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Melting of the Dipalmitoylphosphatidylcholine Monolayer Lu Xu, Gordon Bosiljevac, Kyle Yu, and Yi Y. Zuo Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b00579 • Publication Date (Web): 22 Mar 2018 Downloaded from http://pubs.acs.org on March 22, 2018

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Melting of the Dipalmitoylphosphatidylcholine Monolayer Lu Xu,1 Gordon Bosiljevac,1 Kyle Yu,1 and Yi Y. Zuo 1,2* 1. Department of Mechanical Engineering, University of Hawaii at Manoa, Honolulu, Hawaii 96822, United States 2. Department of Pediatrics, John A. Burns School of Medicine, University of Hawaii, Honolulu, Hawaii, 96826, United States Corresponding Author * 2540 Dole St, Holmes Hall 302, Honolulu, Hawaii, 96822, United States Tel: 808-956-9650; Fax: 808-956-2373; Email: [email protected]

KEYWORDS: Monolayer; Phase transition; Phospholipid; DPPC; Constrained drop surfactometry.

ACS Paragon Plus Environment

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ABSTRACT Langmuir monolayer self-assembled at the air-water interface represents an excellent model for studying phase transition and lipid polymorphism in two dimensions. Compared to numerous studies of phospholipid phase transitions induced by isothermal compression, there are very scarce reports on two-dimensional phase transitions induced by isobaric heating. This is mainly due to technical difficulties of continuously regulating temperature variations while maintaining a constant surface pressure in a classical Langmuir-type film balance. Here, with technological advances in constrained drop surfactometry (CDS) and closed-loop axisymmetric drop shape analysis

(CL-ADSA),

we

studied

the

isobaric

heating

process

of

the

dipalmitoylphosphatidylcholin (DPPC) monolayer. It is found that temperature and surface pressure are two equally important intensive properties that jointly determine the phase behavior of the phospholipid monolayer. We have determined a critical point of the DPPC monolayer at a temperature of 44 ºC and a surface pressure of 57 mN/m. Beyond this critical point, no phase transition can exist in the DPPC monolayer, either by isothermal compression or by isobaric heating. The melting process of the DPPC monolayer studied here provides novel insights into the understanding of a wide range of physicochemical and biophysical phenomena, such as surface thermodynamics, critical phenomena, and biophysical study of pulmonary surfactants.


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INTRODUCTION Spreading a small amount of insoluble phospholipids at the air-water interface produces a self-assembled two-dimensional (2D) Langmuir monolayer.1 The study of phospholipid monolayers, with dipalmitoylphosphatidylcholine (DPPC) being the most studied species,2, 3 has a far-reaching impact on a wide range of scientific disciplines. In physics, the phospholipid monolayer provides an excellent model for studying molecular ordering and condensation in two dimensions since the water surface acts as an ideally smooth substrate for supporting the phospholipid monolayer.1,

4

In chemistry, the phospholipid monolayer serves as important

building blocks for thin film materials and smart surfaces.5 With effective layer-by-layer Langmuir-Blodgett (LB) transfer, self-assembled monolayers have applications in wetting, adhesion, lubrication, and sensors.6,

7

In biology, the phospholipid monolayer can be loosely

considered as a single leaflet of the biomembrane.8 Together with its associated surfactant reservoir,9 the phospholipid monolayer has been used as a well-accepted model for studying the biophysics of natural pulmonary surfactant.10-12 To date, almost all physicochemical and biophysical studies of the phospholipid monolayer rely on the classical Langmuir-type film balance.1 Upon manipulating the surface pressure of the monolayer by regulating its surface area at constant temperature in a Langmuir balance, most phospholipid molecules exhibit three states of ordering or phases in the monolayer.1, 8, 10 At a low surface pressure, because of the weak van der Waals attractions, phospholipid molecules in a dilute gaseous phase tend to remain separated while their fatty acid chains keep waving in air. With increasing surface pressure, the phospholipid molecules start interacting with each other as a consequence of increasing molecular density at the interface. However, in this so-called liquidexpanded (LE) phase, the presence of kinked trans-gauche configuration makes the fatty acid


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chains remain largely mobile. With further increasing surface pressure, the phospholipid molecules are transformed into a more ordered tilted-condensed (TC) phase, in which all acyl groups are in the extended trans configuration. The acyl group of phospholipid molecules in the TC phase shows a strong side-by-side ordering that results in a smaller tilting angle than that in the LE phase. The compression-induced LE-TC phase transition at constant temperature has been proven to be a first-order main phase transition by not only thermodynamic analysis of the compression isotherms but also direct microscopy observation of phospholipid domain formation in the monolayer.7, 13 Compared to the numerous studies of phospholipid phase transitions induced by isothermal compression, it is equally important to understand the phospholipid phase behavior at constant surface pressure, i.e., the isobaric phase transitions.14 However, there are very scarce reports on phospholipid phase transitions induced by isobaric heating. To the best of our knowledge, this is mainly due to technical limitations of the classical Langmuir balance. It is extremely difficult to continuously regulate temperature variations while maintaining a constant surface pressure in a Langmuir balance. Alternatively, Hall and co-workers used the captive bubble surfactometer (CBS) to study the phase behavior of phospholipid and pulmonary surfactant monolayers upon isobaric heating.15-18 Although providing novel insights, the CBS is very difficult to operate and lacks the automatic isobaric control system, thus preventing a comprehensive understanding of isobaric phase transitions in the phospholipid monolayer. Here we report a detailed study of isobaric heating induced phase transitions in the DPPC monolayer using a newly developed technique called the constrained drop surfactometry (CDS). The CDS is a new generation of droplet-based tensiometry technique.19 We have demonstrated its capacity as a miniaturized Langmuir-type film balance in studying phospholipid phase


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transitions induced by isothermal compression.13 Here for the first time, we demonstrate the capacity of the CDS in precisely controlling temperature as a ramp function while maintaining a constant surface pressure in the monolayer, using closed-loop axisymmetric drop shape analysis (CL-ADSA) recently developed in our laboratory.20 We show that these technological advances allow us to study isobaric heating induced phase transitions in the DPPC monolayer. Together with direct film imaging using atomic force microscopy (AFM), our results provide novel insights into the understanding of isobaric phase transitions in phospholipid monolayers.

EXPERIMENTAL SECTION Constrained Drop Surfactometry (CDS). The CDS is a novel experimental methodology recently developed in our laboratory.19 It uses the air-water interface of a sessile drop (~10 µL in volume, ~3 mm in diameter, and ~0.2 cm2 in surface area) to accommodate the spread Langmuir monolayer. The droplet is “constrained” on a carefully machined pedestal using its knife-sharp edge to prevent film leakage and to keep the droplet integrity even at near-zero surface tensions. The spread monolayer can be compressed and expanded, quasi-statically or dynamically, via regulating the surface area of the droplet by controlling liquid into and out of the droplet with a motorized syringe. The surface tension (or surface pressure) as well as the surface area of the monolayer are determined simultaneously using axisymmetric drop shape analysis (ADSA).21 Control of Temperature Ramp in CDS. Owing to system miniaturization, in comparison with the classical Langmuir film balance, the CDS ensures a rigorous control of experimental conditions using an environmental control chamber. The experimental temperature can be maintained at constant with an accuracy of ±0.1 oC using an electrothermostatic controller.


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Furthermore, we have developed a novel thermal profiling technique that allows the ramp function control of the temperature at a quasi-static rate of 1 oC/min or slower. Control of Surface Pressure in CDS. We have developed a novel closed-loop ADSA (CLADSA) in which the surface pressure (or surface tension) of a Langmuir monolayer can be controlled by regulating the surface area of the monolayer in real-time with a customized proportional-integral-derivative (PID) control system.20 In the present study, CL-ADSA was used to maintain a constant surface pressure of the DPPC monolayer while ramping up the environmental temperature. Specifically, a spread DPPC (Avanti Polar Lipids, Alabaster, AL) monolayer on a water droplet (Millipore, Billerica, MA) was first allowed evaporation of the solvent (i.e., chloroform). The DPPC monolayer was then quasi-statically compressed to a series of surface pressures of 10, 20, 30, 40, 50, and 60 mN/m, respectively. Once the target surface pressure was reached, the environmental temperature was quasi-statically ramped up from 10 to 50 ºC at a rate of 1 ºC/min. During temperature increase, the surface pressure of the DPPC monolayer was maintained to be constant with an accuracy of ±0.5 mN/m using CL-ADSA. The temperature and surface area of the DPPC monolayer at the constant surface pressure were recorded simultaneously to determine the heating isobars of the DPPC monolayer. These heating isobars are counterparts of the compression isotherms obtained at constant temperature using the classical Langmuir balance. Atomic Force Microscopy (AFM) Imaging. We have developed an in situ Langmuir-Blodgett (LB) transfer technique that allows transfer of the DPPC monolayer from the surface of a droplet to a solid substrate under controlled environmental conditions.13 Briefly, the in situ LB transfer was performed by lifting a small piece of freshly peeled mica sheet across the air-water interface of the droplet at a rate of 1 mm/min. An Innova AFM (Bruker, Santa Barbara, CA) was used to


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scan the immobilized monolayers in air with the tapping mode using an antimony doped silicon cantilever with a spring constant of 42 N/m and a tip radius of 8 nm. Image analysis was performed with Nanoscope Analysis (version 1.5).

RESULTS AND DISCUSSION Surface tension measurements with continuous temperature variation. We first demonstrate the feasibility and accuracy of real-time surface tension measurements while continuously ramping up the environmental temperature in CDS. Figure 1 shows the surface tension of a water droplet determined as the temperature was continuously ramped up from 10 to 50 oC. As demonstrated in Figure S1 of the Supporting Information (SI), the environmental temperature was precisely controlled to increase linearly at a rate of 1 oC/min, while the volume of the droplet was controlled to be constant using CL-ADSA. The constant drop volume maintained during this process helps rule out potential artifacts caused by droplet evaporation with increasing time and temperature. Reproducibility of the surface tension measurements as a function of temperature can be found in Figure S2 of the SI. As shown in Figure 1, the surface tension of water decreases with increasing temperature. Our measurements are in excellent agreement with the literature value.22 It should be noted that our surface tension measurements were performed in real-time with a single droplet by continuously varying the environmental temperature. However, all previous measurements were performed at discrete individually controlled environmental temperatures.22,

23

Therefore,

compared to existing methods, the CDS provides an accurate, easy-to-use and much more powerful method in determining continuous surface tension dependence on temperature.


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75

CDS measurement Literature value22

74

Surface tension of water (mN/m)

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73

72

71

70

69

68

67 10

15

20

25

30

35

40

45

50

Temperature (°C)

Figure 1. Surface tension measurements of a water droplet when continuously varying temperature with the constrained drop surfactometry (CDS). Good agreement is found between the measurements and literature values. Heating isobars of the DPPC monolayer. Upon proving the accuracy and feasibility of temperature control and surface tension measurements, we study the isobaric heating induced phase transitions in the DPPC monolayer. Figure 2a shows the isobaric expansion of the DPPC monolayer while increasing the temperature from 10 to 50 oC. Heating isobars at six representative surface pressures, 10, 20, 30, 40, 50, and 60 mN/m, are displayed. Representative experimental data of the surface area, surface pressure, and temperature as a function of time are shown in Figure S3 of the SI. It can be seen that the surface pressure was maintained at constant with an accuracy of ±0.5 mN/m and the temperature was controlled to ramp up linearly. Reproducibility of these isobars is demonstrated in Figure S4 of the SI. To make these heating isobars directly comparable with the compression isotherms of the DPPC monolayer, we replot the isobars in temperature against the molecular area of the DPPC monolayer. As shown in Figure 2b, between surface pressures 10 and 50 mN/m, all isobars clearly show a nearly horizontal plateau at which the molecular area increases dramatically with


8

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a narrow increase of temperature. For example, the DPPC monolayer at the surface pressure 10 mN/m only slowly increases the molecular area from 51.4 to 54.2 Å2/molecule when increasing temperate from 10 to 25 oC. However, further increasing temperature by 1 oC causes a significant monolayer relaxation, indicated by a sudden increase in the molecular area from 54.2 to 73.2 Å2/molecule. After passing this plateau region, the molecular area again increases slowly with increasing temperature. The plateau range of the isobars, as shown in the T-A diagram, therefore indicates a first-order phase transition of the DPPC monolayer from a highly packed TC phase to a loosely structured LE phase, i.e., “melting” of the DPPC monolayer. It should be noted that during the first-order phase transition, similar to the compression isotherms, the heating isobars display a nonhorizontal rising plateau. The nonzero slope of the phase transition plateau was usually attributed to impurities or nonequilibrium effects such as the finite rate of film compression in the case of compression isotherms,24, 25 or similarly the finite rate of temperature rising in the present case of heating isobars. It is known that the kinetic effect plays an important role in phase transitions and various self-assembly processes.16, 17, 26-28 More recent structural evidence indicates that the nonzero slope is most likely caused by the two-dimensional mixing entropy of small molecular aggregates or domains formed in the monolayer.13 Our results are in good agreement with those produced with the CBS, in which a DPPC monolayer at surface pressure 10 mN/m starts melting at 25 oC, 54 Å2/molecule and ends at 26 o

C, 73 Å2/molecule.15, 17 However, it should be noted that the DPPC monolayer at high surface

pressures (> 50 mN/m) quickly collapsed in the CBS when the temperature was increased above 44 ºC.15, 17 In contrast, the DPPC monolayer retains the film stability in CDS even at high surface pressure and high temperature. The elimination of even partial film collapse or the so-called film leakage in CDS has been proven with two independent means. First we have conducted quasi-


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static cyclic measurements and found no hysteresis between the heating and cooling isobars (unpublished data). Second, we have directly visualized the lateral structure of the DPPC monolayer and found no bilayered or multilayered collapsed phase even at high surface pressures (see Figure 5). 90 50

80 75

mN/m mN/m mN/m mN/m mN/m mN/m

45

a

70 65 60 55

b

40

Temperature (° C)

10 20 30 40 50 60

85

Molecular area (Å2 /molecule)

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35

30 25

10 mN/m 20 mN/m 30 mN/m 40 mN/m 50 mN/m 60 mN/m Saturation dome Critical point

20

50 15

45 10

40 10

15

20

25

30

35

40

45

50

40

Temperature (° C)

50

60

70

80

90

Molecular area (Å2 /molecule)

Figure 2. Heating isobars of the DPPC monolayer at surface pressures 10, 20, 30, 40, 50, and 60 mN/m, respectively, obtained with CDS upon linearly increasing the temperature from 10 to 50 °C. (a) Isobars shown as the molecular area vs. temperature. (b) Isobars shown as the temperature vs. molecular area of the monolayer. Lining up the starting and ending points of the phase transition plateaus at various surface pressures forms a dome, closely mimicking the saturation dome of a bulk substance such as pure water. To the left of the saturation dome, the DPPC monolayer is in a single ordered TC phase. To the right of the saturation dome, the DPPC monolayer is in a single disordered LE phase. Within the saturation dome, the TC and LE phases coexist in the DPPC monolayer. The apex of the saturation dome defines a critical point at which the differences between TC and LE phases disappear. As shown in Figure 2b, with increasing surface pressure, the phase transition takes place at a higher temperature and spans a smaller molecular area across the phase transition region, which


10

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indicates decreasing entropy and enthalpy for the phase transition. At the surface pressure 60 mN/m, the phase transition plateau disappears. Lining up the starting and ending points of the phase transition plateaus at various surface pressures forms a dome closely mimicking the saturation dome of a bulk substance such as pure water. To the left of the saturation dome, the DPPC monolayer is in a single ordered TC phase. To the right of the saturation dome, the DPPC monolayer is in a single disordered LE phase. Within the saturation dome, the TC and LE phases coexist in the DPPC monolayer. The apex of the saturation dome defines a critical point at which the differences between TC and LE phases disappear. The first-order phase transition can be further studied by determining the isobaric thermal ଵ ௗ஺

expandability ߙగ = ஺ ቀௗ் ቁ , where A and π are the molecular area and surface pressure of the గ

DPPC monolayer, respectively. The isobaric thermal expandability (απ) determined from the T-A diagram is a counterpart of the isothermal compressibility (κT) determined from the π-A diagram.13 As shown in Figure 3, the peak of απ is indicative of the phase transition temperature (Ttr), i.e., the melting point of the DPPC monolayer at a controlled surface pressure. To the left of the peak, the DPPC monolayer is in an ordered TC phase with a low απ of 0.05 1/K. To the right of the peak, the DPPC monolayer is melted into a disorder LE phase with a higher απ of 0.42 1/K. 4.5

10 20 30 40 50

4.0

Thermal Expandability (K-1)

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3.5 3.0

mN/m mN/m mN/m mN/m mN/m

2.5 2.0 1.5 1.0 0.5 0.0 10

15

20

25

30

35

40

45

50

Temperature (°C)


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Figure 3. Thermal expandability of the DPPC monolayer at surface pressures 10, 20, 30, 40, and 50 mN/m, respectively, obtained with CDS upon linearly increasing the temperature from 10 to 50 °C. The location of thermal expandability peaks defines the phase transition temperature at the corresponding surface pressure. Thermodynamics of 2D phase transitions in the DPPC monolayer. The entropy (∆Str) and enthalpy (∆Htr) of the TC-LE phase transition can be determined with the 2D ClausiusClapeyron equations: ௗగ

ሺ‫ܣ‬௅ா − ‫்ܣ‬஼ ሻ ∆ܵ௧௥ = ௗ்

(1)

೟ೝ

ௗగ

ሺ‫ܣ‬௅ா − ‫்ܣ‬஼ ሻ ∆‫ܪ‬௧௥ = ܶ௧௥ ∆ܵ௧௥ = ܶ௧௥ ௗ்

(2)

೟ೝ

where ATC and ALE are the molecular areas at the starting and ending points of the TC-LE phase transition, respectively. All thermodynamic properties pertinent to the phase transition in the DPPC monolayer at various surface pressures are summarized in Table 1.

Table 1. Thermodynamic quantities pertinent to TC-LE phase transitions in the DPPC monolayer derived from the experimental isobars. π (mN/m)

Ttr (°C)

ATC (Å2/mol.)

ALE (Å2/mol.)

∆Str (J/mol·K)

∆Htr (kJ/mol)

10

25.9 ± 0.1

56.1 ± 0.3

73.2 ± 0.5

36.9 ± 1.3

11.0 ± 0.4

20

32.3 ± 0.3

53.7 ± 0.6

65.1 ± 0.3

24.6 ± 2.1

7.5 ± 0.2

30

35.4 ± 0.1

52.1 ± 0.9

59.8 ± 0.4

16.6 ± 1.6

5.1 ± 0.3

40

41.1 ± 0.2

50.2 ± 0.6

56.4 ± 0.7

13.4 ± 1.6

4.2 ± 0.2

50

44.6 ± 0.2

47.2 ± 0.8

49.2 ± 1.1

4.3 ± 0.6

1.4 ± 0.1


12

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As shown in Figure 4, Ttr, ∆Str, and ∆Htr are all linearly correlated with the surface pressure, which is also indicative of a typical first-order phase transition. One characteristic parameter that can be determined from Figure 4 is the critical surface pressure (πc) at which the differences between the TC and LE phases disappear.1, 29 Therefore, πc defines the highest possible surface pressure at which the TC and LE phases may coexist in the DPPC monolayer, i.e., the apex of the saturation dome shown in Figure 2b. πc for the DPPC monolayer is determined to be 57 mN/m by extrapolating the phase transition enthalpy toward zero. Our previous study on isothermal compression induced phase transitions has already determined the critical temperature (Tc) of the DPPC monolayer to be 44 ºC.13 Together with πc determined in this study, we report a critical point of the DPPC monolayer, i.e., (Tc, πc) = (44 ºC, 57 mN/m). Beyond this critical point, no phase transition can exist, either by isothermal compression or by isobaric heating, in the DPPC monolayer. 50

Ttr (°C)

40

30

20

∆Str (J/mol K)

40

30

20

10

0 12

∆Htr (kJ/mol)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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10 8 6 πc=

4

57 mN/m 2 0 10

20

30

40

50

60

Surface pressure (mN/m)


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Figure 4. Temperature (Ttr), entropy (∆Str), and enthalpy (∆Htr) of the main phase transition in the DPPC monolayer as a function of surface pressure. Linear extrapolation of ∆Htr toward zero determines the critical surface pressure (πc). From the point of view of surface thermodynamics, our results suggest that 2D phase transitions and polymorphism phenomena in the phospholipid monolayer can be induced not only by isothermal compression but also by isobaric heating. Most previous physicochemical studies of Langmuir monolayers only focused on pressure-induced phase transitions at constant temperature. This is mainly due to technical limitations of the classical Langmuir balance. After overcoming the technical limitations using the CDS, it is clear that temperature and surface pressure are two equally important intensive properties that jointly determine the phase behavior of monolayers and thin-film materials. The isobaric heating processes studied here may provide novel insights into the understanding of a wide range of physicochemical and biophysical phenomena. For instance, it is unclear how heterothermic animals maintain low surface tensions (i.e., high surface pressures) in their lungs, while varying their body temperature in a large range from as low as 3 oC during hibernation to 37 oC during the summer-active period.30, 31 Although having a reduced level of disaturated phospholipids, the pulmonary surfactant of hibernating animals, such as 13-lined ground squirrels, still contains at least 40% DPPC.30, 31 The current study shows that the DPPC monolayer at a high physiologically relevant surface pressure (> 50 mN/m) is capable of maintaining a single ordered TC phase for a large temperature range from 10 to 45 oC (Figure 2b). Therefore, our study supports the hypothesis that the DPPC monolayer in a single TC phase is responsible for the low surface tension in the lungs.


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domains, and the electrostatic repulsion between the lipid headgroups, which works in the opposite direction.34, 35 Upon increasing surface pressure, the molecular area is reduced (Table 1), thus resulting in larger electrostatic repulsions. Meanwhile, the line tension is expected to decrease as demonstrated by the morphological deformation of the domains with increasing surface pressure (Figure 5). Consequently, both effects lead to the smaller domains upon increasing surface pressure (Figure 6). At 60 mN/m and 45 oC, which is beyond the critical point of the DPPC monolayer, the TCLE phase coexistence appears as a cross-linked network rather than individual domains. This morphological variation is a strong indication of the critical phenomena at which the line tension at the domain boundaries is reduced to minimum, and consequently the boundaries between the two phases become blurry and are largely affected by system fluctuation and random perturbation.36, 37

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Size of TC domains (µm )

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14

12 10

8 6

4 2 0 10

20

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40

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Surface pressure (mN/m)

Figure 6. Quantification results of the size of the TC domains in the DPPC monolayer at surface pressures 10, 20, 30, 40, and 50 mN/m, respectively. It is clear that the TC domains reduce sizes upon increasing surface pressure.


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CONCLUSIONS With technological advances in constrained drop surfactometry (CDS) and closed-loop axisymmetric drop shape analysis (CL-ADSA), we studied the isobaric heating process of the DPPC monolayer. It is found that temperature and surface pressure are two equally important intensive properties that jointly determine the phase behavior and lipid polymorphism of the phospholipid monolayer. Together with our previous studies of the isothermal compression process, we have determined a critical point of the DPPC monolayer, i.e., (Tc, πc) = (44 ºC, 57 mN/m). Beyond this critical point, no phase transition can exist in the DPPC monolayer, either by isothermal compression or by isobaric heating. The melting process of the DPPC monolayer studied here provides novel insights into the understanding of a wide range of physicochemical and biophysical phenomena, such as surface thermodynamics, 2D critical phenomena, and biophysical study of pulmonary surfactants.


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ASSOCIATED CONTENT Supporting Information. Results of Volume-Area-Temperature vs. Time of a water droplet upon increasing temperature. Reproducibility of the surface tension measurements. Results of Surface area-Surface pressure-Temperature vs. Time of the DPPC monolayer maintained at constant surface pressures. Reproducibility of the heating isobars of the DPPC monolayer. The Supporting Information is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * Yi Y. Zuo, [email protected] The authors declare no competing financial interests. ACKNOWLEDGMENT This work was supported by NSF Grant No. CBET-1254795 (Y.Y.Z.).


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Langmuir

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TOC GRAPHICS


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Melting of the Dipalmitoylphosphatidylcholine ... - ACS Publications

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