl, T. L.; Majewski, J.; Howes, P. B.; Kjaer, K.; von Nahmen, A.; Lee, K. Y. C.; Ocko, B.; Israelachvili, J. N.; Smith, G. S. J. Am. Chem. Soc. 1999, 121, 7682-7688. Chou, T. H.; Chu, I. M. Colloids Surf., B 2003, 27, 333-344.
2.1. Materials. The synthetic lipids used to form the lipid monolayer shell (DPPC, DiC22PC, DSPC, DSPE-PEG2000, and DSPE-PEG2000-biotin) were purchased from Avanti Polar Lipids (Alabaster, AL). The fluorescent membrane dyes (DiIC18 and DiOC18) were purchased from Molecular Probes (Eugene, OR). PFB was purchased from SynQuest (Alachua, FL). FITC-NA was purchased from Pierce (Rockford, IL). PEG40S, propylene glycol, glycerin, chloroform, and deuterated chloroform were obtained from Sigma (St. Louis, MO). Milli-Q water (Millipore) was used as the suspension medium. 2.2. Microbubble Preparation. Microbubbles were produced according to the shaking method as previously described.18 Briefly, lipids were premixed in chloroform, dried under nitrogen gas and then vacuum, resuspended in aqueous solution (80 vol % water, 10 vol % glycerin & 10 vol % propylene glycol) via bath sonication and freeze-thaw cycling, placed in serum vials (Wheatley) in 2 mL aliquots, and sealed. The air headspace of each vial was purged with 10 mL of PFB prior to refrigerated storage. A mechanical shaker (courtesy of ImaRx Therapeutics, Tucson, AZ) was used to form the microbubbles from the premicrobubble lipid suspension. Washing of excess lipid and other aqueous components was achieved via flotation, a process whereby mild centrifugation (99.99% of all water-soluble components and concentrated into a cake. The cake was lyophilized overnight to form a dry powder and resuspended in deuterated chloroform for analysis. Standard 1H NMR experiments were performed using 5 mm o.d. NMR tubes on a Bruker DRX-500 spectrometer at room temperature with a 5 mm 1H Bruker probehead. A 90° pulse length of 8 µs was used to collect spectra with 32k points at an acquisition bandwidth of 20 ppm and a 1.0 s relaxation delay. Processing of the spectra was achieved with 1 Hz exponential apodization, where the data were zero filled once. Peak areas were determined by using the integration feature in the XWIN NMR software. A known DSPE-PEG2000 concentration was plotted as a function of the ratio of the poly(ethylene glycol) peak (3.7) to that of the acyl chain peak (1.32 ppm). This calibration, along with peak integral ratios, was used to determine the lipopolymer ratio in the samples. The calibration fit is shown in Supporting Information. (18) Unger, E. C.; Lund, P. J.; Shen, D. K.; Fritz, T. A.; Yellowhair, D.; New, T. E. Radiology 1992, 185, 453-456. (19) Takalkar, A. M.; Klibanov, A. L.; Rychak, J. J.; Lindner, J. R.; Ley, K. J. Controlled Release 2004, 96, 473-482.
Phase Separation in Microbubble Shells 2.4. Fourier Transform Infrared (FTIR) Spectroscopy. Freshly formed (n ) 4) or 1-day-old (n ) 2) microbubbles were extracted from their sealed serum vials and diluted into 3 mL of purified water. A single centrifugation step was used to concentrate the bubbles into a cake, and the infranatant was discarded. Samples of cake were spread onto CaF2 windows for infrared analysis. Spectra were obtained with a Perkin-Elmer 2000 FTIR spectrometer interfaced to a PC with Spectrum 2000 software (Perkin-Elmer, Norwalk, CT). The instrument was purged of water vapor with a dry-air generator (Balston, Haverhill, MA). The sample temperature was controlled with a Peltier device and monitored with a thermocouple. Temperature was ramped at a rate of 2 °C/min for all samples during scanning. The ramp rate has been shown not to affect the transition temperatures of the lipids significantly.20 Spectra were obtained as a function of temperature, with a total of 16 spectra averaged for each temperature point. The CH2 symmetric stretching region, from 3000 to 2800 cm-1, was analyzed. Band positions were determined by taking the second derivatives of the original spectra and averaging intercepts at 80% peak intensity. Phase transitions were determined by plotting the CH2 symmetric stretching position as a function of temperature. 2.5. Freeze-Fracture Transmission Electron Microscopy (ff-TEM). Freeze-fracture transmission electron microscopy samples were prepared and imaged by NanoAnalytical Laboratories (San Francisco, CA) according to a standard test method (STM-01). Samples of DPPC/DPPA/DPPE-PEG5000-coated, perfluoropropanefilled microbubbles in aqueous solution with triacetin (ImaRx Therapeutics, Tucson, AZ) were taken directly from the serum vial and quenched using the sandwich technique and liquid-nitrogencooled propane. Using this technique, a cooling rate of 10 000 °C/s was reached in order to avoid ice crystal formation and artifacts possibly caused by the cryofixation process. The cryofixed samples were stored in liquid nitrogen for less than 2 h before processing. The fracturing process was carried out in a JEOL JED-9000 freezeetching instrument, and the exposed fracture planes were shadowed with Pt for 30 s at an angle of 25-35° and then with carbon for 35 s (2 kV/60-70 mA, 1 × 10-5 Torr). The replicas produced this way were cleaned with concentrated, fuming HNO3 for 24 h, followed by repeated agitation with fresh chloroform/methanol (1:1 v/v) at least five times. The sample replicas cleaned in this way were examined on a JEOL 100 CX electron microscope. The images shown are of lipid-monolayer shelled microbubbles that did not exhibit the thick triacetin layer characteristic of acoustically activated lipospheres.21 2.6. Fluorescence Microscopy. Epifluorescence microscopy of the microbubble shells was performed on an inverted microscope (Nikon, Japan) equipped with a digital video camera (Orca, Hamamatsu, Japan) and image capture and processing software (CImaging, Compix). Visualization of domain growth was performed on a custom upright microscope (Mikron, San Marcos, CA) with a digital video camera (Roper, Tucson, AZ) and image capture, processing, and analysis software (Image Pro, Media Cybernetics). Heating and cooling on the microscope stage was achieved with a Peltier junction (Carl’s electronics, Sterling, MA) controlled with a standard 12 V power supply. Confocal fluorescence microscopy was performed on an upright microscope (Nikon PCM 2000) with a 60× oil-immersion objective and software for z-axis control, image capture, and processing (C-Imaging). Image sequences were stacked to form 2-D maximum projection montages. Microbubbles were suspended in high glycerol to reduce thermal motion, and this did not affect the microstructure. Image analysis was performed with ImageJ by converting the images to 8-bit gray scale and then 32-bit gray scale.
3. Results and Discussion 3.1. Shell Composition. NMR spectroscopy was used to determine the average ratio of lipopolymer to PC in the (20) Ricker, J. V.; Tsvetkova, N. M.; Wolkers, W. F.; Leidy, C.; Tablin, F.; Longo, M.; Crowe, J. H. Biophys. J. 2003, 84, 3045-3051. (21) May, D. J.; Allen, J. S.; Ferrara, K. W. IEEE Trans. Ultrason. Ferr. 2002, 49, 1400-1410.
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Figure 2. Percentage of lipopolymer in the shells of washed DSPC/ DSPE-PEG2000-coated microbubbles (mean ( s.d.) as determined by 1H NMR spectroscopy. The line is theoretical result of equivalent shell and bulk fractions.
Figure 3. Typical FTIR thermal profile of the CH2 symmetric stretch of concentrated microbubbles coated with 90 mol % DSPC and 10 mol % DSPE-PEG2000. The wavenumber (υ-1) is shown on the left axis (raw data points), and the first derivative of the profile (δυ-1/δT) is labeled on the right axis (unsmoothed gray line).
microbubble shells as a function of the bulk concentration of the lipid suspension prior to microbubble formation. This was achieved by measuring the peak-area ratios of PEG (δ ) 3.7 ppm) to hydrocarbon chains (δ ) 1.32 ppm) of the shell contents from washed microbubbles (containing
