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Mechanical Properties and Microstructure of Polycrystalline Phospholipid Monolayer Shells: Novel Solid Microparticles Dennis H. Kim,† M. Joseph Costello,§ P. Brent Duncan,‡ and David Needham*,‡ Department of Mechanical Engineering & Materials Science, Duke University, Durham, North Carolina 27708, and Department of Cell Biology and Anatomy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 Received May 7, 2003. In Final Form: July 17, 2003 W This paper contains enhanced objects available on the Internet at http://pubs.acs.org/ journals/langd5. A polycrystalline phospholipid monolayer self-assembled at the surface of an air microbubble in aqueous solution represents a novel material structure: in essence, a solid shell of wax with micrometer-scale dimensions and a thickness of only a single molecule. Micropipet manipulation of these microparticles revealed the dependence of the mechanical properties of the lipid shells, specifically, yield shear and shear viscosity, on the composition, grain microstructure, and thermal processing of the material, in particular the cooling rate of the shells from the melt. Properties were measured as a function of the (1) lipid composition at a fixed cooling rate and (2) cooling rate at a fixed lipid composition. Epifluorescent microscopy and transmission electron microscopy revealed that the morphology of the 1,2-distearoyl-sn-glycero-3phosphatidylcholine monolayer microstructure, which develops upon freezing from the melt, is dependent on the cooling rate through the lipid transition temperature Tm, with larger micrograins being formed at slower cooling rates. Mechanical properties of the lipid shell follow micrograin size, with the coarse grain structure exhibiting a higher resistance to shear deformation than the fine grain structure does, which is behavior consistent with that of more traditional bulk crystalline materials.
Introduction The same principles of self-assembly that describe the formation and structure of a lipid monolayer on a flat trough of water also apply to the spontaneous organization of amphiphilic material at a curved gas-liquid interface such as that posed by the surface of an air microbubble in water.1-3 It is well-known that at sufficient concentrations the surface contaminants on a “dirty” bubble in water can effectively immobilize the air-water interface and cause the bubble to behave as a rigid particle. A relatively recent addition to the family of experimental techniques for studying monolayer properties known as axisymmetric drop shape analysis (ADSA) was employed by Kwok and co-workers as a film balance, albeit on a curved droplet surface, to obtain surface pressure-surface area (Π-A) isotherms for monolayer films of 1-octadecanol.4,5 ADSA experiments have demonstrated the equivalence of curved monolayers and flat monolayers with respect to surface phase states and transitions. Until recently, however, the direct measurement of the mechanical properties of * To whom correspondence may be addressed. † Current address: MEMPHYS Center for Biomembrane Physics, Department of Physics, University of Southern Denmark, Odense, Denmark. ‡ Duke University. § University of North Carolina. (1) D’Arrigo, J. S. J. Colloid Interface Sci. 1984, 100, 106-111. (2) Singhal, S.; Moser, C. C.; Wheatley, M. A. Langmuir 1993, 9, 2426-2429. (3) Leighton, T. G. The Acoustic Bubble; Academic Press: San Diego, 1994. (4) Kwok, D. Y.; Vollhardt, D.; Miller, R.; Li, D.; Neumann, A. W. Colloids Surf., A 1994, 88, 51-58. (5) Kwok, D. Y.; Tadros, B.; Deol, H.; Vollhardt, D.; Miller, R.; Cabrerizo-Vilchez, M. A.; Neumann, A. W. Langmuir 1996, 12, 18511859.
monolayers spread at micrometer-scale curved interfaces has eluded investigators; furthermore, extant work on monolayers, whether on flat or curved interfaces, has been mostly confined to the liquid-expanded or liquid-condensed-expanded coexistence regimes of their phase diagrams. The current study presents the first mechanical property measurements made for solid lipid monolayer shells of well-defined composition and micrograin structure formed on the surfaces of gas microbubbles, thus essentially comprising gas microparticles stabilized by a solid wax monolayer. The precedent for the investigation was found in studies done on solid phospholipid bilayer vesicles; the surface shear viscosity and yield shear of gel-phase dimyristoylphosphatidylcholine (DMPC, diC14:0 PC) bilayer vesicles (diameter ∼ 20 µm) have been measured by micropipet manipulation methods and found to be on the order of 1 surface poise and 10-3 mN m-1, respectively.6 The objective of the present study was to characterize the surface viscoelastic shear properties of lipid monolayer shells formed at the air-water interface of air microbubbles using a methodology similar to that used in the Evans and Needham experiment on a vesicle system. Instead of the liquid-expanded-state monolayers of traditional monolayer studies, we focused on lipid monolayers comprised of saturated diacyl phospholipids varying homologously with the acyl chain length (from 18 to 24 carbons per chain) and, as such, forming highly rigid, solid monolayers at room temperature. The mechanical properties of such monolayers have been largely inaccessible to more traditional measurement techniques but are measurable by micropipet manipulation. Furthermore, using epifluorescent microscopy and transmission electron (6) Evans, E.; Needham, D. J. Phys. Chem. 1987, 91, 4219-4228.
10.1021/la034779c CCC: $25.00 © 2003 American Chemical Society Published on Web 09/06/2003
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Kim et al.
Table 1. Phospholipids Used as the Main Component of the Monolayer Shellsa
a
lipid
abbreviations
carbons per acyl chain
Tm (°C)
1,2-distearoyl-sn-glycero-3-phosphatidylcholine 1,2-diarachidoyl-sn-glycero-3-phosphatidylcholine 1,2-dibehenoyl-sn-glycero-3-phosphatidylcholine 1,2-dilignoceroyl-sn-glycero-3-phosphatidylcholine
diC18:0 PC, DSPC diC20:0 PC, DAPC diC22:0 PC, DBPC diC24:0 PC, DLgPC
18 20 22 24
55.1 ( 1.5 64.5 ( 0.5 74.0 ( 1.4 80.1
Tm is the main bilayer gel-to-liquid crystalline phase-transition temperature. Values are as cited by Marsh.18
microscopy (TEM), we examined the cooling-rate-dependent microstructure of the lipid shell and studied the influence of micrometer-scale shell grain structure on the material properties. Historically, the measurement of lipid monolayer properties has been geometrically constrained to flat, twodimensional systems, and, therefore, a preponderance of studies in this area have involved the Langmuir trough and its variants.7-9 A series of temperature- and composition-dependent surface phase transitions have been mapped for various lipid monolayers in this fashion.10,11 The most common measurement made with the trough technique has been that of surface pressure (Π) as a function of surface molecular area (A) at a constant temperature; the two-dimensional surface compressional modulus is derived directly from the slope of the isotherm. Another material property, the surface shear viscosity, is defined as the ratio of tangential force per unit length of the surface to the rate of strain of the surface produced by the stress. The surface shear viscosity of monolayers has been measured on various configurations of surface viscometers, some of which are essentially modified versions of the Langmuir trough. These methods have been reviewed by Miller et al.12 The canal method has been regarded as the most accurate method of measuring the surface viscosity of insoluble films, but the range of accurately measurable values is low, typically 10-5-10-3 surface poise.7,13 The rotating wall knife-edge surface viscometer has a range of measurable viscosities with an upper limit only around 10-1 surface poise.7,12,13 Higher monolayer surface viscosities have thus far been beyond the ability of conventional trough-based techniques to measure. The surface yield shear is another property whose measurement can be obtained by either the canal or the rotational torsional viscometer. As the term suggests, the surface yield shear is the stress that a monolayer can sustain before undergoing two-dimensional shear flow. The surface yield values of a limited number of substances have been measured in this manner. For example, an equilibrated monolayer of lauryl alcohol (LA) adsorbed from a saturated solution at 25 °C was found to have a yield shear of approximately 0.12 dyn cm-1.14 In general, the yield of monolayers is higher for condensed phases than that for expanded phases.7 Again, it is worth noting that the mechanical properties of highly condensed, solidstate monolayers have not been measured by these (7) Davies, J. T.; Rideal, E. K. Interfacial Phenomena, 2nd ed.; Academic Press: New York, 1961. (8) Gaines, G. L., Jr. Insoluble Monolayers at Liquid-Gas Interfaces; Interscience Publishers: New York, 1966. (9) Pallas, N. R.; Pethica, B. A. Langmuir 1985, 1, 509-513. (10) Phillips, M. C.; Chapman, D. Biochim. Biophys. Acta 1968, 163, 301-313. (11) Albrecht, O.; Gruler, H.; Sackmann, E. J. Phys. (Paris) 1978, 39, 301-313. (12) Miller, R.; Wustneck, R.; Kragel, J.; Kretzschmar, G. Colloids Surf., A 1996, 111, 75-118. (13) Edwards, D. A.; Brenner, H.; Wasan, D. T. Interfacial transport processes and rheology; Butterworth-Heinemann: Boston, 1991. (14) Brown, A. G.; Thuman, W. C.; McBain, J. W. J. Colloid Sci. 1953, 8, 491-507.
Figure 1. (Top) Chemical structure of a generic saturated diacyl PC molecule. 〈n〉 is the total number of carbons per chain. (Middle) Chemical structure of PEG 40 stearate. (Bottom) Chemical structure of BODIPY FL diC16 PE.
methods because of physical limitations of the measurement techniques. Materials and Methods Lipid-Shell Preparation. Saturated diacyl phosphatidylcholines (PCs) constituted the major component of the lipid shells tested in the current study. All phospholipids were obtained from Avanti Polar Lipids, Inc. (Alabaster, AL), as lyophilized powder of greater than 99% purity and were used without further purification. A homologous series of PCs varying in acyl chain length were used in the current study; these lipids are listed in Table 1, and their generic structure is given in Figure 1. A second, minor component of the lipid shell was poly(ethylene glycol) stearate (PEG stearate, Myrj 52, Sigma, St. Louis, MO), a nonionic surfactant commonly used as an emulsifier (see Figure 1). The PEG moiety consisted of 40-mers (approximate molecular weight 1760 daltons), attached to the stearic acid portion of the molecule via an ester linkage. PEG stearate served as a steric stabilizer to the shell, preventing coalescence of microbubbles with each other and possibly with the gas-liquid interface of the suspension (because microbubbles rise in an aqueous medium).15-17 The PC to PEG stearate molar ratio was 10:1. For fluorescence micrography, approximately 0.1 mol % of a fluorescently labeled lipid, N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3propionyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (BODIPY FL diC16:0 PE, polyethylene, Molecular Probes, Eugene, OR), was included to label the shell (see Figure 1). The excitation and emission wavelengths were 505 and 511 nm, respectively. The shell components were weighed in dry-powder form (total lipid mass approximately 25 mg) into a 20-mL borosilicate glass (15) Wheatley, M. A.; Peng, S.; Singhal, S.; Goldberg, B. B. U.S. Patent 5,352,436, 1994. (16) Wheatley, M. A.; Singhal, S. React. Polym. 1995, 25, 157-166. (17) Wang, W.; Moser, C. C.; Wheatley, M. A. J. Phys. Chem. 1996, 100, 13815-13821.
Polycrystalline Phospholipid Monolayer Shells scintillation vial and codissolved in chloroform. The solvent was then removed in a vacuum trap desiccator, and the remaining lipid film was rehydrated with 5 mL of phosphate buffered saline (PBS). After the addition of PBS to the dried film, the vial was tightly capped, swirled briefly, and sonicated in a Branson 1200 bath sonicator (Danbury, CT) for approximately 20 s to reclaim lipid from the vial walls as well as promote dispersion of the lipid. The vial was then incubated in an oven at a temperature at least 10 °C above the main phase-transition temperature Tm of the main lipid component for 2-3 h (see Table 1). Following incubation, the sample was removed from the oven and the suspended lipid aggregates were dispersed using a probe sonicator (model XL2020, Misonix, Farmingdale, NY) equipped with a 12.7mm flat tip. The probe was first lowered into the vial and positioned close to the bottom of the vial; the sample was then sonicated at a low output level (3 on a range of 1-10, or approximately 82.5 W) for 3 min. This procedure reduced the large aggregates of phospholipid, as indicated by the reduced turbidity of the sample. The sonicator was then used again to produce microbubbles of air in the lipid suspension.19 To perform this task, the probe tip was positioned within the vial until it just made contact with the surface of the lipid suspension (i.e., airwater interface). The sample was then sonicated at the maximum output level (level 10, or approximately 104.5 W) for a short burst of 30 s. This procedure produced a milky polydisperse suspension of microbubbles and foam from the entrained ambient air. The probe was then withdrawn, and the sample vial was recapped. The lipid composition of the microbubble precursor was assayed by thin-layer chromatography and found to be identical before and after the sonication procedure; that is, the procedure did not produce any lipid degradation products. The probe sonication technique increased the temperature of the lipid suspension. After the first step in the sonication procedure, the suspension temperature was typically 80-85 °C, as measured by a thermocouple probe (Omega model DP116, Stamford, CT). Therefore, during the second step of the sonication procedure, the lipid vesicles in suspension spread onto the newly created water-air interfaces (i.e., the surfaces of the microbubbles) from the liquid state, and the resulting liquid-phase monolayers were subsequently cooled to T < Tm to form a frozen, solid, polycrystalline monolayer shell encapsulating the gas microbubble, in effect creating a gas-filled lipid microparticle. It should be noted that no apparent effects on the gel-phase transition arise from the presence of the PEG stearate, as assayed by differential scanning calorimetry of the microparticle samples (data not shown). Microbubbles coated in this fashion were stabilized because the high surface pressure characteristic of a solid lipid monolayer shell offsets the Laplace pressure that drives bubble dissolution;20 others have shown that when the gas concentration in solution is below saturation, the lipid shell also retards the diffusion of gas from the bubble interior to the surrounding medium, although it does not completely arrest this diffusion.21 Several methods were used to achieve a range of cooling rates spanning 4 orders of magnitude (100-103 °C/min) and thereby control the crystallization rate of the adsorbed lipid monolayer shell from the melt. The slowest cooling rates used in the current study were obtained by placing the sample vial into a small, loosely capped thermos and allowing it to cool gradually through the lipid bilayer liquidto-gel transition and down to room temperature (23-25 °C) over the course of 20-30 min (cooling rate 2-3 °C/min). A second cooling protocol consisted of placing the sample vial in 25 mL of tap water at room temperature in a 50-mL beaker; in this manner, room temperature was typically reached in 5-10 min (cooling rate 6-12 °C/min). In a third method, the vial was cooled in a cold circulating water bath thermostated at 5 °C. Room temperature was typically reached in 30-40 s (cooling rate 100-120 °C/min). Last, the fastest cooling rates were obtained by drawing (18) Marsh, D. CRC Handbook of Lipid Bilayers; CRC Press: Boca Raton, FL, 1990. (19) Hilmann, J.; Hoffmann, R.-R.; Muetzel, W.; Zimmermann, I. U.S. Patent 4,466,442, 1984. (20) Epstein, P. S.; Plesset, M. S. J. Chem. Phys. 1950, 18, 15051509. (21) Borden, M. A.; Longo, M. L. Langmuir 2002, 18, 9225-9233.
Langmuir, Vol. 19, No. 20, 2003 8457 a microparticle aliquot into a heated steel syringe barrel and subsequently plunging the needle into ice water. The metal walls of the needle provided improved heat conduction from the sample, resulting in cooling rates on the order of 2200 °C/min. After formation, the sample of microparticles self-segregated from the infranate by flotation and stratified by size, with the largest particles rising most rapidly to the top of the suspension. The sample, therefore, consisted of three regions: an upper layer of foam; a size-graded middle layer of microparticles; and a lower, less turbid layer of nanoparticles (on the order of 200 nm) and unincorporated lipid aggregates dispersed throughout the aqueous phase. Microparticles were carefully extracted by a Gilson precision microliter pipet (Woburn, MA) from the middle layer with as little agitation of the sample as possible and injected directly into the micromanipulation chamber for micropipet experimentation on single microparticles. Cryofixation of Microparticles for TEM. Formed microparticle samples, after preparation by the above sonication and cooling techniques, were prepared for TEM by a standard cryofixation technique involving ultrarapid plunge freezing to produce cooling rates in excess of 10 000 °C/s.22 Freeze fracture was performed in a Cryofract 190 (Reichert-Jung) at -150 °C under a 10-7-Torr vacuum. The complementary fracture surfaces were replicated with 15 Å of Pt/C, followed by 100 Å of C; the sample was then coated with collodion.22-24 The sample replica was retrieved by dissolving the copper sample support with chromic acid, digesting the sample with Clorox bleach, and dissolving the collodion with methanol. The replicas were mounted on 50-mesh gold grids and viewed on a JEOL 200CX transmission electron microscope at 80 kV. Micromanipulation Technique and Data Acquisition. The micromanipulation technique has been described in detail elsewhere.6,25-28 Briefly, pipets were fashioned from standard glass capillary tubing, pulled to create a suitable taper using a vertical pipet puller (model 730, David Kopf Instruments, Tujunga, CA) and given a flat tip using a custom-made microforge device. Three micropipets were required for the experiment: two pipets (designated the holding and measuring pipets) of approximately the same internal diameter (4-6 µm), mounted coaxially and facing each other, and a third pipet (the transfer pipet) of a large internal diameter (40-50 µm) used as a protective sheath for the holding pipet during transfer of a microparticle between the two chambers. For sensitive pressure application to a microparticle, a micropipet was filled with PBS and connected to a water-filled manometer system containing in-line pressure transducers. Suction pressure was applied by a syringe connected to the system. The dual transducer system made possible the measurement of pressures ranging from the on order of 10-1104 Pa (10-6-10-1 atm). The pipets and chamber surfaces were incubated in an aqueous 0.2 g % solution of bovine serum albumin (BSA, fatty acid free, Sigma) for 15-30 min to prevent the lipidcoated bubbles from sticking to the glass.29 The BSA solution was removed and replaced by PBS prior to the experiment. The micromanipulation chambers were fashioned from cut glass slides and cover slips; a schematic is shown in Figure 2A. Each chamber had an “open” design: the cover slips formed the floor and ceiling of the chamber, with the chamber fluid held in place by surface tension, and the pipets entered the chamber from the open sides. The entire assembly was placed on a modified two-dimensional-translatable stage on a Nikon Diaphot-TMD inverted microscope equipped with differential interference contrast optics. Images were obtained with 40× (air) magnification. Experiments were recorded on videotape, and the elapsed (22) Costello, M. J.; Fetter, R.; Corless, J. M. Proceedings of the 4th Pfefferkorn Conference; AMF O’Hare: Chicago, 1985; pp 105-115. (23) Costello, M. J.; Fetter, R. D.; Frey, T. G. Proceedings of the 4th Pfefferkorn Conference; AMF O’Hare: Chicago, 1985; pp 95-101. (24) Fetter, R. D.; Costello, M. J. J. Microscopy 1986, 141, 277-290. (25) Evans, E. A.; Skalak, R. Mechanics and Thermodynamics of Biomembranes; CRC Press: Boca Raton, FL, 1980. (26) Kwok, R.; Evans, E. Biophys. J. 1981, 35, 637-652. (27) Needham, D.; Zhelev, D. V. In Vesicles; Rosoff, M., Ed.; Marcel Dekker: New York, 1996; pp 373-444. (28) Needham, D.; Zhelev, D. In Giant Vesicles; Luisi, P. L., Walde, P., Eds.; John Wiley & Sons, Ltd.: Chichester, 2000; pp 103-147. (29) Meltzer, H.; Silberberg, A. J. Colloid Interface Sci. 1988, 126, 292-303.
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Figure 2. Schematic of a standard micromanipulation chamber with pipets. (A) Three-dimensional view. The length and width of the chamber base are 7.6 and 2.5 cm, respectively (i.e., the dimensions of a standard microscope specimen slide). The volume of a full chamber is approximately 400 µL. (B) Plane view. During the transfer process of the experiment, the positions of the pipets are fixed and the chamber is translated by a movable stage. time and pipet suction pressure were displayed directly on the monitor screen by a video multiplexing system (Colorado Video, Inc., Boulder, CO; Vista Electronics, La Mesa, CA). All experiments were recorded on videotape for subsequent analysis. A recorded stage micrometer provided the calibration for distance measurements, which were made from the videotaped images using a video caliper system (Vista Electronics). To ensure a constant temperature environment for the microparticles, experiments were conducted in a special variant of the standard micromanipulation chamber described previously, referred to as the temperature control chamber. The chamber itself is housed in a stainless steel jacket connected to a thermostated water bath that allows precise regulation of the chamber temperature. During the course of the experiment, the temperature in the chamber was monitored with a thermocouple inserted into one corner of the chamber. Degassed Buffer Solutions. Any micropipet-induced deformation of the initially spherical lipid monolayer shells involves the dilational, shear, and bending modes of deformation.25 To deform the shell solely in the shear mode, excess surface area must be produced via volume reduction (i.e., deflation) of the underlying gas content, with the implicit assumption that surface shell material is conserved. To provide an air-depleted environment to which the microparticles could be transferred for controlled deflation, a separate volume of PBS solution was degassed under reduced pressure with sonication30 and kept sealed until the time of the experiment. An isolated dissolved oxygen meter (World Precision Instruments, Sarasota, FL) was used to measure the change in O2 content within a fluid-filled micromanipulation chamber with time; after 40 min, the O2undersaturated solution nearly returned to saturation levels. Therefore, during the course of the experiment, the degassed chamber solution was periodically replaced with aliquots taken from the sealed reservoir. Fluorescent Microscopy for Domain-Boundary Visualization. Epifluorescence was used to observe the morphology of crystalline domains in the 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC) shells. The method was inspired by the filmbalance technique for visualizing the distribution of crystalline (30) Suslick, K. S. Ultrasound: its chemical, physical, and biological effects; VCH Publishers: New York, 1988; pp xiii, 336.
Kim et al. and liquid regions in a phospholipid monolayer in their coexistence regime of surface pressure; in the current study, however, the monolayer is at maximum surface pressure and, therefore, almost entirely in the condensed state. In both cases, impurities such as dye probes are excluded from the condensed gel phase and demarcate the boundary regions of crystalline domains.31,32 The DSPC microparticle batch was washed by bucket rotor centrifugation to remove the background fluorescence from unincorporated fluorescent material lingering in the sample. A drop of microparticles (1-2 µL) was mounted in approximately 10 µL of buffer on a standard glass slide and placed on the stage of a Nikon Diaphot 200 inverted microscope equipped with a 100× oil immersion plan-apochromatic objective. An Optronics VI-470 CCD camera (Goleta, CA) was used to acquire images of microparticles under both bright-field and epifluorescent illumination, and the images were recorded on videotape with a Sony SVO-9500MD super-VHS VCR. Bright-field and epifluorescent images were captured from videotape onto an Apple Power MacIntosh 7200/90 equipped with a Data Translation DT3155 frame grabber card (Marlboro, MA) and image-processing software (NIH Image 1.61, Research Services Branch, NIMH). Surface Yield Shear and Shear Viscosity Measurement. The experiment to measure the yield shear and shear viscosity of lipid monolayer microparticle shells is conceptually similar to micropipet manipulation experiments performed previously on gel-phase phospholipid bilayer vesicles.6 As in the vesicle experiment, the phase state of the lipid monolayer shell was solid (gel) at room temperature. The frozen DMPC vesicle possessed excess surface area in the form of surface ripples that when “pulled out” by micropipet tension resulted in a projection of the bilayer supported in the suction micropipet; for the gasmicroparticle experiments, excess monolayer surface area was produced by partial microparticle deflation (volume loss) effected by a brief and controlled degassing of an individual microparticle under a supporting tension applied by the micropipet. The procedure by which excess area was produced on a single microparticle will now be described. A small drop (∼1 µL) of the sample containing microparticles was injected into one end of the chamber containing regular air-saturated PBS, where the microparticles were stable. One of the small bore pipets was used as a holding pipet to capture a microparticle of suitable size (typically 10-20-µm diameter) and sphericity (Figure 3A). The microparticle was then transferred under a low holding pipet suction pressure (on the order of 10 Pa) to the second, degassed chamber. This process required moving the captured test microparticle through the interfaces and the air gap spanning the space between the two liquid-filled chambers. To protect the microparticle during this translocation, the microparticle and the end of the pipet supporting it were inserted into the largebore transfer pipet prior to transfer from one chamber to another. During the transfer process, the pipets are kept stationary while the stage translates along the axes of the pipets (see Figure 2B). Upon exposure to the degassed environment, the captured microparticle rapidly underwent volume loss at a constant surface area; typically, a 15-25% volume reduction was achieved in 2-5 s. Although evaporation and the diffusion of other gases across a monolayer-coated interface appear to be retarded relative to diffusion across a bare interface,33,34 the presence of the monolayer did not provide enough of a permeability barrier to impede the (air) deflation of the microparticle in the air-undersaturated medium on the time scale of the experiment.35 As the gas microparticle deflated, the applied micropipet suction pressure was sufficient to support the shell; the reduction in microparticle volume was, thus, accompanied by the production of excess surface area and manifested in an increase of the projection length in the pipet (Figure 3B). As in the case of gel-phase liposomes, (31) Peters, R.; Beck, K. Proc. Natl. Acad. Sci. U.S.A. 1983, 80, 71837187. (32) Losche, M.; Mohwald, H. Eur. Biophys. J. 1984, 11, 35-42. (33) Hawke, J. G.; Alexander, A. E. In Retardation of Evaporation by Monolayers: Transport Processes; La Mer, V. K., Ed.; Academic Press: New York, 1962; pp 67-73. (34) Blank, M. In Retardation of Evaporation by Monolayers: Transport Processes; La Mer, V. K., Ed.; Academic Press: New York, 1962; pp 75-95. (35) Duncan, P. B.; Needham, D. To be submitted for publication, 2003.
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Figure 3. Videomicrographs of the experimental sequence. The pipet inner diameter is 5.2 µm. The main lipid component of the microparticle shell is diC18 PC. (A) A single microparticle is captured. The captured microparticle is then inserted into a wide bore pipet and transferred into an adjacent chamber containing degassed (i.e., air-undersaturated) solution. (B) In this environment, the microparticle undergoes a controlled deflation under a holding pressure. Some of the excess surface area thus created is drawn into the pipet as a supported projection. During deflation, the shell transiently assumes an irregular, faceted appearance, characteristic of a solid-phase polycrystalline monolayer. (C) Following deflation, the suction pressure is increased to smooth out the surface monolayer, taking up all the excess surface area into a well-defined geometry, and the microparticle is returned to the starting chamber and brought into the proximity of the measuring pipet. (D) The holding pressure is released as a capture pressure is exerted by the measuring pipet, and the gas microparticle is extracted from the holding pipet. The microparticle shell retains a projection that is a molded replica of the pipet, and the aspirated particle geometry is characteristic of a solid shell with near 0 tension and only residual elasticity in the surface. The pressure is increased incrementally until a projection equal to one pipet radius in length is produced. (E) This point is the threshold of shear-elastic entry. At a pressure in excess of the yield threshold, the projection increases steadily and the shell is sheared and flows into the pipet. (F) The motion of the shell in this manner is tracked by a reduction of the projection outside the pipet and an increase in the projection inside the pipet. W A movie clip of the experimental sequence in Quicktime format is available. the rigidity of the deflated microparticle shell and the reduction of tension in the surface were demonstrated by the nonspherical shape assumed upon deflation and the persistence of the excess area projection upon release of the supporting pipet pressure. (The projection did not, however, maintain its shape indefinitely as a result of residual tension in the surface. The persistence of the deformed shape in fact varied with the composition of the lipid monolayer, with lipids with longer acyl chains maintaining the projection for the longest periods.) To perform the yield shear and shear viscosity experiments, the deflated microparticle was returned to the first chamber environment, where it was transferred to the second small bore pipet, designated the measuring pipet (Figure 3C,D). The suction pressure applied by the measuring pipet was then increased in small increments; up to a threshold pressure, the shell underwent elastic deformation. The threshold of entry occurred when the projection length of the shell was equal to one pipet radius Rp, as predicted by theory (Figure 3E).36 From the threshold entry pressure P0, the yield shear τs was calculated (see the appendix for a derivation of the membrane constitutive equations):
τs )
P0Rp 4 ln(R0/Rp)
Figure 4. Typical time course of pipet suction pressure application and the corresponding projection length changes of a gas microparticle during the experiment. Letters D, E, and F correspond to the panels in Figure 3. The pipet aspiration pressure and the projection length are scaled by the entry threshold pressure P0 and the radius of the pipet Rp, respectively. The pressure is increased stepwise until the monolayer yield is reached at P ) P0, indicated by L ) Rp. The pressure is then stepped up to a level in excess of P0 (e.g., three times the threshold); the effect on the projection length is a steady increase as the microparticle shell flows into the pipet under viscoplastic deformation (indicated by the linear increase in L/Rp between E and F). where R0 is the radius of the spherical portion of the microparticle outside the pipet. After the threshold pressure was determined, the suction pressure was preset to a value approximately 3 times in excess of this threshold level, and the microparticle was made to flow into the pipet (Figure 3F) until the portion of the microparticle outside the pipet once again formed a smooth spherical shape, that is, the point at which area dilation (or reduced-pressure volume reduction) was necessary for shell expansion and further entry. This flow into the pipet represents the shear of the spherical body of the microparticle at a constant shell area, that is, positive shear near the mouth of the pipet, negative shear distal to the pipet, and plug flow of the monolayer in the pipet and the nose. A typical “schedule” of the pipet suction pressures to which a microparticle was subjected and the corresponding changes in the projection length are diagrammed in Figure 4. The shear flow of the shell into the pipet under aspiration pressure is indicated by the projection length increase inside the pipet coupled with the decrease of the projection outside the pipet. The surface shear viscosity ηs of the microparticle shell is given by
ηs )
∆PRp2 4(dL/dt)[1 - (Rp/R0)2]
(2)
where ∆P is the difference between the excess level of suction pressure Pxs and the threshold entry pressure P0 and dL/dt is the rate of change of the projection length (cm s-1) under the application of excess suction pressure (Figure 4). In the first set of experiments, a single cooling method, namely, cooling of the sample vial in a beaker of tap water to effect a cooling rate on the order of 101 °C/min, was applied to all the coated microparticle preparations. The main PC component was varied between different microparticle samples by selecting saturated diacyl PCs with varying acyl chain lengths (18, 20, 22, or 24 carbons per chain). PEG stearate was present in all compositions as a stabilizer; without it, no stable microparticles were formed (see Materials and Methods). Surface shear properties were also measured for microparticles of a single lipid composition (diC18:0 PC) formed at different cooling rates. For convenience, a modified protocol for producing the requisite shell geometry was employed: to accommodate a single wide microchamber for oil immersion optics, a method of creating excess microparticle surface area was needed that did not require transfer into an undersaturated solution chamber. With the use of a single pipet, a spherical microparticle was captured and brought to the floor of the chamber containing gas-saturated solution, and a sufficiently high aspiration pressure (on the order of 104 Pa) was then applied to reduce the pressure on the gas and produce area dilation of the microparticle surface.
(1) (36) Yeung, A.; Evans, E. Biophys. J. 1989, 56, 139-149.
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Figure 5. Surface yield shear τs of phospholipid monolayercoated microparticle shells as a function of reduced temperature Tr of the lipid. The chamber temperature is on average 24 °C (297 K). Each point represents the mean of 30-40 measurements. Under this suction pressure, the resulting volume deflation was accompanied by the production of a projection length supported by the aspirating pipet. After the “nose” configuration was achieved (equivalent to the geometry in Figure 3C), the pressure was dropped to a low holding level (on the order of 10 Pa), and the shell was allowed to come to equilibrium. Subsequent inversion of the microparticle orientation with a coaxial measuring pipet (as in the standard technique) demonstrated the stability of the shell after deflation of the microparticle. With the desired experimental geometry obtained, the surface yield shear and shear viscosity measurements were conducted as in the standard technique previously described.
Results Surface Shear Properties. The experimental sequence described in Materials and Methods for measurement of the surface shear properties of the lipid monolayer is summarized as capture, transfer, deflation, transfer, threshold entry, and (plastic shear) flow induction. The entire sequence was conducted for each test microparticle, and 30-40 microparticles, taken from 2-4 different batches, were tested for each shell composition. The mean and standard error were compiled for each composition. All microparticles were tested at room temperature (24 °C, which varied by as much as (2 °C). At a given temperature T, each lipid species was defined by a characteristic reduced temperature, Tr, defined as
Tr )
Tm - T Tm
(3)
where T is the temperature in the micromanipulation chamber (i.e., the environment of the coated microparticle) and Tm is the main (gel-to-liquid crystalline) phasetransition temperature of the lipid shell (see Table 1). The reduced temperature is, therefore, an index of how far below the main phase transition a lipid monolayer shell is at T and provides a normalization for the comparison of lipids with different transition temperatures. (It should be noted, however, that this does not necessarily imply the mechanical equivalence of different lipid shells at the same reduced temperature.) Lipid shells that are farther below their Tm’s have a higher reduced temperature and in general exhibit more “gel-like character” than do shells that are closer to their Tm’s. The yield shear values are plotted as a function of the reduced temperature in Figure 5 and reflect this trend. Among the lipid species tested, diC18:0 PC formed shells that displayed the lowest yield shear. The absolute difference between room temperature and the Tm of diC18:0 PC was approximately 30 °C. In contrast, diC24:0 PC was 55 °C below Tm and, hence, at a greater reduced temperature. Shells of this type were much more resistant to shear
Figure 6. Surface shear viscosity ηs of phospholipid monolayercoated microparticle shells as a function of reduced temperature Tr of the lipid. The chamber temperature is on average 24 °C (297 K). The surface shear viscosity for diC16 PC was taken from Kragel et al.40 Each point represents the mean of 30-40 measurements.
stress than the other shells and were characterized by the highest yield shear values observed. The surface shear viscosity values corresponding to the samples in Figure 5 are given in Figure 6. As with the surface yield shear data, the abscissa is the reduced temperature for the lipid monolayers at room temperature, and the surface shear viscosity increases monotonically with increasing reduced temperature. The deformation of the stiffer lipid shells (those composed of diC20:0, diC22: 0, and diC24:0 PCs) required high suction pressures that produced some slight deflation (loss of volume) of the microparticle concurrently with the shearing of the shell and, therefore, deviation from the ideal model of surface shear at a constant microparticle volume. This deviation may account for the relatively large error bars for the lipids with longer acyl chains. Note that an equivalent bulk viscosity can be obtained by dividing the surface value by the approximate thickness of the monolayer (∼3 nm): for diC18:0 PC the equivalent bulk viscosity is, therefore, on the order of 107 P. By comparison, the shear viscosity of window glass is on the order of 105 P at 800 °C and 1013 P at 515 °C,37 and the typical shear viscosity range for plastics is 106-1012 P.38 The melt viscosity of PE, which is dependent upon molecular weight, temperature, and pressure, is on the order of 103-104 P.39 It is reasonable to expect that the lipid monolayer shell, very roughly approximated as a crystalline array of short PE chains, exhibits a greater viscosity than the melt. Domain Morphology and Its Relation to Monolayer Properties. Fluorescent videomicrography of microparticle shells formed from diC18:0 PC and doped with BODIPY FL diC16:0 PE revealed for the first time the existence of a polycrystalline domain microstructure and its accompanying network of grain boundaries in a micrometer-scale phospholipid monolayer shell. During formation of crystalline microdomains in the predominantly diC18:0 PC shell, the BODIPY PE marker was excluded from the microdomains because of orientational differences between the PC and fluorescently labeled PE molecules imposed by their headgroups.41 Fluorescently labeled lipid-coated microparticles were prepared with the different cooling rates previously described and examined on the microscope stage, where epifluorescent illumination and oil immersion optics revealed the microstructural organization in the monolayer shell. (37) Van Vlack, L. H. Elements of Materials Science Engineering, 6th ed.; Addison-Wesley Publishing Company: Reading, MA, 1989. (38) Lenk, R. S. Plastics rheology; mechanical behaviour of solid and liquid polymers; Wiley-Interscience: New York, 1968. (39) Brandrup, J.; Immergut, E. H. Polymer Handbook, 3rd ed.; John Wiley & Sons: New York, 1989. (40) Kragel, J.; Li, J. B.; Miller, R.; Bree, M.; Kretzschmar, G.; Mohwald, H. Colloid Polym. Sci. 1996, 274, 1183-1187. (41) Kaganer, V. M.; Mohwald, H.; Dutta, P. Rev. Mod. Phys. 1999, 71, 779-819.
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Figure 7. Microparticle shells, doped with BODIPY FL DPPE lipid dye, viewed under epifluorescence. The microparticle diameters are both approximately 25 µm. The monolayer shell lipid is diC18:0 PC. Crystalline diC18:0 PC domains are dark, nonfluorescing regions. (A) Microparticle cooled at a rate on the order of 102 °C/min. (B) Microparticle cooled at a rate on the order of 103 °C/min. Scale bar ) 10 µm.
Figure 9. TEM micrograph of an air-filled diC18:0 PC microparticle cooled at a rate on the order of 100 °C/min. The microparticle diameter is approximately 5 µm. Scale bar ) 1 µm.
Figure 8. Maximum mean linear grain size as a function of the monolayer shell cooling rate. The mean linear grain size is defined as the reciprocal of the linear grain density (the number of grain boundaries per unit length). The data are compiled from epifluorescent microscopy experiments. Each point represents the mean of 4-11 measurements.
Videomicrographs of microparticles cooled at different rates are shown in Figure 7. Under bright-field illumination (no contrast enhancement), there was no evidence of microstructure in the microparticle shells. For epifluorescent images, the focal plane was selected near the “bottom” of the particle, that is, near the contact of the particle with the slide, where the particle was slightly flattened. This convention allowed observation of maximal surface area of the microparticle. As Figure 7 illustrates, epifluorescent illumination of the microparticle revealed an inhomogeneous distribution of solid (dark) regions in the monolayer shell, where individual domains of gelphase lipid had dimensions on the order of micrometers and were surrounded by dye probe-enriched boundaries. Figure 7A shows the domain microstructure of a microparticle cooled at a rate on the order of 102 °C/min. In contrast, the particle in Figure 7B was cooled more rapidly at a rate on the order of 103 °C/min, and the surface exhibits smaller average domain sizes relative to that of the comparably sized particle in Figure 7A. To quantify this further, we evaluated the maximum mean linear grain size, defined as the reciprocal of the grain density, that is, the number of grain boundaries occurring per unit length. This parameter is plotted as a function of the cooling rate of the monolayer shell in Figure 8 and illustrates the dependence of microstructure on the rate at which the overall lipid monolayer crystallizes. The particular domain microstructure that evolves for a lipid monolayer as it freezes reflects the interplay between the concurrent processes of domain nucleation and growth (which are mapped in the temperature-time transformation, or TTT, diagrams familiar to materials scientists). For microparticles on the same 20-30-µm-diameter size scale (and, therefore, of approximately the same curva-
ture), the faster the cooling rate, the smaller the domains in the polycrystalline monolayer. In terms of classical nucleation theory, in the high-cooling-rate regime, nucleation sites are abundant but experience low growth, resulting in small domains. Fully formed and equilibrated gas microparticles were also subjected to a standard cryofixation technique and examined by TEM. Micrographs revealed multiple fracture surfaces of deflated, nonspherical microparticles (Figure 9). The most striking feature of the micrographs was the appearance of well-defined surface domains that resembled the domains observed under epifluorescent illumination. Furthermore, the shells exhibited multiple folds, dimples, and creases that are hallmarks of a buckling instability in the shell during deflation, consistent with observations that were made in the optical microscope. When measured at maximum resolution, the width of these raised edges was on the order of 4 nm, the thickness of a lipid bilayer. Therefore, it appeared that the grain boundaries were in fact elevated from the surface, again consistent with their being in a state of compression when the gas and shell reach equilibrium. As the lipid-coated gas microparticle was cooled from the lipid melt through the main transition, the lipid monolayer necessarily condenses by about 25%.42 This condensation of the monolayer then exposes or produces an interface that still has an interfacial tension, which as a linear average of 75% gel (0 mN/m) and 25% uncoated (72 mN/m) would be ∼18 mN/m. As with all free, surfactant-coated, and liquidlipid-coated microbubbles, any finite interfacial tension gives rise to a Laplace pressure that drives gas out of the gas microparticle even in saturated solution.35 As the gas leaves, the volume of the particle reduces along with its surface area, and eventually the solid-phase monolayer area matches the underlying gas microparticle area and the tension in the surface approaches 0, reaching a stable (reduced Laplace pressure), state. In reaching this state, the monolayer was, therefore, likely to be in slight compression when the gas microparticle stopped shrinking, leading to our postulation that the raised boundaries are a direct result of a slight compressive stress. It should also be noted that the TEM microparticles were smaller than those used in the fluorescence experiments, and so the absolute grain size was much smaller than that for the larger particles, consistent with the fact that smaller (42) Needham, D.; Evans, E. Biochemistry 1988, 27, 8261-8269.
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Figure 10. Surface yield shear of diC18:0 PC monolayer shells as a function of the cooling rate applied during the postproduction stage. All the microparticles were measured at room temperature. Each point represents the mean of 30-40 measurements. Figure 12. Videomicrographs of lipid monolayer shells incorporating BODIPY FL PE, viewed under epifluorescence. The scale bar is 5 µm. Parts A and B show two different microparticles from a batch formed by cooling at 100 °C/min. Parts C and D are images of the microparticles in parts A and B, respectively, following the application of a single annealing cycle (heat to 50 °C, hold at this temperature for 180 s, and cool to 25 °C).
Figure 11. Surface shear viscosity of diC18:0 PC monolayer shells as a function of the cooling rate applied during the postproduction stage. All the microparticles were measured at room temperature. Each point represents the mean of 30-40 measurements.
microparticles possess higher curvatures that limit the extent of domain growth. The appearance of the domain organization provided direct evidence that grain structure was indeed affected by the cooling rate. Application of the yield shear and shear viscosity tests, administered by micromanipulation techniques, then demonstrated how the structural changes produced by processing variations translated to differences in material properties. The fixed lipid-shell composition of diC18:0 PC and PEG 40 stearate allowed comparisons to be made of sample shell properties as a function of the processing conditions. The yield shear of the lipid shells as a function of the cooling rate is given in Figure 10 and shows a decrease in the yield shear with increasing cooling rate, that is, smaller grain size; the corresponding shear viscosities as a function of the cooling rate are given in Figure 11 and show a similar decrease in shear viscosity with increasing cooling rate and smaller grain size. With the crystalline microstructure of the lipid shells clearly identified via epifluorescent labeling of the grain boundaries, annealing of the microstructure was performed by heating and subsequently cooling the microparticles and observing the change in grain boundary density before and after the cycle. Microparticles were initially formed at a 100 °C/min cooling rate and observed with epifluorescent videomicroscopy (Figure 12A,B). The chamber was heated to approximately 50 °C, which is 5 °C below the main phase (melting) transition of this lipid, held at this temperature for 180 s, and then cooled at a rate of 5 °C/min, more than an order of magnitude slower than the cooling rate applied to initially form the microparticles. The same microparticles that were viewed prior to the heating were observed after the cooling and are shown in Figure 12C,D. The videomicrographs reveal a new domain pattern with a lower density of grain boundaries or, in other words, larger crystalline domains.
Figure 13. Plot of grain boundary density PL (inverse grain size) versus microparticle diameter. The open markers indicate boundary densities for microparticles prior to the annealing cycle, and the filled markers indicate boundary densities for microparticles after the annealing cycle.
This trend is shown in a plot of the boundary density versus particle size for 14 microparticles as formed before heating and for 17 microparticles after annealing (Figure 13). Clearly, the grain boundary density is higher prior to the annealing cycle than it is following the cycle. Again, the behavior observed here, namely, the crystal growth of an initial microstructure upon application of a heat treatment in which the temperature is raised to just below Tm of the material, is that of an essentially twodimensional polycrystalline material that is consistent with the annealing behavior of “conventional” bulk crystalline materials. Discussion The principle of amphiphilic self-assembly dictates that at the surface of an air microparticle in an aqueous suspension, phospholipids adopt a configuration of headgroups facing the aqueous medium and tails facing the more hydrophobic, gaseous interior of the particle. In an earlier study, we reported the success of receptor-ligandmediated specific adhesion between a phospholipid-coated microbubble and a coated glass bead substrate.43 Biotin was conjugated to the lipid headgroup via an aminohexanoyl or PEG linker, and avidin was either physisorbed
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or conjugated to the bead surfaces. The specific adhesion effected between the microparticle and the bead confirmed, among other results, the orientation of lipid headgroups toward the exterior of the coated microbubble. In experiments using the canal method to measure the surface viscosity of monolayers formed from a homologous series of saturated long-chain alcohols, Huhnerfuss observed viscosity increasing with chain length, albeit at only low and moderate surface pressures (3 and 20 mN m-1) corresponding to relatively expanded states.44 In a later work, Huhnerfuss demonstrated that, relative to straight-chain parent compounds, derivatives containing sterically bulky groups exhibited lower monolayer viscosities because of the attenuation of cohesive hydrophobic interactions between the monolayer constituents.45 More recently, Sacchetti and co-workers employed a twodimensional analogue of the Doolittle equation for polymer viscosity to correlate the shear viscosity within a lipid monolayer with the molecular area.46 Theory and experiment, with stearic acid, diC16:0 PC, and poly(tert-butyl methacrylate) monolayers, demonstrated that the surface shear viscosity increases with decreasing molecular free area. The model, therefore, predicted the maximum shear viscosity exhibited by monolayers in the crystalline state. While the initial molecular orientation of the lipid and some level of cohesion of the monolayer at the gas-liquid interface arise from the hydrophobic effect, the origin of the higher cohesive strength of the phospholipid monolayer shell, especially as a solid material, resides in the van der Waals interactions between the hydrocarbon-chain moieties. These interactions are particularly strong because of the high degree of order imposed on the long acyl chains of the insoluble PCs in their thermotropic gel state: on average, the saturated chains are in a nearly all-trans conformational state, indicating maximum longitudinal extension and minimum cross-sectional area. Such a configuration allows the most efficient packing of lipid molecules within a prescribed area, such as the air-water interfacial area afforded by the surface of an air microparticle of a given size in an aqueous medium. This effect is also the basis for lipid bilayer cohesion in, for example, gel-phase DMPC and cholesterol-containing bilayers.6,47 Within the crystalline matrix, lipid molecules can be envisioned as rodlike or cylindrical moieties that are mutually oriented and pack in a hexagonal array. This highly condensed “nearest neighbor” arrangement allows each lipid molecule to participate in a maximum number of cohesive interactions with other lipid molecules. The resulting cohesion of the matrix and its resistance to shear deformation is predicted by continuum mechanics, which models shear deformation in crystals as a diffusion phenomenon involving the internal local movement of atomic constituents into vacancies in the matrix. In order for a lipid molecule to displace in this fashion, cohesive interactions that are accumulated along the length of the lipid acyl chains in a crystalline lattice must be overcome. Although some intragrain deformation is required to accommodate the overall shear deformation of a polycrystalline monolayer that is topologically constrained to the curved surface of a microparticle, it is by no means the sole mode of deformation. Intergrain deformation occurs via the movement of crystalline domains past one another along their mutual grain boundaries, and this mode can
dominate when the material is relatively close to its melting transition. Mouritsen and Zuckermann employed computer simulations based on Monte Carlo techniques and a combination of the multistate models of Pink and Potts to predict the temperature- and pressure-dependent phase transitions and microstructure of condensed monolayers on an air-water interface.48,49 Among the findings was the simulations’ prediction that upon heating and approaching the gel-liquid crystalline phase transition from below, interfacial melting occurs preferentially at domain boundaries. During deformation, shear-induced melting and refreezing of the lipid at the boundaries takes place, analogous to the slip motion in two-dimensional molecularly thin lubrication layers.50 The dependence of intergrain deformation on the melting temperature, coupled with the acyl chain length dependence of vacancy displacement for intragrain deformation, therefore, accounts for the trends in the surface shear properties observed in the current study. Phospholipids with long acyl chains have high main transition (melting) temperatures and strong intracrystalline cohesive interactions, hence the relatively high values of yield shear and shear viscosity of diC24:0 PC monolayer shells. Relatively shorter acyl chains such as those of diC18:0 PC melt at lower temperatures and participate in relatively fewer intermolecular van der Waals attractions, which is reflected in the lower yield shear and shear viscosity of the monolayer. A further implication of the intergrain deformation concept is that the density of grain boundaries within the lipid shell influences the resulting mechanical properties of the shell. This dependence between the microstructure and properties has long been known in the disciplines of materials science and metallurgy. For example, in 1948 King et al. recognized the dependence of shear deformation on crystal size and boundary sliding in polycrystalline tin.51 In the current study, (1) epifluorescence revealed the location of labeled lipid probes that had been excluded from the crystalline domains and accumulated in the grain boundaries, and (2) the application of different postproduction cooling regimens to the microparticle samples allowed control of the microstructure of the lipid shells. The cue for the latter technique was again provided by materials science and metallurgy, namely, by heat treatment, the application of a certain temperature history to a material (such as steel) to regulate the phase transitions, phase separations, and relative thermotropic rates of domain nucleation and domain growth to thereby generate a particular microstructure. The data presented in Figures 10 and 11 appear to bear out this prediction. At a fixed lipid-shell composition, rapidly cooled microparticle shells exhibit smaller grains, higher grain boundary densities, and a measurably lower resistance to shear deformation via lower yield shear and shear viscosity relative to microparticle shells cooled at lower rates. To render a full account of the interactions between lipid molecules, we recognize that, in addition to the van der Waals attraction between the lipid acyl chains, the assumption of a close-packed array of lipid molecules places the zwitterionic headgroups in a similarly regular arrangement, with the dipolar groups (the phosphate group and choline nitrogen) of one lipid molecule in proximity to their counterparts on neighboring molecules.
(43) Kim, D. H.; Klibanov, A. L.; Needham, D. Langmuir 2000, 16, 2808-2817. (44) Huhnerfuss, H. J. Colloid Interface Sci. 1985, 107, 84-95. (45) Huhnerfuss, H. J. Colloid Interface Sci. 1987, 120, 281-282. (46) Sacchetti, M.; Yu, H.; Zografi, G. Langmuir 1993, 9, 2168-2171. (47) Needham, D.; Nunn, R. S. Biophys. J. 1990, 58, 997-1009.
(48) Mouritsen, O. G.; Zuckermann, M. J. Phys. Rev. Lett. 1987, 58, 389-392. (49) Mouritsen, O. G.; Zuckermann, M. J. Chem. Phys. Lett. 1987, 135, 294-298. (50) Thompson, P. A.; Robbins, M. O. Science 1990, 250, 792-794. (51) King, R.; Cahn, R. W.; Chalmers, B. Nature 1948, 161, 682.
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These electrostatically unfavorable interactions can be alleviated to some extent by ionization of the dipolar groups or even by the limited conformational changes allowable within the headgroup.52 In general, the presence of the charged headgroup region contributes a net repulsive pressure between phospholipid molecules that acts to reduce the overall cohesion of the monolayer. For the current study, however, the lipid headgroup and ionic environment, choline and PBS, respectively, were identical for all compositions examined, and so the repulsive contributions to the interaction energy between molecules were kept constant for all acyl chain lengths. The shear property trends that we have observed, therefore, primarily reflect changes in the van der Waals attractionderived cohesive energy induced by changes in the chain composition of the monolayer and the detailed microstructure of the monolayer as determined by the cooling rate and, concomitantly, grain size. The values of yield shear (1-6 mN m-1) and shear viscosity (5-25 mN s m-1) obtained by the micromanipulation technique for phospholipid shells of different lipid compositions are on orders of magnitude that are inaccessible to other techniques but are consistent with the results of studies on different surfactant monolayer systems obtained by other methods. Surface yield shear values of mixed surfactant monolayers of sodium lauryl sulfate (SLS) with LA at the air-water interface of aqueous suspensions of the surfactants14 are on the order of 0.01-0.1 mN m-1, which is approximately 1 order of magnitude lower than the yield (0.87 ( 0.14 mN m-1) for diC18:0 PC at room temperature (Figure 5). These results are qualitatively consistent with each other because of the expanded phase state of the SLS-LA monolayer, in comparison to the solid, maximally condensed crystalline monolayers of saturated diacyl PCs. The surface shear viscosities measured for the various phospholipid shells by micropipet are also consistent with other results in the literature. Kragel et al.53 investigated monolayers of diC16:0 PC with a surface shear rheometer and observed a marked dependence of viscosity values on the surface pressure of the monolayer. The maximum value reported, 0.6 surface poise, was obtained at maximum compression of the monolayer, achieved at 45 mN m-1 of surface pressure. This viscosity value of diC16:0 PC compares favorably with the micromanipulation values because the monolayer constrained to the particle surface is also under maximum compression as a result of the contractile surface tension inherent in the underlying interface (Figure 6). That Kragel and co-workers were able to measure the surface shear viscosity of diC16:0 PC is particularly fortuitous because diC16:0 PC shells were not sufficiently rigid to retain the deformed geometry required by the micromanipulation technique for more than a few seconds, and so direct measurement of their viscosity by micropipet was not possible. The micropipet method, therefore, constitutes a valuable tool for measuring surface shear properties because of its ability to measure the high-range yield shears (100-101 mN m-1) and shear viscosities (100101 surface poise) exhibited by gel-phase phospholipid monolayers and thereby complements the low-range (yield shear , 100 mN m-1 and shear viscosities from 10-5 to 10-1 surface poise) methods previously described. Although the lipid monolayer shell has been treated theoretically as a two-dimensional material, the structure obviously possesses a finite thickness. For a gel-phase
phospholipid monolayer, this thickness h is on the order of 28 Å. The conversion of surface yield and viscosity to bulk (three-dimensional) values can be approximated by dividing the surface values by h. The corresponding bulk yield shear strength and shear viscosity of PC monolayer shells are, therefore, on the order of 0.1-1 MPa and 107 P, respectively. The equivalent bulk yield strength thus obtained is 1-2 orders of magnitude lower than the yield shear strength of PE (6-20 MPa),54 while the equivalent bulk viscosity of PC monolayers is in the range of softened window glass and plastics.37,38 The viscosity of the shell is also 9 orders of magnitude higher than that of water at 20 °C; this difference supports a posteriori the assumption that the drag imposed by the vicinal water layer surrounding the microparticle provides a negligible contribution to the observed monolayer shear viscosity.
(52) Nagle, J. F.; Wilkinson, D. A. Biophys. J. 1978, 23, 159-175. (53) Kragel, J.; Kretzschmar, G.; Li, J. B.; Loglio, G.; Miller, R.; Mohwald, H. Thin Solid Films 1996, 285, 361-364.
(54) Shackelford, J. F.; Alexander, W.; Park, J. S. CRC materials science and engineering handbook, 2nd ed.; CRC Press: Boca Raton, FL, 1994.
Concluding Remarks In summary, the material properties of phospholipid monolayer shells in shear deformation exhibit a composition dependence that is explicable on the basis of fundamental intermolecular forces, such as van der Waals interactions, electrostatic repulsion, and hydration forces, as well as the lipid domain size and distribution that comprise the specific microstructure of the monolayer shell. Resistance to shear deformation of a monolayer shell increases with the length of the lipid acyl chains and is dependent on temperature. For the intragrain deformation mode, short-range (local) displacements within crystalline domains are rendered more difficult for longer-chain lipids because greater intermolecular attractions must be overcome. For the intergrain deformation mode, longer-chain lipids undergo melting at greater reduced temperatures and are, therefore, more resistant to grain boundary sliding than their shorter-chain counterparts at a given temperature. These observations underscore the utility of employing composition-structure relationships to engineer the properties of the material. The micropipet technique complements the current literature by extending the range of measurable surface shear properties by orders of magnitude beyond those accessible to existing methods and, thus, constitutes a new and valuable tool in the study of monolayer films. Acknowledgment. We thank Evan A. Evans for invaluable discussions regarding the modeling and Alexander L. Klibanov for guidance and expertise with the microparticle preparation. This work was supported by NIH Grant GM40162 and Mallinckrodt, Inc. (Hazelwood, MO). Appendix The derivation of the constitutive equations relating membrane surface shear properties to the pressures and geometry of the system is based on a two-dimensionalization of the system and neglect of acceleration and bending considerations.25 A representation of this model system is shown in Figure 14A. The model is based on a polar coordinate system centered on the opening of the pipet; in the (two-dimensional) plane of the membrane, the curvilinear s coordinate is equivalent to the radial r coordinate. A force balance on a membrane element
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where Rp is the inner (effective) radius of the pipet and P is the pipet suction pressure. The shear force resultant is defined by
τˆ s ≡
τm - τφ 2
(A-8)
Rearranging the equation of equilibrium A-6 and substituting the definitions A-7 and A-8 leads to the expression
P)
Figure 14. (A) Schematic of the model geometry. The subscripts m and φ designate meridional and azimuthal components, respectively, of the polar coordinate system centered on the opening of the pipet. In the plane of the membrane, the curvilinear coordinate s is equivalent to the radial coordinate r. (B) The flow geometry and flow vectors of the system.
τˆ s (A-1)
∂(τmmr) ∂τφm ∂r + - τφφ + σmr ) 0 ∂s ∂φ ∂s
(A-2)
τmm τφφ + ) ∆P Rm Rφ
(A-3)
where τ denotes principal tensions; σ denotes shear resultants; the m and φ subscripts designate the meridional and azimuthal components, respectively; Rm and Rφ are the principal radii of curvature; and ∆P is the pressure difference across the membrane. For an axisymmetric system, the system of equations reduces to two equations containing the principal tensions τm and τφ.
(A-4)
τφ τm + ) ∆P Rm Rφ
(A-5)
where η is the shear viscosity and Vs is the shear rate. Equations A-9 and A-10 combine to give the expression
4τ0s R0 P0 ) ln Rp Rp
(A-11)
which relates the threshold pressure of entry to the surface yield shear.6 An expression for surface shear viscosity can also be derived for the two-dimensional planar model. The relevant flow vectors are shown in Figure 14B. From mass conservation relations, the flow rate of the surface material must satisfy the relation
dL Rp ) vr dt r
(A-12)
vr ) constant
(A-13)
The shear rate is given by25
(A-6)
For the boundary conditions, the meridional tension is assumed to be 0 at the outer boundary because the tensions drop off inversely as the square of the radial distance, consistent with the essentially unchanged shape of the equatorial region of the microparticle during the experiment,25 and at the pipet entrance is defined by the force balance
τpm2πRp ) PπRp2
(A-10)
therefore
For the aspirated membrane problem, the region local to the mouth of the pipet is considered approximately flat, and the equation of equilibrium A-4 in the surface reduces to
∂ (rτ ) - τφ ) 0 ∂r m
{
τ0s τˆ s ) τ0s + 2ηVs (flow)
-
∂(τmr) ∂r - τφ + σmr ) 0 ∂s ∂s
(A-9)
Experiments show that the monolayer behaves as a Bingham plastic; therefore, there are three regimes of behavior for the flow of the material under stress. Below some threshold yield shear τˆ s, the deformation is elastic, and no flow occurs. At the yield shear, which corresponds to a threshold applied pressure P0, the material undergoes viscoplastic flow into the pipet. Above the yield shear level, flow occurs and the shear force resultant in excess of the yield value depends on the shear viscosity and shear rate. These results are summarized as follows:
provides a set of three equations of mechanical equilibrium25
∂τφφ ∂(τφmr) ∂r + τφm + σφr ) 0 + ∂φ ∂s ∂s
4τˆ s R0 ln Rp Rp
(A-7)
2Vs )
∂vs vs ∂r ∂s r ∂s
(A-14)
In the plane of the surface, r ) s. Combination of the expression for shear rate A-14 and the conservation relation A-12 leads to the relation
Vs )
dL Rp dt r2
(A-15)
Vs can be substituted into the definition A-10 for the shear regime in excess of the yield given by
τˆ s ) τ0s + 2ηsVs
(A-16)
In turn, the expression for the surface shear force resultant
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A-16 can be substituted into the equation of equilibrium A-6, resulting in an expression that can be integrated as before to obtain the relation
[ ( )]
PRp Rp R0 ηs dL ) τ0s ln + 14 Rp Rp dt R0
2
pressure in excess of the threshold value (∆P), the pipet radius, the radius of the spherical portion of the microparticle, and the rate of entry of surface material into the pipet. Equation A-11 can be rearranged to the form
(A-17) τ0s )
where the boundary conditions have been applied. Rearranging eq A-17 and using the definition established earlier for the threshold pressure A-11 gives the expression
ηs )
∆PRp2 4(dL/dt)[1 - (Rp/R0)2]
(A-18)
which relates the surface shear viscosity to the applied
P0Rp 4 ln(R0/Rp)
(A-19)
Equations A-18 and A-19 describe the surface shear viscosity and surface yield shear, respectively, and are expressed entirely in terms of measurable quantities. LA034779C
