Langmuir 2006, 22, 6939-6943
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On the Wettability of Lipid DPPC Films Jens Gu¨nster*,†,‡ and Ryutaro Souda§ Laser Application Center, Clausthal UniVersity of Technology, D-38678 Clausthal-Zellerfeld, Germany, International Center for Young Scientists, National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan, and AdVanced Materials Laboratory, National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan ReceiVed March 12, 2006. In Final Form: May 22, 2006 By employing temperature-programmed desorption and time-of-flight secondary ion mass spectroscopy, the adsorption of water on the hydrophilic and hydrophobic surfaces of a lipid (DPPC) film has been investigated. It could be shown that it is possible to prepare lipid films ex situ with a preferential orientation of the lipid molecules on a solid support and to retain their specific properties under ultrahigh vacuum conditions. The water adsorption and desorption kinetics on the hydrophilic and hydrophobic surfaces provided by a lipid film are discussed in terms of their structural and chemical properties.
Introduction Living cells are encircled by a membrane that separates the cell’s interior, the cytoplasm, from its surrounding. The membrane lets water, certain ions, and substances into the cell and is vital to the cell’s energy budget, and it excretes substances. The most common constituents of a cell membrane are phospholipids. Lipid molecules consist of a hydrophilic (polar) headgroup (the phosphate group with the rest attached) and a hydrophobic fatty acid tail. In an aqueous environment, lipid molecules have a tendency to self-assembly into larger structures such as vesicles or liposomes. Vesicles are stable as individual units and are equipped with a simple intercellular skeleton; they even reflect basic morphological properties of a living cell.1 Once adsorbed on a solid surface, vesicles can spontaneously rupture and fuse to form a supported lipid bilayer (SLB).2 Because they are useful biomimetic systems, a significant body of literature has recently been devoted to SLBs; see ref 3 for a review. Typically, a thin water layer (1 to 2 nm thickness) is trapped between the support and the lower leaflet of the SLB. This water interlayer enables a certain mobility of the adsorbed molecular assembly, which in turn allows the SLB to retain many of the properties of the free vesicle. In contrast, lipid films transferred by a film-balance system (Langmuir-Blodgett trough) to a solid support and then desiccated in vacuum are directly linked to the supporting surface. By controlling the surface pressure in the film balance system, the lateral density of the lipid molecules and thus their state of aggregation is determined. Because of the direct link between the lipid film and the support, this structure is retained after the transfer. With this approach, it is possible to prepare lipid films ex situ on a solid support whose molecular density is comparable to the density of those self-assembled in an aqueous environment. The lipid molecules in a vesicle membrane are preferentially oriented in such a way that all polar hydrophilic headgroups of * Corresponding author. E-mail:
[email protected]. † Clausthal University of Technology. ‡ International Center for Young Scientists, National Institute for Materials Science. § Advanced Materials Laboratory, National Institute for Materials Science. (1) Hotani, H.; Inaba, T.; Nomura, F.; Takeda, S.; Takiguchi, K.; Itoh, T. J.; Umeda, T.; Ishijima, A. BioSystems 2003, 71, 93. (2) Cremer, P. S.; Boxer, S. G. J. Phys. Chem. B 1999, 103, 2554. (3) Hamai, C.; Yang, T.; Kataoka, S.; Cremer, P. S.; Musser, S. M. Biophys. J. 2006, 90, 1241.
the lipid molecules are pointing in one direction, such as in the case of a lipid bilayer pointing toward the lipid-water interface. The present work sheds some light on the water-lipid-film interaction. It will be shown that the hydrophilic/hydrophobic character of a lipid film is retained in the ultrahigh vacuum environment, which in turn enables a detailed investigation of these two surfaces by surface-sensitive analytical tools. It is found that the hydrophilic surface of a dipalmitoyl-sn-glycero-3phosphocholine (DPPC) film is very effectively wetted by water. A prerequisite for this high wettability is a moderate long-range surface-water attraction that is comparable in strength to the water-water attractive interaction. Experimental Section For the present study, lipid films from 1,2-dipalmitoyl-sn-glycero3-phosphocholine (DPPC), with the fatty acid chains of the lipid molecules containing 16 carbon atoms, have been prepared. DPPC of greater than 99% purity was obtained as a chloroform stock solution from Avanti Polar Lipids, Inc. and was transferred without further purification at a constant surface pressure of 35 mN/m in a KSV Instruments, Ltd Langmuir-Blodgett trough. DPPC was spread on deionized water, serving as the subphase, and compressed to the desired surface pressure. To prepare a hydrophobic DPPC surface, a Si(100) substrate covered with its native oxide was pulled out of the subphase at a speed of 2 mm/min. As confirmed by our own and previously published time-of-flight secondary ion mass spectroscopy (TOF-SIMS) studies, this procedure provides DPPC films with the polar headgroup of the lipid molecule preferentially pointing toward the silicon oxide substrate and its hydrophobic moiety pointing toward the substrate-vacuum interface.4 To obtain lipid films with the opposite orientation, a sample prepared as previously described has been pushed into the subphase at a speed of 2 mm/min. This procedure provides lipid double layers whose surface is hydrophilic. After placing the so-prepared samples into an ultrahigh-vacuum apparatus (base pressure 1 × 10-10 mbar) and cooling by means of a closedcycle helium refrigerator, high-purity water (D2O) from Fisher Scientific was dosed by backfilling the vacuum chamber. From the fact that the DPPC monolayer has been prepared at temperatures well below its gel-liquid-crystalline phase-transition temperature (Tm ) 314 K), no major structural changes are expected to occur while cooling the films.5 All water adlayers were condensed at a water partial pressure of 1 × 10-7 mbar, which corresponds to an (4) Pacholski, M. L.; Cannon, D. M., Jr.; Ewing, A. G.; Winograd, N. J. Am. Chem. Soc. 1999, 121, 4716. (5) Lee, S.; Kim, D. H.; Needham, D. Langmuir 2001, 17, 5544.
10.1021/la060676t CCC: $33.50 © 2006 American Chemical Society Published on Web 06/27/2006
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Figure 1. TOF-SIMS spectra taken from a DPPC film with the polar headgroup of the lipid molecules preferentially pointing toward the surface-vacuum interface (hydrophilic surface) and vice versa (i.e., with the nonpolar fatty acid chains toward the surface-vacuum interface (hydrophobic surface)). No signal from sputtering the Si/SiO2 surface could be detected, confirming that a closed DPPC layer is formed on the supporting silicon substrate. exposure of 0.1 Langmuirs (L) per second. In the present experimental setup, a water exposure of approximately 3.25 L is required to form one geometric water bilayer on a Ni(111) surface held at 100 K (approximately 2 × 1015 molecules cm-2). Assuming that the sticking coefficient, that is, the probability of an impinging water molecule to be captured by the Ni(111) surface, is close to unity, this result provides, for the present setup, a relation between the measured water partial pressure and the rate of molecules impinging the sample surface. The corresponding amount of water adsorbed has been calibrated via temperature-programmed desorption spectroscopy. To facilitate a convenient discussion of the various water adsorption experiments on DPPC films, water coverages are presented in terms of water bilayer equivalents (WBE). One WBE corresponds to a coverage of approximately 2 × 1015 molecules cm-2. To investigate the water adsorption kinetics, water condensation has been monitored in situ by time-of-flight secondary ion mass spectroscopy (TOF-SIMS) during water exposure. For TOF-SIMS, He+ ions produced in an electron-impact-type ion source were accelerated to 2 keV, chopped into 60 ns (fwhm) pulses by means of an electrostatic deflector, and finally scattered at the sample surface. Positively charged sputter fragments detached from the sample surface, because of He+ ion impact, were measured by floating the sample with a bias voltage of +500 V. A stainless steel mesh placed 4 mm in front of the sample surface facilitates the extraction of the positively charged sputter fragments into the field-free drift region of a time-of-flight (TOF) tube. Even though TOF-SIMS cannot be considered to be truly nondestructive, in the present experiment the surface damage was minimized by keeping the incident He+ flux without chopping below 0.1 nA/cm2. In the present setup, damage to the investigated surfaces becomes noticeable after excessive, typically >2 h, exposure to He+ ions. For temperature-programmed desorption spectroscopy (TPD), a quadrupole mass spectrometer (Hiden Analytical, HAL 301 S/2) attached to a differential pumping stage was employed. TPD measurements made of the D2O+ ion m/z ) 20 were collected at a linear heating rate of 0.085 K/s. Temperature ramping of the sample, mounted to the coldfinger of a closed-cycle He refrigerator, was controlled by a standard PID controller.
Results TOF-SIMS Results. Figure 1 compares TOF-SIMS spectra obtained from DPPC films of both orientations, that is, either with the nonpolar fatty acid chains or with the polar phosphoric acid ester group of the DPPC molecules preferentially pointing toward the substrate-vacuum interface. In the following text,
Gu¨nster and Souda
Figure 2. TOF-SIMS signal from the hydrophilic and hydrophobic surface of a DPPC film and the SiO2/Si(100) surface exposed to a water partial pressure of 1 × 10-7 mbar as a function of the substrate temperature. The hydrophobic DPPC lipid film is represented by the C2H3+ sputter fragment; the hydrophilic, by NCH3+; SiO2, by Si+; and the adsorbed water, by D+.
the first orientation will be referred to as the hydrophobic DPPC surface, and the second, as the hydrophilic DPPC surface. In the TOF-SIMS spectra, the intensity of fragments sputtered from the lipid film by He+ ion impact are plotted versus their massto-charge ratio. Because most sputter fragments are singly charged, the abscissa can be read as the mass of the sputter fragments. Because of the low penetration depth of the He+ projectiles into the substrate surface, TOF-SIMS is highly surfacesensitive, and thus a preferential orientation of the adsorbed DPPC molecules should be noticeable in Figure 1. For an interpretation of the spectra presented in Figure 1, it is important to note that because of their short lifetime some charged sputter fragments that might be detached in a He+ collision event do not reach the detector at the end of the TOF tube. Basically, the TOF-SIMS spectrum of the hydrophobic DPPC film reflects the intensity distribution found in electron impact fragmentation tables for long open-chain alkanes, which confirms the character of this surface. The most striking difference between the TOF-SIMS spectra of the hydrophobic and hydrophilic films is the absence of masses 28 and 45 in the spectrum of the hydrophobic film. Assuming that the mass 45 signal originates exclusively from the NC2H7+ fragment of the lipid molecule’s polar headgroup, the degree of molecular orientational disorder in the films is, according to the data presented, below 5%. To study the adsorption kinetics of water on the different DPPC films, we have investigated the water uptake by TOFSIMS (Figure 2). Because of the high surface sensitivity of TOFSIMS, a water monolayer is sufficient to suppress the signal of the underlying DPPC film significantly. Therefore, TOF-SIMS provides useful insights into the water adsorption kinetics by monitoring the signal obtained from a DPPC film as a function of water exposure. For the detection of the hydrophobic DPPC film surface, we have chosen the C2H3+ sputter fragment; for the hydrophilic, the NCH3+; and for the adsorbed water, D+. Because we have highly oriented films, for the first two species the corresponding lines in Figure 1 are masses 27 and 29, respectively. During the adsorption experiment, the substrate temperature was reduced linearly from 170 K down to 20 K at an ambient water partial pressure of 1 × 10-7 mbar. Additionally, in a similar experiment the water uptake of a clean SiO2 surface (i.e., the Si(100) surface covered with its native oxide) has been investigated. In Figure 2, the water exposure starts at a temperature (170 K) at which no significant water adsorption is expected for
On the Wettability of Lipid DPPC Films
the given water partial pressure of 1 × 10-7 mbar. From 170 to 70 K, the most important temperature range, the sample has been cooled at an almost linear rate of 0.056 K/s. Below 70 K, the rate slightly increases, which, however, is not relevant to the experiment. The time required for passing an arbitrary temperature interval corresponds, because of the constant water partial pressure, to a certain water exposure. The total water exposure is plotted in Figure 2 at the right ordinate of the graph. In Figure 2, an abrupt decline in the substrate intensity marks the onset of water adsorption. Concomitant with the attenuation of the substrate signals, intensity from adsorbed water evolves. For the SiO2 surface, the onset of water adsorption is found at a temperature threshold of about 140 K; for the hydrophilic DPPC surface, at 133 K; and for the hydrophobic DPPC surface, at 106 K. Besides the different temperature thresholds, the rate at which the substrate signal declines seems to differ for the investigated surfaces as well. The decreasing substrate signal corresponds to the subsequent covering of the respective surface by water. Though it is tempting to correlate the temperature-dependent evolution of the substrate signals to the water adsorption kinetics, there exist no direct relation between substrate intensity and water coverage. The different sputter fragments, such as Si+, C3H3+, and NCH3+ for the three investigated surfaces, most likely have different mean free paths in the water adlayer. Hence, the much higher rate with which the signal from the lipid films disappears relative to that for the signal from the SiO2 surface might be attributed to a greater mean free path of the Si+ fragments. However, it is safe to assume that the persistent signal from the hydrophobic DPPC film at the lowest temperatures originates from its poor wetting by water. On the SiO2 surface, some intensity from adsorbed water molecules is already noticeable by TOF-SIMS at temperatures higher than the threshold temperature for the onset of significant water adsorption at 140 K. It appears reasonable to assume that minority sites on the SiO2 surface are populated by water molecules in this temperature interval between 160 and 140 K. TPD Results. The following procedure was carried out with TPD. To study the desorption kinetics of water (D2O) molecules adsorbed on lipid DPPC films, defined amounts of water have been dosed onto the films held at a temperature of 70 K. At this temperature, even for the hydrophobic DPPC film a sticking coefficient of the water molecules close to unity has been confirmed.6 After water dosage, the temperature of the substrate has been ramped from 70 to 300 K at a linear heating rate of 0.085 K/s. During temperature ramping, the flux of desorbing molecules was monitored by a mass spectrometer. The so-obtained sets of temperature-programmed desorption (TPD) traces are presented in a temperature interval from 140 to 195 K in Figure 3. The kinetics of desorption, as obtained from the desorption trace profiles and the coverage dependence of the desorption characteristics, give information on the state of aggregation of the adsorbed species. The position of the peak desorption rate is related to the enthalpy of adsorption (i.e., to the strength of binding to the surface). The integral trace intensity is proportional to the respective amount of water adsorbed. Because for the hydrophilic and hydrophobic DPPC surface the same water exposures do result in the same integral trace intensities, a water sticking coefficient close to unity can be safely assumed for both surfaces. Even though the TPD traces shown in Figure 3 merely reveal one distinct desorption feature, the interpretation of the data is rather complex. Depending on the respective water coverage, different desorption kinetics must be considered. For water
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Figure 3. Sequence of temperature-programmed desorption traces (m/z ) 20) obtained for various amounts of water (D2O) adsorbed on the hydrophilic and hydrophobic surfaces of a Si/SiO2-supported lipid DPPC film. Maximum water exposure occurs in (a) 52 and (b) 14 L.
exposures higher than 15 L, both surfaces, hydrophilic as well as hydrophobic, basically reveal zeroth-order desorption kinetics indicative of water desorption from a multilayer environment. According to these desorption kinetics, increasing water exposure results in a shift of the peak desorption rate to higher temperatures with the desorption traces showing a common low-temperature (leading) edge (Figure 3a). In the case of the hydrophobic DPPC surface, a common leading edge is, however, not really well established, which rather suggests pseudo-zeroth-order desorption kinetics. These desorption kinetics are typically found for desorption from 3D clusters formed by a strong attractive adsorbate-adsorbate interaction, relative to the adsorbatesubstrate interaction. At exposures below 15 L, zeroth-order desorption kinetics persist in the case of the hydrophobic surface but not in the case of the hydrophilic (Figure 3b). Whereas the hydrophobic surface still reveals desorption from a multilayer water environment, the hydrophilic surface shows first-order desorption kinetics at water exposures of 5 to 14 L. According to first-order desorption kinetics, the peak desorption rate appears at a constant temperature (172 K). The exposure range between 5 and 14 L corresponds to water coverages between 1.5 and 3.5 water bilayer equivalents (WBE). First-order desorption kinetics are indicative of desorption from interfacial sites on the surface.
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In the exposure range below 5 L, the hydrophobic surface reveals water desorption traces whose leading edge is, with decreasing water exposure, consecutively shifted toward lower substrate temperatures. This so-called undercutting has been previously observed for water adsorption on hydrophobic surfaces and is attributed to a subtle decrease in the binding energy of the adsorbed water molecules confined in small clusters.7 According to this model, the water activation desorption energy is reduced by a few percent toward the lowest coverages. At exposures below 3 L, this undercutting is observed, to a small extent, for the hydrophilic DPPC surface as well. It is noteworthy, though not critical for the present study, that below an exposure of 19 L the TPD traces obtained from the hydrophobic surface do not reveal the local desorption rate maximum at 164 K, which is already observed in the case of the hydrophilic surface at 10 L. This feature is typically attributed to a thermally activated structural transformation of the adsorbed amorphous-to-crystalline water ice.8,9 In the many desorption experiments done, we have observed this feature concomitantly with the opening of a closed water adlayer (i.e., at the moment of the surface dewetting). Therefore, a morphological change in the water surface should be reconsidered to be a possible origin of this feature.10
Discussion Our understanding of the interaction of water molecules with solid surfaces is based on a wealth of experiments in which water has been exposed to more or less hydrophilic inorganic substrates, and conclusions drawn from those experiments have led to a picture in which the initial stages of water condensation are governed by the interaction of water monomers with a respective surface rather than by the water-water interaction itself; see refs 11, 12, and 13 for a review. However, the binding energy of water monomers to organic hydrophobic surfaces is typically below 100 meV7,14 and thus about 1 order of magnitude smaller than the molecular water-water interaction. Because of this low binding energy, the condensation mechanism of water on hydrophobic surfaces is somewhat more complex. In the temperature range relevant for the onset of water adsorption (between 100 and 140 K), hydrophobic surfaces are not able to capture water monomers directly. Quickly captured in a diffusive adsorption precursor, a water monomer will detach from the surface as long as it does not accidentally meet other monomers and is then stabilized by the formation of a small water cluster acting as a condensation nucleus;7,14 see also Figure 4. The probability of capturing a molecule in such a water cluster depends on the density of the diffusive adsorption precursors on the respective surface, which is, in turn, related to the respective ambient water partial pressure and substrate temperature. The onset of water adsorption on the hydrophobic surface is observed at 30 K lower temperature than on the hydrophilic (Figure 2). However, after the onset of water adsorption the substrate signal decreases at a slope that is comparable to the (6) Gu¨nster, J.; Souda, R. Phys. ReV. B 2005, 71, 041407-1. (7) Dubois, L. H.; Zegarski, B. R.; Nuzzo, R. G. J. Am. Chem. Soc. 1990, 112, 570. (8) Lo¨fgren, P.; Ahlstro¨m, P.; Chakarov, D. V.; Lausmaa, J.; Kasemo, B. Surf. Sci. 1996, 367, L19. (9) Smith, R. S.; Huang, C.; Wong, E. K. L.; Kay, B. D. Surf. Sci. 1996, 367, L13. (10) Sack, N. J.; Baragiola, R. A. Phys. ReV. B 1993, 48, 9973. (11) Henderson, M. A. Surf. Sci. Rep. 2002, 46, 1. (12) Glebov, A.; Graham, A. P.; Menzel, A.; Toennies, J. P. J. Chem. Phys. 2000, 112, 11011. (13) Girardet, C.; Toubin, C. Surf. Sci. Rep. 2001, 44, 159. (14) Linderoth, T. R.; Zhdanov, V. P.;, Kasemo, B. Phys. ReV. Lett. 2003, 90, 156103.
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Figure 4. (Left) DPPC molecule, with its polar head (the phosphate group with a choline attached) and its hydrophobic fatty acid chains containing 16 carbon atoms. (Right) The adsorption of water on a DPPC film is governed by three consecutive steps: (1) impingement of the water molecule, (2) surface diffusion, and (3) capture in a water cluster.
hydrophilic surface. This observation strongly suggests that after the formation of the first water nuclei the water adsorption kinetics on both surfaces are comparable. Bearing in mind that the hydrophilic and hydrophobic DPPC surfaces are terminated by methyl groups (Figure 4), it is no surprise that water adsorption follows similar kinetics on both surfaces. To both surfaces, water was offered at the same partial pressure. Therefore, the different temperatures for the onset of water adsorption must be explicable in terms of a significantly longer lifetime of the adsorption precursor on the hydrophilic surface. This longer lifetime is naturally associated with a stronger attractive interaction between the hydrophilic DPPC surface and water molecules. The origin for this attraction can be found in the DPPC molecules’ polar headgroups that interact with the polar water molecules via a van der Waals-type force. In the initial stages of water adsorption on the hydrophilic SiO2 surface, a feature distinctive to inorganic surfaces comes into play: Inorganic solid surfaces typically offer specific adsorption sites. Some of these adsorption sites, minority sites, are characteristic surface defects that do interact rather strongly with water molecules. This explains the subtle, though significant, water adsorption on the SiO2 surface starting from 160 K. In Figure 2, the population of minority sites on the SiO2 surface is noticeable as a weak D+ signal in the temperature range from 160 to 140 K. Then, at 140 K adsorption on the defect-free surface (majority sites) results in an abruptly declining substrate intensity. After the onset of water adsorption, a subsequent converging of the entire surface by water is governed not only by the water adsorption rate but also by the ability of the water molecules to spread on the surface laterally. This wettability depends on the strength and type of surface-adsorbate interactions, localized versus delocalized. The slope with which the substrate intensity declines in Figure 2 might be indicative of the ability of the water molecules to wet a respective surface. However, in TOFSIMS the substrate signal also depends on the mean free path of the detected surface fragments in the water adlayer. Nonetheless, the hydrophobic DPPC surface is not really wetted by water as seen from the persistent substrate signal even at the lowest temperatures. In the case of the hydrophilic DPPC surface, in TPD three distinct coverage regimes are noticeable. At water coverages below 1 WBE, the adsorption seems to result in the formation
On the Wettability of Lipid DPPC Films
of small water clusters as deduced from a slight undercutting of the corresponding desorption traces. Then, between 1 and 3 WBE the peak desorption rate does not depend on the actual water coverage. First-order desorption kinetics suggest desorption from water molecules at interfacial surface sites. On a flat surface, such as the Ni(111), essentially 1 WBE is sufficient to form a closed water adlayer. However, the lipid surface provides a somewhat more open structure. Therefore, more water molecules are required to cover the lipid surface. Because of an anisotropic dipole-dipole interaction, the probability of capturing a water molecule is not uniformly distributed over the hydrophilic DPPC surface. Assuming more or less well-oriented DPPC molecules at the vacuum-lipid interface, comparable to Figure 4, sites between the polar headgroups should be most favorable. However, in line with the lipid dipole moment the water attraction should be negligible. Water adsorption then starts at distinguished surface sites. From these sites, the water adlayer spreads over the hydrophilic lipid surface. The final stage at coverages higher than 3 WBE follows zeroth-order desorption kinetics indicative of the sublimation of multilayer water. On the hydrophobic surface, only two desorption stages are clearly distinguishable in TPD, that is, a significant undercutting at coverages below 1.5 WBE followed by pseudo-zeroth-order
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desorption kinetics at coverages greater than 1.5 WBE. The first coverage regime can be attributed to the formation of small water clusters acting as condensation nuclei. In the second regime, these nuclei merely grow in size with increasing coverage. Thus, both sets of desorption kinetics suggest, according to the hydrophobic nature of the substrate, the formation of individual water droplets on the substrate surface.
Conclusions On the basis of the presented data, it can be concluded that a lipid (DPPC) surface with the lipid molecules preferentially oriented with their polar headgroups toward the lipid-vacuum interface (hydrophilic DPPC surface) is effectively wetted by water molecules. No uniquely strong bound water species is found on this surface. Water adsorption starts at 133 K (water partial pressure: 1 × 10-7 mbar). On the contrary, on the hydrophobic surface water adsorption starts at 106 K and results in the formation of water droplets, which merely grow in size upon increasing coverage. The adsorption kinetics on both surfaces do not differ very much. On both organic surfaces, water must form condensation nuclei in the initial stages of adsorption. LA060676T
