Hydrostatic Pressurization of Lung Surfactant Microbubbles

Subscriber access provided by READING UNIV

Article

Hydrostatic Pressurization of Lung Surfactant Microbubbles: Observation of a Strain-Rate Dependent Elasticity Alec N. Thomas, and Mark Andrew Borden Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b03307 • Publication Date (Web): 24 Oct 2017 Downloaded from http://pubs.acs.org on November 2, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Langmuir is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 39

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

Langmuir

1

Hydrostatic Pressurization of Lung Surfactant

2

Microbubbles: Observation of a Strain-Rate

3

Dependent Elasticity

4

Alec N. Thomas1 and Mark A. Borden*1,2

5 6

1

Department of Mechanical Engineering, 2Materials Science and Engineering Program, University of Colorado, Boulder, Colorado 80309, USA

7 8 9 10 11 12 13 14 15 16

*Corresponding Author Mark A. Borden, PhD University of Colorado 1111 Engineering Drive Boulder, CO 80309-0427 Phone: 303.492.7750 Fax: 303.492.3498 Email: [email protected]

17 18

KEYWORDS: Survanta, DPPC, perfluorobutane, dissolution, monolayer collapse

19

ACS Paragon Plus Environment

1

Langmuir

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

Page 2 of 39

20

Abstract

21

The microbubble offers a unique platform to study lung surfactant mechanics at physiologically

22

relevant geometry and length scale. In this study, we compared the response of microbubbles

23

(~15 µm initial radius) coated with pure dipalmitoyl-phosphatidylcholine (DPPC) versus

24

naturally derived lung surfactant (Survanta®) when subjected to linearly increasing hydrostatic

25

pressure at different rates (0.5 to 2.3 kPa/s) at room temperature. The microbubbles contained

26

perfluorobutane gas and were submerged in buffered saline saturated with perfluorobutane at

27

atmospheric pressure. Bright-field microscopy showed that DPPC microbubbles compressed

28

spherically and smoothly, whereas Survanta microbubbles exhibited wrinkling and smoothing

29

cycles associated with buckling and collapse.

30

collapse amplitude was constant, but collapse rate increased with pressurization rate.

31

analysis of the pressure-volume curves indicated that the dilatational elasticity increased during

32

compression for both shell types. The initial dilatational elasticity for Survanta was nearly twice

33

that of DPPC at higher pressurization rates (>1.5 kPa/s), producing a pressure drop of up to 60

34

kPa across the film prior to condensation of the perfluorobutane core. The strain-rate dependent

35

stiffening of Survanta shells likely arises from its composition and microstructure, which

36

provides enhanced in-plane monolayer rigidity and lateral repulsion from surface-associated

37

collapse structures. Overall, these results provide new insights into lung surfactant mechanics

38

and collapse behavior during compression.

Seismograph analysis showed that Survanta An

39


2

Page 3 of 39

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

Langmuir

40

Introduction

41

The pulmonary system is a complex network of interconnected structures terminating at

42

microscopic alveolar sacs lined with lung surfactant (LS) at the air/water interface.1

43

comprises lipids and proteins working in concert to maintain a low surface tension throughout

44

the dynamic breathing cycle.1–3 Low surface tension is necessary to prevent alveolar collapse and

45

reduce the required work to expand the lungs by the thoracic muscles. A hallmark of acute

46

respiratory distress syndrome (ARDS) is the inactivation of LS, rendering the film incapable of

47

dynamic mechanical stability and leading to alveolar collapse and low blood oxygenation.4,5

48

Direct instillation of lung surfactant replacement therapy (LSRT) has substantially improved

49

survival rates for neonatal respiratory distress syndrome (NRDS), and it is the current standard

50

practice of care.6,7 More recently, researchers have attempted to formulate LSRTs to treat ARDS

51

more generally, and to mitigate the risks of animal-derived LSRT by creating purely synthetic

52

surfactant formulations.8,9,2

LS

53

The typical human lung contains ~170 alveoli per mL of lung parenchyma,10 and each alveolus

54

is quasi-spherical in shape with an average diameter of ~100 µm.11 Owing to the complexity of

55

the lung and difficulty imaging it without inducing artifacts, the exact physiological structure of

56

LS and the mechanics at the alveolar air/water interface during inflation/deflation are not yet

57

fully understood.11,12 This makes it challenging to strictly define physiological values for strain

58

and strain rate. A recent review lists values for the linear strain ( = − /, where

59

and are initial and final radii) to be ~5-25% during normal breathing.11 A recent in vivo x-ray

60

imaging study in mice reported a similar range of strains.13 Given the respiratory rate for an

61

adult human of ~12-20 breaths/min, the alveolar strain rate is estimated to be ~1-10 %/s. This


3

Langmuir

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

Page 4 of 39

62

value can vary significantly between different regions of the lung, and for different tidal volumes

63

and respiratory rates.

64

Development and application of a functional LSRT relies on an understanding of LS

65

mechanics during compression.14–16 During inhalation, the diaphragm and intercostal muscles

66

expand the chest cavity to induce a negative pressure that draws air in. During exhalation, the

67

muscles induce a positive pressure that drives air out, thus compressing the alveolar surface area

68

and collapsing the surfactant film. Langmuir trough experiments have shown that natural and

69

synthetic LS films can achieve high surface pressure (near zero surface tension) prior to

70

collapse.17–19 Collapse is evident as a plateau in the surface pressure-area isotherm, indicating a

71

very low film elasticity. Unfortunately, the Wilhelmy plate method of measuring surface tension

72

loses accuracy as the film transitions into a solid.20

73

measurements have shown similar results for LS films between spreading and collapse.21,22

74

However, the mechanical properties of LS films during compression beyond the onset of

75

collapse is poorly understood and rarely investigated. The post-collapse mechanics may be

76

critical to the development of efficacious LSRTs since alveoli experience a wide range of strains

77

dependent on their location in the lung.

Captive bubble and pendant drop

78

A microbubble is a small, gas-filled particle (1-100 µm diameter) stabilized by a surfactant

79

shell that inhibits coalescence and surface tension-induced dissolution. Microbubbles have been

80

widely studied for use as ultrasound contrast agents, drug/gene delivery vehicles and injectable

81

oxygen carriers.23–25

82

microbubbles for these applications.26–28 In this study, we used a custom microscopy chamber to

83

monitor the response of individual microbubbles coated with Survanta® (a commercially

84

available LSRT), versus those coated with pure dipalmitoyl-phosphatidylcholine (DPPC) (the

Our group has previously demonstrated the synthesis and use of LS


4

Page 5 of 39

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

Langmuir

85

main component of LS), to a linear pressure increase. In this sense, we use the microbubble as a

86

sort of film balance to study LS mechanics. This idea is in part inspired by the original work of

87

R.E. Pattle,29 who discovered LS by the observation of highly stable microbubbles liberated from

88

the aspirated lung.30 We caution that an individual microbubble does not completely mimic the

89

complex interconnected nature of the pulmonary system. However, many of the properties of the

90

microbubble are similar to those of an isolated lung alveolus. For example, the spherical shape

91

of a microbubble provides a similar geometry and curvature. This curvature is relevant because

92

the surfactant molecules are known to assemble into microdomains within the monolayer

93

plane,31 and alveolar deformation is on the order of microns during normal breathing. By

94

following the response of a microbubble to pressurization, one may determine physiologically

95

relevant physical properties, such as surface elasticity and gas permeability.

96

In this study, we first observed the bubble shape as it compressed under the hydrostatic load

97

and analyzed collapse events by plotting the corresponding seismograms. We then analyzed the

98

pressure-volume behavior of each microbubble with an analytical model that accounts for both

99

compression and dissolution. The analysis provided an estimation of the initial dilatational

100

elasticity of the shell ( ), which serves to characterize the mechanical response of Survanta and

101

DPPC shells.

102 103

Theory

104

Here, an analytical model is derived for the compression and dissolution of a microbubble

105

subjected to a linear increase in hydrostatic pressure. Fick’s law describes the accumulation of

106

gas in the bubble over time,

107

= − ,

(1)


5

Langmuir

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

Page 6 of 39

108

where is the number of moles, is time, is the mass transfer coefficient, is the bubble

109

surface area, is the dissolved gas concentration at the bubble surface, and is the dissolved

110

gas concentration in the bulk medium. Henry’s law relates the dissolved gas concentration to the

111

partial pressure for dilute solutions at equilibrium, =

112

,

(2)

113

where is Henry’s constant (in this form, the “Ostwald coefficient”) and is the dissolved gas

114

concentration. The molar gas concentration, = , is determined by the ideal gas law, where

115

is the gas partial pressure,

is the gas constant, ! is the temperature. Substitution gives,

116

=

"#$

− .

(3)

117

Before pressurization begins, the medium is equilibrated with the gas at atmospheric pressure .

118

For a single gas component, the partial pressure is equal to the total pressure inside the bubble

119

(),

120

=

"#$

− .

(4)

121

The shell interfacial rheology has been shown to significantly influence the microbubble

122

response to pressure changes.32,33 Here, we adopt the exponential elasticity model by Paul et

123

al.34 to describe the surface dilatational elasticity, , of the shell:

= ∙ exp−)*,

124

(5)

" /

125

where is the initial dilatational elasticity, * = +," . − 11 is the fractional change in area

126

from the unstrained state, and ) is a constant equal 1.5.34 The surface tension, 2, of the film is

127

then determined by,

128

-

2 = 2 + ∙ *,

(6)


6

Page 7 of 39

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

Langmuir

129

where 2 is the equilibrium surface tension. The microbubble is modeled as a sphere on which

130

the interfacial stresses cause a jump (pressure drop) in the normal stress at the interface. Sarkar

131

et al.35 previously defined this pressure jump at the interface as the dynamic boundary condition:

132

78 9:78 /< /= 7 / = 4 + 46 + ; + - + +, . − 11, 7

7

7

7

7-

(7)

133

where 4 is the hydrostatic pressure, 6 is the medium viscosity and > is the surface dilatational

134

viscosity. For the pressurization rates used in this study, the viscous normal stress in the medium

135

(water) and the dilatational viscosity contribution of the lipid shell (of order ~µN/m36) can be

136

neglected. Additionally, the initial surface tension is set to zero for an initially stable (non-

137

dissolving) microbubble.37,38 The dynamic boundary condition then reduces to the well-known

138

Laplace pressure equation with the surface dilatational elasticity contribution,

139

= 4 +

/= 7

7 /

+,7 . − 11 . -

(8)

140

Equations (5), (6) and (8) was solved numerically using the backward-difference method with

141

as the single fitting parameter to the experimental data using a least-squares regression.

142 143

Experimental Section

144

Materials. Survanta® (beractant, AbbVie Inc., Chicago, IL, USA) was purchased and stored

145

at 4 °C, as directed. Special care was taken to ensure sterility during the use of the product.

146

Survanta is a naturally derived bovine lung surfactant that is fortified to standardize each batch

147

with the following components: DPPC, palmitic acid and tripalmitin. The concentrations of

148

components are listed on the package insert as 25 mg/mL phospholipid (primarily DPPC), 0.5-

149

1.75 mg/mL triglycerides, 1.4-3.5 mg/mL fatty acids and less than 1.0 mg/mL proteins (SP-B,

150

SP-C). Though Survanta is naturally derived, the extraction and purification process alters the


7

Langmuir

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

Page 8 of 39

151

composition to reduce hydrophobic surfactant proteins SP-B and SP-C and eliminate cholesterol

152

and the hydrophilic surfactant proteins. DPPC was purchased from Avanti (Alabaster, AL) in

153

lyophilized dry powder form of greater than 99% purity and stored at -20°C with a nitrogen gas

154

head space. Perfluorobutane (PFB) was purchased from FluoroMed (Round Rock, TX). PFB

155

was chosen as the filling gas because it generated a higher yield of microbubbles and was less

156

susceptible to dissolution during pressurization than air microbubbles. Supplemental Figure 1

157

shows that even at the lowest pressurization rate an air microbubble dissolves rapidly, yielding

158

strain rates beyond the physiological range and making it difficult to analyze the surfactant

159

mechanics. Phosphate buffered saline (PBS) 137 mM NaCl, pH 7.4 (ThermoFischer Scientific,

160

Fair Lawn, NJ) was purchased and used as directed.

161

Microbubble Synthesis. Survanta microbubbles were produced as follows: the 20-mL stock

162

vial of Survanta was warmed to room temperature and gently swirled to homogenize the

163

contents.

164

phospholipid in 0.2-µm filtered PBS (137 mM NaCl) made from purified water (18.2 MΩ cm

165

resistivity; Direct-Q, Millipore, Billerica, MA, USA). To ensure a well-mixed solution, each

166

batch was bath sonicated for 10 min at 37 oC. Then 2-mL aliquots were dispensed in 3-mL glass

167

serum vials, immediately capped, the air headspace was exchanged with PFB, and the vial was

168

stored at 4 oC.

The Survanta material was extracted by sterile syringe and diluted to 2 mg/mL

169

DPPC microbubbles were made as follows: Lipid was rehydrated in PBS to a concentration of

170

2 mg/mL and then homogenized by tip sonication until the solution appeared translucent.

171

During sonication, the temperature was periodically monitored to ensure the temperature did not

172

exceed 10 °C past the main phase transition temperature (41 °C). 2-mL aliquots were dispensed


8

Page 9 of 39

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

Langmuir

173

into 3-mL glass serum vials, the air headspace was exchanged with PFB, and the vial was stored

174

at 4 oC.

175

All microbubbles were produced by securing a serum vial in a shaker device (VialMix, Bristol-

176

Myers Squib) and activating for 45 s. The suspension was extracted with a 3-mL syringe, and

177

larger microbubbles were isolated by inverting the syringe for 10 s, discarding the sub-phase, and

178

refilling the syringe with fresh PFB-saturated PBS to dilute the microbubbles.

179

Pressurization Chamber. Survanta and DPPC microbubbles were observed at six separate

180

linear pressurization rates (0.5, 0.8, 1.1, 1.5, 1.9 and 2.3 kPa/s) using a custom chamber fastened

181

to a microscope stage (Olympus BX52, Center Valley, PA, USA) at room temperature (21 °C ±

182

1 °C). The chamber was outfitted with a pressure transducer (Transducers Direct TDH40

183

Cincinnati, OH, USA) and a type-K thermocouple insulated wire (Omega HSTC-TT-K-24S-36,

184

Stamford, CT, USA), which was tethered and sealed through the thermocouple port.

185

performed experiments at room temperature to establish a baseline response of the surfactant

186

films undergoing compression during microbubble pressurization. Several prior studies have

187

also investigated lung surfactant films at room temperature,39–42 and the results obtained at this

188

temperature remain relevant to technologies employing these types of surfactant films, such as

189

microbubbles used for drug delivery and oxygenation.29 The chamber was outfitted with a

190

pressure transducer (Transducers Direct TDH40 Cincinnati, OH, USA) and a type-K

191

thermocouple insulated wire (Omega HSTC-TT-K-24S-36, Stamford, CT, USA), which was

192

tethered and sealed through the thermocouple port. The thermocouple tip was secured adjacent

193

to the optical window to obtain an accurate reading of the microbubble temperature. Images

194

were captured in bright-field mode on an upright microscope with a digital camera (Q-Imaging

195

Q-Click, Surrey, BC, Canada) at 10 Hz sampling frame rate. All data were acquired through a

We


9

Langmuir

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

Page 10 of 39

196

data acquisition device (USB-6000, National Instruments, Austin, TX, USA) by a custom

197

LabVIEW (National Instruments) program.

198

199 200

Figure 1. Illustration of the chamber design. The chamber pressure was controlled by a syringe

201

pump (Harvard Apparatus, Holliston, MA) programmed to deliver a calibrated volumetric flow

202

rate. A metal syringe (KD Scientific, Holliston, MA) filled with PFB-saturated PBS was used to

203

ensure uniform pressurization during the entire experiment.

204 205

The experiment commenced by first filling the chamber with PFB-saturated PBS to maintain a

206

saturated environment. Then microbubbles were injected into the chamber and floated to the top

207

coverslip. The outlet valve was shut off and the inlet valve was opened to the metal syringe.

208

The syringe pump was programmed to achieve the desired pressurization rate, and data

209

acquisition was started approximately one minute before the onset of pressurization to verify that

210

bubble volume remained constant before the onset of pressurization.

211

Image Analysis. Digital images were processed through a custom MATLAB (Mathworks,

212

Natick, MA) program. Briefly, each acquired frame was converted to a binary image and

213

analyzed to measure the cross sectional area, perimeter and eccentricity of the microbubble


10

Page 11 of 39

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

Langmuir

214

during pressurization. The grayscale pixel threshold was automatically generated using the

215

MATLAB function graythresh.

216

deviation, unless otherwise noted. The largest microbubble radius change was 8 µm (mean 4 ± 2

217

µm) before the onset of condensation. The imaging plane was not altered during the experiment,

218

and the depth of focus of our imaging system was ~17 µm. Thus, each microbubble remained in

219

focus during the experiment.

All measured values are presented as mean ± standard

220

Statistical Analysis. A two-way Analysis of Variance was performed on the fitted initial

221

dilatational elasticities for Survanta and DPPC. The population means for the initial dilatational

222

elasticity of the shell types were compared against each other at each pressurization rate using

223

the Tukey method in order to quantify the comparisons. A significance level of p < 0.05 was

224

used. The statistical results are reported in Supplemental Table 1.

225 226 227

Results and Discussion

228

Microbubble Morphology. The spherical microbubble shape is a consequence of surface

229

tension acting to minimize surface area. In our experiments, all microbubbles were initially

230

observed to have a spherical shape (n ≥ 20 microbubbles for each shell type and pressurization

231

rate). Examples are shown in Figure 2. The initial tension of these microbubbles must have

232

been quite low, however, or they would have spontaneously dissolved under their own Laplace

233

pressure.37,38,43 Rather, we observed that the bubbles were stable in size for at least several

234

minutes prior to hydrostatic pressurization.

235

Figure 2A shows typical microscope images of Survanta microbubbles during pressurization at

236

the slowest and fastest rates. In general, the cross section remained highly circular during the


11

Langmuir

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

Page 12 of 39

237

initial compression. After a linear strain of ~9 ± 2 %, however, Survanta shells deviated from

238

circularity and appeared to wrinkle and smooth in cycles. This wrinkling is seen on the image at

239

249.2 s on the top panel, and at 86.7 s on the bottom panel of Figure 2A. Within one video

240

frame (0.1 s), the microbubble rapidly smoothed and regained circularity.

241

wrinkling/smoothing behavior was observed for Survanta microbubbles at all pressurization

242

rates. This behavior is reminiscent of the “clicking” behavior observed by Pattle on liberated

243

lung bubbles44 and by Schürch et al. on the captive bubble surfactometer,45 as well as the “jerks”

244

observed on Langmuir films40 of model lung surfactant. The wrinkling of Survanta shells

245

suggested that the surface was rigid enough to deform out-of-plane as a mechanism to sustain the

246

increasing surface stress. The shape change indicated that the shells achieved states of very low,

247

perhaps even negative, surface tension. Furthermore, the sudden shape change suggested that

248

Survanta shells experienced a buckling instability and collapsed.42,46 The return to a more

249

circular shape at these collapse transitions was consistent with a transient increase in surface

250

tension.

251

perfluorobutane gas core condensed at a hydrostatic pressure of 272 ± 9 kPa (mean ± standard

252

deviation), which was ~60 kPa above the equilibrium saturation curve.47

Similar

The wrinkling/smoothing process repeated itself during pressurization until the

253


12

Page 13 of 39

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

Langmuir

254 255

Figure 2. Images acquired for Survanta (A) and DPPC (B) microbubbles during pressurization at

256

0.5 kPa/s (top row of each panel) and 2.3 kPa/s (bottom row of each panel). The scale bar is 10

257

µm.

258 259

Figure 2B shows a similarly sized DPPC bubble undergoing pressurization at the same rates.

260

In general, DPPC microbubbles remained circular throughout the pressurization process, for all

261

pressurization rates. These microbubbles were not observed to wrinkle. The smooth, spherical


13

Langmuir

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

Page 14 of 39

262

shape of DPPC shells indicated that they were not able to buckle and collapse in the same

263

manner as Survanta shells. On average, DPPC microbubbles condensed at a slightly lower

264

hydrostatic pressure of 262 ± 8 kPa, which was ~50 kPa above the equilibrium saturation curve,

265

Collapse Phenomena. Figure 3 shows typical plots of microbubble cross sectional area as a

266

function of time for the slowest and fastest pressurization rates. The Survanta microbubbles

267

initially had a relatively smooth and steep area reduction until the onset of collapse (denoted by

268

the first arrow in Fig. 3A). These microbubbles continued to experience buckling/collapse

269

cycles until just before condensation (denoted by the second arrow in Fig. 3A). The sharp

270

collapse events indicated a series of elastic compression and singular failure events.

271

supports our assumption that the dilatational elasticity dominates over dilatational viscosity.

This

272

273


14

Page 15 of 39

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

Langmuir

274

Figure 3. Cross-sectional areas of typical Survanta (black curve) and DPPC (red curve)

275

microbubbles, along with the chamber pressure (blue curve). (A) Pressurization rate of 0.5 kPa/s

276

and (B) pressurization rate of 2.3 kPa/s. The arrows indicate the first and last buckling events for

277

the Survanta microbubbles.

278 279

Typical DPPC microbubbles are shown in Figure 3B. These microbubbles exhibited a smooth

280

reduction in cross sectional area, absent of discontinuities, throughout the pressurization process.

281

Similar to Survanta, the cross section of DPPC microbubbles initially decreased rapidly and then

282

plateaued, and an abrupt decrease in area was observed upon condensation of the core.

283

Figure 4 compares seismograms for the Survanta and DPPC microbubbles shown in Figure 3.

284

This analysis follows that by Gopal et al.40 for Langmuir films and by Kwan et al.48 for

285

microbubbles coated with long acyl-chain lipids. Here, we define a collapse event as a spike in

286

the cross-sectional area velocity (−∆A /∆). The cross-sectional area velocity of DPPC never

287

exceeded the threshold value of 15 µm2/s. Survanta shells, on the other hand, showed several

288

spikes in the seismograms, indicating buckling/collapse transitions. The left arrow denotes the

289

first collapse event for Survanta, while the right arrow marks the final collapse event before

290

condensation. The first collapse event occurred at a linear strain of 9 ± 2 % (mean ± standard

291

deviation), independent of pressurization rate. Interestingly, the seismogram did not exhibit a

292

trend for either collapse frequency or amplitude as a function of time (and therefore size).

293


15

Langmuir

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

Page 16 of 39

294 295

Figure 4. A representative seismogram for compression of Survanta (black) and DPPC (red)

296

shelled microbubbles at 0.5 kPa/s, corresponding to the area-time curves in Figure 3. The dashed

297

line indicates the area velocity threshold arbitrarily assigned to define a buckling event. The blue

298

arrows correspond to the first and last buckling events.

299 300

Figure 5A shows the collapse amplitude for Survanta microbubbles at each pressurization rate.

301

Collapse amplitude was calculated as the surface area difference between consecutive collapse

302

events (−∆ ) to indicate the amount of ejected material. Prior research suggests this material

303

remains associated to the microbubble surface as folds and vesicles.28 The average value of ∆

304

was 20.7 ± 3.1 µm2 (mean ± standard deviation) at all pressurization rates. The magnitude of

305

collapse was found to be independent of both microbubble size and compression rate.

306

Figure 5B shows the average collapse frequency at each pressurization rate for Survanta

307

microbubbles. To determine the collapse frequency (BA ), the total number of collapse events

308

exceeding the threshold was divided by the pressurization time, up to condensation. As might be

309

expected from the constant collapse amplitude, the frequency monotonically increased with

310

pressurization rate. The trend was approximately linear, which is unexpected for isothermal


16

Page 17 of 39

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

Langmuir

311

compression of an ideal gas. This result indicated that microbubble pressurization at these rates

312

is a complex process involving both compression and dissolution.

313

314 315

Figure 5. (A) The average surface area reduction between consecutive collapse events versus

316

pressurization rate for Survanta shelled microbubbles. The dashed line indicates the average

317

surface area loss. (B) Collapse frequency versus pressurization rate. The red line is a linear

318

regression trend-line (R2=0.98).

319 320

Pressure-Volume Behavior. To examine the relative degree of compression and dissolution,

321

we compared the experimental data to theoretical curves for each process, respectively (Figure

322

6). The experimental normalized volume, C/C, is shown for each shell at the slowest and

323

fastest pressurization rates (mean ± standard deviation). The upper curves in each plot were


17

Langmuir

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

Page 18 of 39

324

calculated from the ideal gas law assuming isothermal compression without dissolution of the

325

gas core and that the bubble pressure was equal to the hydrostatic pressure, = 4 .

326

Surprisingly, the experimental data for Survanta shells were above the isothermal compression

327

curves for the first ~50 s at the slowest rate and ~20 s at the fastest rate. This result cannot be

328

explained by gas diffusion into the bubble because the chemical potential gradient in this case

329

favors a net transfer of gas molecules in the opposite direction. Thus, the pressure inside the

330

gas core must have been less than the measured hydrostatic pressure for these microbubbles. A

331

pressure drop suggests that Survanta shells are capable of sustaining stress (i.e., negative surface

332

tension) under these conditions.

333

simulations, which showed that model lung surfactant membranes are capable of sustaining a

334

negative surface tension under compression.49

335

Eventually, the experimental normalized volume dropped below the isothermal compression

336

curve, indicating that gas dissolution is significant for both Survanta and DPPC microbubbles at

337

each pressurization rate.

This effect is supported by previous molecular dynamics

This effect was not observed for DPPC.


18

Page 19 of 39

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

Langmuir

338 339

Figure 6. Normalized volume-time curves. A) Survanta (left) and DPPC (right) microbubbles at

340

the slowest pressurization rate (n ≥ 20). B) Similar plots at the fastest pressurization rate (n ≥

341

20). The upper (red) curves were calculated assuming isothermal compression without

342

dissolution. The bottom (green) curves were calculated by use of equation (4) for pure

343

dissolution without shell resistance to gas permeation.

344 345

The lower curves in each plot of Figure 6 were calculated from equation (4) assuming that the

346

mass transfer coefficient is equal to that for a purely diffusing sphere with no shell resistance:

347

= D/, where D is the diffusivity of perfluorobutane in water (7×10-10 m2/s); and bubble

348

pressure was set equal to the hydrostatic value. The experimental data were always above these

349

“pure dissolution” curves, indicating that gas dissolution was hindered by the shell.

Two


19

Langmuir

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

Page 20 of 39

350

mechanisms may explain this result: First, the shell may have been able to sustain stress (as

351

indicated above), leading to a pressure drop across the shell that diminished the concentration

352

gradient. Second, the shell material may have impeded gas transport. These mechanisms are

353

investigated in detail below.

354

Model analysis. The results above suggest that Survanta and DPPC microbubbles compressed

355

and dissolved as the surrounding hydrostatic pressure was increased. We therefore modeled the

356

bubble response to pressurization using equations (3)-(8), which explicitly account for

357

dissolution. Unfortunately, the model failed to fit the experimental data accurately with the mass

358

transfer coefficient set to a constant value. To better fit the data, we introduced an empirical

359

relation for the mass transfer coefficient to account for the effect of convection. The relation

360

forced a linear increase in with increasing rate of change in the radius E8 E:

361

= + E8 E,

362

where is the mass transfer coefficient in the quasi-static case and is an empirical constant.

363

The quasi-static mass transfer coefficient is given by the equation:

364

1/ = /D + F4GHH ,

(9)

(10)

365

where D is the diffusivity of PFB in water and F4GHH is the shell resistance to gas permeation

366

(107 s/m).25 The empirical constant was set to 103.

367


20

Page 21 of 39

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

Langmuir

368 369

Figure 7. Model results from representative Survanta shelled microbubbles at (A) the slowest

370

pressurization rate and (B) the fastest pressurization rate. The first column shows the theoretical

371

volume of the bubble using E0 as the fitting parameter. The second column is the elasticity of

372

the shell during the pressurization experiment using the exponential elasticity model. The third

373

column compares the hydrostatic pressure to the apparent bubble pressure.

374 375

Figure 7 shows results from representative Survanta microbubbles at the slowest (top) and

376

fastest (bottom) pressurization rates. The calculated volume fits the experimental data well.

377

According to the model, the surface dilatational elasticity monotonically increased with time and

378

then plateaued prior to condensation (Fig. 7B). The elasticity values fall within the range of

379

experimentally reported surface dilatational elasticity values for DPPC of 0.03 – 2.5 N/m.35,50–53

380

The model also predicted an increasing pressure drop across the shell (negative surface tension)

381

that followed the increasing elasticity.


21

Langmuir

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

Page 22 of 39

382

Figure 8 shows the results of representative DPPC microbubbles. The general shape of the

383

elasticity-time curves were similar for DPPC and Survanta, but the pressure drop across the

384

DPPC shell was significantly less. At the slowest pressurization rate, the estimated pressure drop

385

was 40.4 ± 11.1 kPa (mean ± standard deviation) for Survanta and 21.8 ± 12.7 kPa for DPPC. At

386

the fastest rate, the pressure drop was 62.7 ± 9.9 kPa for Survanta and 27.3 ± 11.3 kPa for DPPC.

387

Mountford et al.47 reported condensation for PFB gas inside DPPC microbubbles at 25–75 kPa

388

greater than the equilibrium saturation curve across a wide temperature range.

389

observed condensation at similarly high hydrostatic pressures, providing confidence in the

390

theoretical model applied here to analyze compression and dissolution. Taken together, these

391

results suggest that Survanta shells can absorb more stress than their pure DPPC counterparts.

Here, we

392

On the other hand, Survanta shells did not significantly affect gas transport. At the end of

393

compression, prior to condensation, the model estimated that the fraction of gas remaining in the

394

bubble compared to that at the onset of pressurization (/ ) was 0.60 ± 0.027 and 0.61 ± 0.031

395

for Survanta and DPPC, respectively, at the slowest pressurization rate. At the fastest rate, the

396

ratio was 0.66 ± 0.030 and 0.66 ± 0.025 for Survanta and DPPC, respectively, indicating that

397

mass transport was nearly identical for the two shell compositions.

398


22

Page 23 of 39

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

Langmuir

399 400

Figure 8. Model results from representative DPPC shelled microbubbles at (A) the slowest

401

pressurization rate and (B) the fastest pressurization rate. The first column shows the theoretical

402

volume of the bubble using E0 as the fitting parameter. The second column is the elasticity of

403

the shell during the pressurization experiment using the exponential elasticity model. The third

404

column compares the hydrostatic pressure to the apparent bubble pressure.

405 406

Figure 9 shows a comparison of the initial dilatational elasticity values for both shell types.

407

Survanta shells gave statistically similar values to DPPC at the three slowest rages. At the

408

three fastest pressurization rates, however, the value of for Survanta was significantly larger

409

than for DPPC (p < 0.05). This difference can be explained by the multi-component composition

410

of Survanta, which contains both saturated and unsaturated lipids as well as hydrophobic

411

surfactant proteins SP-B and SP-C. This composition provides microstructure that enables the

412

shell to better sustain in-plane stress.42 Additionally, Survanta microbubbles have been shown to

413

form surface associated folds after a compression event, and lung surfactant at the alveolar


23

Langmuir

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

Page 24 of 39

414

air/water interface is believed to form lipid reservoirs in the hypophase connected to the film

415

surface as multilamellar lipid stacks. Patashinski et al.54 showed that a monolayer must form a

416

multilayered structure to sustain negative surface tension. Survanta is uniquely capable of

417

forming and sustaining the necessary multilayers because of the presence of the hydrophobic

418

proteins SP-B and SP-C. We therefore speculate that the increased for Survanta at higher

419

compression rates resulted from both in-plane monolayer rigidity and lateral repulsion between

420

surface-associated structures.

421

422 423

Figure 9. The initial dilatational elasticity, E0, for the two shell types versus pressurization rate

424

(mean I standard error, n ≥ 20). The black dashed line indicates the average elasticity value for

425

DPPC shells. The red line is a sigmoidal fit of the Survanta data, and is shown to help guide the

426

eye. The difference of the means between DPPC and Survanta at the three fastest pressurization

427

rates is significant (p < 0.05).

428 429

Conclusion


24

Page 25 of 39

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

430

Langmuir

In this study, we examine LS mechanics by the use of individual microbubbles subjected

431

to linear pressurization at room temperature.

432

remained

433

wrinkling/smoothing cycles indicative of monolayer buckling and collapse. Pressure-volume

434

results suggested that Survanta shells support a negative surface tension, and that both Survanta

435

and DPPC microbubbles dissolve as they are pressurized. We employed an empirical model to

436

account for the simultaneous effects of isothermal compression and gas dissolution, in order to

437

better analyze the effects of surfactant film mechanics.

438

dilatational elasticity of Survanta shells diverges from that of DPPC shells as strain rate

439

increases. We speculate that the higher elasticity of Survanta shells is a consequence of in-plane

440

monolayer rigidity and interactions between surface-associated folds and vesicles. Overall, this

441

technique reveals new physical insights into surfactant film mechanics and collapse behavior

442

during compression.

443

Acknowledgements. This work was funded by NSF grant DMR-1409972 and NIH grant R21

444

RHL129144.

spherical

during

pressurization,

We observed that pure DPPC microbubbles while

Survanta

microbubbles

exhibited

Analysis showed that the initial

445 446

Supporting Information. The Supporting Information is available free of charge on the ACS

447

Publications website: (i) normalized volume-time pressurization curve for an air-filled

448

microbubble, (ii) PFB microbubble condensation pressures, (iii) onset pressures for the first

449

Survanta buckling-collapse event, (iv) histograms for initial elasticity , (v) normalized

450

volume-time curves for Survanta and DPPC, and (vi) curve-fitting statistics for the model fit to

451

experimental data.

452


25

Langmuir

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

453

References

454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496

(1) (2) (3) (4) (5) (6)

(7)

(8)

(9)

(10)

(11) (12)

(13)

(14) (15) (16)

(17)

Page 26 of 39

Scarpelli, E. M. Physiology of the Alveolar Surface Network. Comp. Biochem. Physiol. A. Mol. Integr. Physiol. 2003, 135 (1), 39–104. Tanaka, Y.; Takei, T.; Aiba, T.; Masuda, K.; Kiuchi, A.; Fujiwara, T. Development of Synthetic Lung Surfactants. J. Lipid Res. 1986, 27 (5), 475–485. Goerke, J.; Clements, J. A. Alveolar Surface Tension and Lung Surfactant. In Comprehensive Physiology; John Wiley & Sons, Inc., 2011. Avery, M. E.; Mead, J. Surface Properties in Relation to Atelectasis and Hyaline Membrane Disease. AMA J. Dis. Child. 1959, 97 (5_PART_I), 517–523. Zambon, M.; Vincent, J.-L. Mortality Rates for Patients With Acute Lung Injury/ARDS Have Decreased Over Time. Chest 2008, 133 (5), 1120–1127. Fujiwara, T.; Chida, S.; Watabe, Y.; Maeta, H.; Morita, T.; Abe, T. ARTIFICIAL SURFACTANT THERAPY IN HYALINE-MEMBRANE DISEASE. The Lancet 1980, 315 (8159), 55–59. Ainsworth, S. B.; Beresford, M. W.; Milligan, D. W.; Shaw, N. J.; Matthews, J. N.; Fenton, A. C.; Ward Platt, M. P. Pumactant and Poractant Alfa for Treatment of Respiratory Distress Syndrome in Neonates Born at 25-29 Weeks’ Gestation: A Randomised Trial. Lancet Lond. Engl. 2000, 355 (9213), 1387–1392. Notter, R. H.; Wang, Z.; Egan, E. A.; Holm, B. A. Component-Specific Surface and Physiological Activity in Bovine-Derived Lung Surfactants. Chem. Phys. Lipids 2002, 114 (1), 21–34. Bernhard, W.; Mottaghian, J.; Gebert, A.; Rau, G. A.; von der HARDT, H.; Poets, C. F. Commercial versus Native Surfactants. Am. J. Respir. Crit. Care Med. 2000, 162 (4), 1524–1533. Ochs, M.; Nyengaard, J. R.; Jung, A.; Knudsen, L.; Voigt, M.; Wahlers, T.; Richter, J.; Gundersen, H. J. G. The Number of Alveoli in the Human Lung. Am. J. Respir. Crit. Care Med. 2004, 169 (1), 120–124. Roan, E.; Waters, C. M. What Do We Know about Mechanical Strain in Lung Alveoli? Am. J. Physiol. - Lung Cell. Mol. Physiol. 2011, 301 (5), L625–L635. Gatto, L. A.; Fluck, R. R.; Nieman, G. F. Alveolar Mechanics in the Acutely Injured Lung: Role of Alveolar Instability in the Pathogenesis of Ventilator-Induced Lung Injury. Respir. Care 2004, 49 (9), 1045–1055. Chang, S.; Kwon, N.; Kim, J.; Kohmura, Y.; Ishikawa, T.; Rhee, C. K.; Je, J. H.; Tsuda, A. Synchrotron X-Ray Imaging of Pulmonary Alveoli in Respiration in Live Intact Mice. Sci. Rep. 2015, 5, 8760. Lee, K. Y. C. Collapse Mechanisms of Langmuir Monolayers. Annu. Rev. Phys. Chem. 2008, 59 (1), 771–791. Rugonyi, S.; Biswas, S. C.; Hall, S. B. The Biophysical Function of Pulmonary Surfactant. Respir. Physiol. Neurobiol. 2008, 163 (1–3), 244–255. Parra, E.; Pérez-Gil, J. Composition, Structure and Mechanical Properties Define Performance of Pulmonary Surfactant Membranes and Films. Chem. Phys. Lipids 2015, 185, 153–175. Gopal, A.; Lee, K. Y. C. Morphology and Collapse Transitions in Binary Phospholipid Monolayers. J. Phys. Chem. B 2001, 105 (42), 10348–10354.


26

Page 27 of 39

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

497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540

Langmuir

(18) Gopal, A.; Lee, K. Y. C. Headgroup Percolation and Collapse of Condensed Langmuir Monolayers. J. Phys. Chem. B 2006, 110 (44), 22079–22087. (19) Zhang, H.; Wang, Y. E.; Fan, Q.; Zuo, Y. Y. On the Low Surface Tension of Lung Surfactant. Langmuir 2011, 27 (13), 8351–8358. (20) Witten, T. A.; Wang, J.; Pocivavsek, L.; Lee, K. Y. C. Wilhelmy Plate Artifacts in Elastic Monolayers. J. Chem. Phys. 2010, 132 (4), 046102. (21) Crane, J. M.; Hall, S. B. Rapid Compression Transforms Interfacial Monolayers of Pulmonary Surfactant. Biophys. J. 2001, 80 (4), 1863–1872. (22) Zuo, Y. Y.; Acosta, E.; Policova, Z.; Cox, P. N.; Hair, M. L.; Neumann, A. W. Effect of Humidity on the Stability of Lung Surfactant Films Adsorbed at Air–water Interfaces. Biochim. Biophys. Acta BBA - Biomembr. 2006, 1758 (10), 1609–1620. (23) Unger, E. C.; Porter, T.; Culp, W.; Labell, R.; Matsunaga, T.; Zutshi, R. Therapeutic Applications of Lipid-Coated Microbubbles. Adv. Drug Deliv. Rev. 2004, 56 (9), 1291– 1314. (24) Feinstein, S. B. The Powerful Microbubble: From Bench to Bedside, from Intravascular Indicator to Therapeutic Delivery System, and Beyond. Am. J. Physiol. Heart Circ. Physiol. 2004, 287 (2), H450-457. (25) Ferrara, K.; Pollard, R.; Borden, M. Ultrasound Microbubble Contrast Agents: Fundamentals and Application to Gene and Drug Delivery. Annu. Rev. Biomed. Eng. 2007, 9 (1), 415–447. (26) Sirsi, S.; Pae, C.; Oh, D. K. T.; Blomback, H.; Koubaa, A.; Papahadjopoulos-Sternberg, B.; Borden, M. Lung Surfactant Microbubbles. Soft Matter 2009, 5 (23), 4835–4842. (27) Garg, S.; Thomas, A. A.; Borden, M. A. The Effect of Lipid Monolayer In-Plane Rigidity on in Vivo Microbubble Circulation Persistence. Biomaterials 2013, 34 (28), 6862–6870. (28) Sirsi, S. R.; Fung, C.; Garg, S.; Tianning, M. Y.; Mountford, P. A.; Borden, M. A. Lung Surfactant Microbubbles Increase Lipophilic Drug Payload for Ultrasound-Targeted Delivery. Theranostics 2013, 3 (6), 409–419. (29) Borden, M. A. Microbubble Dispersions of Natural Lung Surfactant. Curr. Opin. Colloid Interface Sci. 2014, 19 (5), 480–489. (30) Pattle, R. E. Properties, Function and Origin of the Alveolar Lining Layer. Nature 1955, 175 (4469), 1125–1126. (31) Discher, B. M.; Schief, W. R.; Vogel, V.; Hall, S. B. Phase Separation in Monolayers of Pulmonary Surfactant Phospholipids at the Air-Water Interface: Composition and Structure. Biophys. J. 1999, 77 (4), 2051–2061. (32) Marmottant, P.; Meer, S. van der; Emmer, M.; Versluis, M.; Jong, N. de; Hilgenfeldt, S.; Lohse, D. A Model for Large Amplitude Oscillations of Coated Bubbles Accounting for Buckling and Rupture. J. Acoust. Soc. Am. 2005, 118 (6), 3499–3505. (33) Sarkar, K.; Katiyar, A.; Jain, P. Growth and Dissolution of an Encapsulated Contrast Microbubble: Effects of Encapsulation Permeability. Ultrasound Med. Biol. 2009, 35 (8), 1385–1396. (34) Paul, S.; Katiyar, A.; Sarkar, K.; Chatterjee, D.; Shi, W. T.; Forsberg, F. Material Characterization of the Encapsulation of an Ultrasound Contrast Microbubble and Its Subharmonic Response: Strain-Softening Interfacial Elasticity Model. J. Acoust. Soc. Am. 2010, 127 (6), 3846–3857.


27

Langmuir

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

541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585

Page 28 of 39

(35) Sarkar, K.; Shi, W. T.; Chatterjee, D.; Forsberg, F. Characterization of Ultrasound Contrast Microbubbles Using in Vitro Experiments and Viscous and Viscoelastic Interface Models for Encapsulation. J. Acoust. Soc. Am. 2005, 118 (1), 539–550. (36) Kim, K.; Q. Choi, S.; A. Zasadzinski, J.; M. Squires, T. Interfacial Microrheology of DPPC Monolayers at the Air–water Interface. Soft Matter 2011, 7 (17), 7782–7789. (37) Kim, D. H.; Costello, M. J.; Duncan, P. B.; Needham, D. Mechanical Properties and Microstructure of Polycrystalline Phospholipid Monolayer Shells: Novel Solid Microparticles. Langmuir 2003, 19 (20), 8455–8466. (38) Duncan, P. B.; Needham, D. Test of the Epstein-Plesset Model for Gas Microparticle Dissolution in Aqueous Media: Effect of Surface Tension and Gas Undersaturation in Solution. Langmuir ACS J. Surf. Colloids 2004, 20 (7), 2567–2578. (39) Rüdiger, M.; Tölle, A.; Meier, W.; Rüstow, B. Naturally Derived Commercial Surfactants Differ in Composition of Surfactant Lipids and in Surface Viscosity. Am. J. Physiol. Lung Cell. Mol. Physiol. 2005, 288 (2), L379–L383. (40) Gopal, A.; Belyi, V. A.; Diamant, H.; Witten, T. A.; Lee, K. Y. C. Microscopic Folds and Macroscopic Jerks in Compressed Lipid Monolayers. J. Phys. Chem. B 2006, 110 (21), 10220–10223. (41) Stenger, P. C.; Isbell, S. G.; Zasadzinski, J. A. Molecular Weight Dependence of the Depletion Attraction and Its Effects on the Competitive Adsorption of Lung Surfactant. Biochim. Biophys. Acta BBA - Biomembr. 2008, 1778 (10), 2032–2040. (42) Pocivavsek, L.; Frey, S. L.; Krishan, K.; Gavrilov, K.; Ruchala, P.; Waring, A. J.; Walther, F. J.; Dennin, M.; Witten, T. A.; Lee, K. Y. C. Lateral Stress Relaxation and Collapse in Lipid Monolayers. Soft Matter 2008, 4, 2019–2029. (43) Epstein, P. S.; Plesset, M. S. On the Stability of Gas Bubbles in Liquid-Gas Solutions. J. Chem. Phys. 1950, 18 (11), 1505. (44) Pattle, R. E. Properties, Function, and Origin of the Alveolar Lining Layer. Proc. R. Soc. Lond. B Biol. Sci. 1958, 148 (931), 217–240. (45) Schurch, S.; Bachofen, H.; Goerke, J.; Possmayer, F. A Captive Bubble Method Reproduces the in Situ Behavior of Lung Surfactant Monolayers. J. Appl. Physiol. 1989, 67 (6), 2389–2396. (46) Pocivavsek, L.; Dellsy, R.; Kern, A.; Johnson, S.; Lin, B.; Lee, K. Y. C.; Cerda, E. Stress and Fold Localization in Thin Elastic Membranes. Science 2008, 320 (5878), 912–916. (47) Mountford, P. A.; Sirsi, S. R.; Borden, M. A. Condensation Phase Diagrams for LipidCoated Perfluorobutane Microbubbles. Langmuir 2014, 30 (21), 6209–6218. (48) Kwan, J. J.; Borden, M. A. Lipid Monolayer Collapse and Microbubble Stability. Adv. Colloid Interface Sci. 2012, 183–184, 82–99. (49) Baoukina, S.; Monticelli, L.; Amrein, M.; Tieleman, D. P. The Molecular Mechanism of Monolayer-Bilayer Transformations of Lung Surfactant from Molecular Dynamics Simulations. Biophys. J. 2007, 93 (11), 3775–3782. (50) Arriaga, L. R.; López-Montero, I.; Ignés-Mullol, J.; Monroy, F. Domain-Growth Kinetic Origin of Nonhorizontal Phase Coexistence Plateaux in Langmuir Monolayers: Compression Rigidity of a Raft-Like Lipid Distribution. J. Phys. Chem. B 2010, 114 (13), 4509–4520. (51) Anton, N.; Pierrat, P.; Lebeau, L.; Vandamme, T. F.; Bouriat, P. A Study of Insoluble Monolayers by Deposition at a Bubble Interface. Soft Matter 2013, 9 (42), 10081–10091.


28

Page 29 of 39

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

586 587 588 589 590 591 592 593 594

Langmuir

(52) Kotula, A. P.; Anna, S. L. Insoluble Layer Deposition and Dilatational Rheology at a Microscale Spherical Cap Interface. Soft Matter 2016, 12 (33), 7038–7055. (53) Lum, J. S.; Dove, J. D.; Murray, T. W.; Borden, M. A. Single Microbubble Measurements of Lipid Monolayer Viscoelastic Properties for Small-Amplitude Oscillations. Langmuir 2016, 32 (37), 9410–9417. (54) Patashinski, A. Z.; Orlik, R.; Paclawski, K.; Ratner, M. A.; Grzybowski, B. A. The Unstable and Expanding Interface between Reacting Liquids: Theoretical Interpretation of Negative Surface Tension. Soft Matter 2012, 8 (5), 1601–1608.


29

Langmuir

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

Figure 1. Illustration of the chamber design. The chamber pressure was controlled by a syringe pump (Harvard Apparatus, Holliston, MA) programmed to deliver a calibrated volumetric flow rate. A metal syringe (KD Scientific, Holliston, MA) filled with PFB-saturated PBS was used to ensure uniform pressurization during the entire experiment. 66x43mm (300 x 300 DPI)


Page 30 of 39

Page 31 of 39

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

Langmuir

Figure 2. Images acquired for Survanta (A) and DPPC (B) microbubbles during pressurization at 0.5 kPa/s (top row of each panel) and 2.3 kPa/s (bottom row of each panel). The scale bar is 10 µm. 188x174mm (300 x 300 DPI)


Langmuir

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

Figure 3. Cross-sectional areas of typical Survanta (black curve) and DPPC (red curve) microbubbles, along with the chamber pressure (blue curve). (A) Pressurization rate of 0.5 kPa/s and (B) pressurization rate of 2.3 kPa/s. The arrows indicate the first and last buckling events for the Survanta microbubbles. 137x185mm (300 x 300 DPI)


Page 32 of 39

Page 33 of 39

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

Langmuir

Figure 4. A representative seismogram for compression of Survanta (black) and DPPC (red) shelled microbubbles at 0.5 kPa/s, corresponding to the area-time curves in Figure 3. The dashed line indicates the area velocity threshold arbitrarily assigned to define a buckling event. The blue arrows correspond to the first and last buckling events. 77x59mm (300 x 300 DPI)


Langmuir

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

Figure 5. (A) The average surface area reduction between consecutive collapse events versus pressurization rate for Survanta shelled microbubbles. The dashed line indicates the average surface area loss. (B) Collapse frequency versus pressurization rate. The red line is a linear regression trend-line (R¬2=0.98). 138x188mm (300 x 300 DPI)


Page 34 of 39

Page 35 of 39

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

Langmuir

Figure 6. Normalized volume-time curves. A) Survanta (left) and DPPC (right) microbubbles at the slowest pressurization rate (n ≥ 20). B) Similar plots at the fastest pressurization rate (n ≥ 20). The upper (red) curves were calculated assuming isothermal compression without dissolution. The bottom (green) curves were calculated by use of equation (4) for pure dissolution without shell resistance to gas permeation. 158x123mm (300 x 300 DPI)


Langmuir

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

Figure 7. Model results from representative Survanta shelled microbubbles at (A) the slowest pressurization rate and (B) the fastest pressurization rate. The first column shows the theoretical volume of the bubble using E0 as the fitting parameter. The second column is the elasticity of the shell during the pressurization experiment using the exponential elasticity model. The third column compares the hydrostatic pressure to the apparent bubble pressure. 110x59mm (300 x 300 DPI)


Page 36 of 39

Page 37 of 39

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

Langmuir

Figure 8. Model results from representative DPPC shelled microbubbles at (A) the slowest pressurization rate and (B) the fastest pressurization rate. The first column shows the theoretical volume of the bubble using E0 as the fitting parameter. The second column is the elasticity of the shell during the pressurization experiment using the exponential elasticity model. The third column compares the hydrostatic pressure to the apparent bubble pressure. 110x60mm (300 x 300 DPI)


Langmuir

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

Figure 9. The initial dilatational elasticity, E0, for the two shell types versus pressurization rate (mean ± standard error, n ≥ 20). The black dashed line indicates the average elasticity value for DPPC shells. The red line is a sigmoidal fit of the Survanta data, and is shown to help guide the eye. The difference of the means between DPPC and Survanta at the three fastest pressurization rates is significant (p < 0.05). 71x49mm (300 x 300 DPI)


Page 38 of 39

Page 39 of 39

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

Langmuir

TOC figure 41x22mm (300 x 300 DPI)


Hydrostatic Pressurization of Lung Surfactant Microbubbles

Recommend Documents