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Article

Atomic Force Microscopy Imaging of AdsorbedPulmonary Surfactant Films

Lu Xu,1 Yi Yang,1 and Yi Y. Zuo1,2,*1Department of Mechanical Engineering, University of Hawaii at Manoa, Honolulu, Hawaii and 2Department of Pediatrics, John A. BurnsSchool of Medicine, University of Hawaii at Manoa, Honolulu, Hawaii

ABSTRACT Pulmonary surfactant (PS) is a lipid-protein complex that adsorbs to the air-water surface of the lung as a thin film.Previous studies have suggested that the adsorbed PS film is composed of an interfacial monolayer, plus a functionally attachedvesicular complex, called the surface-associated surfactant reservoir. However, direct visualization of the lateral structure andmorphology of adsorbed PS films using atomic force microscopy (AFM) has been proven to be technically challenging. To date,all AFM studies of the PS film have relied on the model of Langmuir monolayers. Here, we showed the first, to our knowledge,AFM imaging of adsorbed PS films under physiologically relevant conditions using a novel, to our knowledge, experimentalmethodology called constrained drop surfactometry. In conjunction with a series of methodological innovations, including sub-phase replacement, in situ Langmuir-Blodgett transfer, and real-time surface tension control using closed-loop axisymmetricdrop shape analysis, constrained drop surfactometry allowed the study of lateral structure and topography of animal-derived nat-ural PS films at physiologically relevant low surface tensions. Our data suggested that a nucleation-growth model is responsiblefor the adsorption-induced squeeze-out of the PS film, which likely results in an interfacial monolayer enriched in dipalmitoyl-phosphatidylcholine with the attached multilayered surface-associated surfactant reservoir. These findings were further sup-ported by frequency-dependent measurements of surface dilational rheology. Our study provides novel, to our knowledge,biophysical insights into the understanding of the mechanisms by which the PS film attains low surface tensions and stabilizesthe alveolar surface.

SIGNIFICANCE A pulmonary surfactant (PS) is a lipid-protein mixture that coats the air-water surface of the lung byadsorption. Although it is generally accepted that the adsorbed PS film at the air-water surface forms multilayers, it istechnically challenging to visualize the lateral structure and topography of the adsorbed PS film. We have developed anovel, to our knowledge, experimental methodology called constrained drop surfactometry. In conjunction with atomicforce microscopy, we studied the lateral structure, topography, and interfacial rheology of animal-derived natural PS filmsat physiologically relevant low surface tensions. Our data suggested an adsorption-induced squeeze-out, which providesnovel, to our knowledge, biophysical insights into understanding the mechanism by which the PS film attains low surfacetensions and stabilizes the alveolar surface.

INTRODUCTION

A pulmonary surfactant (PS) is a lipid-protein complex thatcovers the entire air-water surface of the lung as a thin film(1,2). It consists of �80% phospholipids, 5–10% neutrallipids (mainly cholesterol), and 5–10% surfactant-associ-ated proteins (SPs) by weight. The main biophysical func-tion of the PS film is to lower the alveolar surface tensionto near zero to protect alveoli against collapse, thus main-

Submitted March 25, 2020, and accepted for publication June 26, 2020.

*Correspondence: [email protected]

Editor: Roland Winter.

756 Biophysical Journal 119, 756–766, August 18, 2020

https://doi.org/10.1016/j.bpj.2020.06.033

� 2020 Biophysical Society.

taining a large surface area of the lung for gas exchange(3,4). In addition to surface tension reduction, the PS filmalso acts as the first-line host defense against inhaled parti-cles and pathogens (5,6).

The innate PS film is formed at the alveolar surface viaadsorption from surfactant vesicles synthesized by the alve-olar type II cells (7). Electron microscopy observations havedemonstrated that the innate PS film consists of multilayersof phospholipids (7,8). Using captive bubble surfactometry,Sch€urch et al. have convincingly demonstrated that ad-sorbed PS films contain considerably more highly surfaceactive material than can be accounted for by a single mono-layer, indicating such films must adopt a multilayered

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FIGURE 1 Schematic of the (a) spread film and (b) adsorbed film. The

spread film, often known as the Langmuir film, consists of insoluble surfac-

tant molecules, which are usually formed by spreading, with the aid of an

organic solvent, to the air-water surface via a microsyringe. After

spreading, the Langmuir film usually assumes a monolayer conformation.

The adsorbed film, often known as the Gibbs film, usually consists of wa-

ter-soluble surfactant molecules, which are usually formed by molecular

adsorption from the aqueous subphase to the air-water surface. Although

largely composed of insoluble lipid molecules, the natural pulmonary sur-

factant (PS) film is formed by adsorption of lipid vesicles to the air-water

surface. After adsorption, the PS film assumes a multilayer conformation

consisting of an interfacial monolayer plus a surface-associated surfactant

reservoir (SASR) that is functionally attached to the interfacial monolayer.

To see this figure in color, go online.

Imaging Adsorbed Lung Surfactant Films

conformation (9). Many subsequent investigations showedthat the adsorbed PS film is composed of an interfacialmonolayer at the air-water surface plus a functionallyattached vesicular complex (10–13), i.e., the so-called sur-face-associated surfactant reservoir (SASR) (9). Furtherresearch suggested that the SASR is most likely stabilizedby hydrophobic surfactant proteins (SP-B and SP-C)(14,15). The combined interfacial monolayer and SASRform a dynamically stable complex that favors interex-change of surface active materials between them duringthe highly dynamic inhalation and exhalation cycles ofnormal tidal breathing (16,17). The importance of theSASR in maintaining the normal biophysical function ofthe PS film was demonstrated in recent studies (18–20). Itwas found that impaired multilayer structure or the SASRled to deteriorated in vitro biophysical properties of thePS film and induced symptoms of acute respiratory failurein animal models with nanoparticle inhalation or intratra-cheal instillation (18–20).

Despite its success in visualizing the conformation of thePS film, electron microscopy suffers from a few limitations,such as complications in sample preparation (e.g., require-ments of fixation, staining, and vacuum) and a lack ofthree-dimensional topographic information, which weakenits popularity in studying monolayers and biomembranes.Atomic force microscopy (AFM), on the other hand, hasbeen proven to be an ideal method for visualizing the lateralstructure and topography of the PS film (21–25). AFM hashelped obtain valuable biophysical knowledge about thePS film, such as phospholipid phase separation and transi-tions, lipid-protein interactions, nano-bio interactions, andthe collapse mechanisms of the PS film (1,26).

However, to date, almost all AFM studies of the PS filmrely on monolayers spread at the air-water surface withthe aid of organic solvents. As illustrated in Fig. 1, such aspread film, often known as a Langmuir film, is fundamen-tally different from an adsorbed film, often known as aGibbs film. This difficulty is mainly inherent to the use ofthe classical Langmuir trough, which has intrinsic technicallimitations for studying adsorbed PS films (1,2), such as alow surface area/volume ratio that prevents rapid adsorptionof the PSs, and technical difficulties of performing Lang-muir-Blodgett (LB) transfer of the adsorbed PS film becauseLB transfer in general requires a clean liquid subphase.Otherwise, the solid substrate for LB transfer would becontaminated by surfactant vesicles in the subphase beforethe transfer. To the best of our knowledge, all existingAFM imaging of the PS films was performed on spreadLangmuir films rather than on adsorbed PS films.

Here, we present the first, to our knowledge, AFM imag-ing of adsorbed PS films under physiologically relevant con-ditions using a novel experimental methodology developedin our laboratory, called constrained drop surfactometry(CDS). CDS is a new generation of droplet-based surfacetensiometry capable of high-fidelity biophysical simulations

of the PS film under physiologically relevant conditions(18–20). We have developed a novel subphase replacementtechnique that allows washing and replacing the vesicularsubphase of the PS suspension without disturbing the ad-sorbed interfacial monolayer and SASR. Together with anin situ LB transfer technique integrated with the CDS, thesubphase replacement facilities AFM imaging of de novoadsorbed PS films at physiologically relevant low surfacetensions, i.e., 5–20 mN/m. With these technical advances,we have studied two animal-derived clinical PSs, Infasurfand Curosurf. Differences were found for the lateral struc-ture and topography of these two adsorbed PS films. Suchdifferences in the film structure were further related to theinterfacial rheology of these PS films, which was also deter-mined with the CDS. Our study provides novel biophysicalinsights into the understanding of natural PSs and the

Biophysical Journal 119, 756–766, August 18, 2020 757

Xu et al.

mechanisms by which the PS films attain low surface ten-sions upon compression.

MATERIALS AND METHODS

PSs

Infasurf was a gift from ONY Biotech (Amherst, NY). It is prepared from

lung lavage of newborn calves with centrifugation and organic extraction.

Infasurf contains all of the hydrophobic components of natural bovine sur-

factant, whereas hydrophilic surfactant proteins (SP-A and SP-D) were

removed during the extraction process. Curosurf was donated by Corner-

stone Therapeutics (Cary, NC). It is a modified animal PSs prepared from

minced porcine lung tissue. In addition to organic extraction and centrifu-

gation, an additional procedure for removing all neutral lipids by gel chro-

FIGURE 2 Schematic of the constrained drop surfactometry (CDS). CDS uses

droplet is constrained on a knife-sharp pedestal and is enclosed in an environmen

The surface tension and surface area of the PS film are determined simultaneous

implemented with a coaxial pedestal connected to two motorized syringes, wi

another one simultaneously injecting buffer into the droplet with the same volu

washing and removal of lipid vesicles from the droplet without disturbing the ad

surface to a freshly peeled mica substrate for atomic force microscopy (AFM)


matography is involved in the manufacture of Curosurf. Both surfactants

were stored at �20�C in sterilized vials with a total phospholipid concen-

tration of 35 mg/mL for Infasurf and 76 mg/mL for Curosurf. They were

diluted by a saline buffer (pH 7.0; 0.9% NaCl, 1.5 mM CaCl2, and 2.5

mN HEPES) to a final concentration of 1 mg/mL on the day of the

experiment.

CDS

CDS is a new, to our knowledge, generation of droplet-based tensiometry

technique developed in our laboratory (18–20). As illustrated in Fig. 2,

CDS uses the air-water surface of a sessile droplet (�30 mL in volume,

�5 mm in diameter, and �0.4 cm2 in surface area) to accommodate the ad-

sorbed PS film. The surfactant droplet is constrained on a carefully

machined pedestal using its knife-sharp edge to prevent film leakage

even at low surface tensions. The adsorbed surfactant film can be

the air-water surface of a droplet to accommodate the adsorbed PS film. The

tal control chamber that enables simulation of the physiological conditions.

ly from the shape of the droplet using CL-ADSA. Subphase replacement is

th one withdrawing the vesicle-containing subphase from the droplet and

metric rate. Consequently, the subphase replacement technique facilitates

sorbed PS film. The PS film is subsequently LB transferred from the droplet

imaging. To see this figure in color, go online.


compressed and expanded periodically at physiologically relevant rates and

compression ratios by controlling liquid flow out of and into the droplet us-

ing a motorized syringe. The surface tension and surface area of the PS film

are determined simultaneously using newly developed closed-loop axisym-

metric drop shape analysis (CL-ADSA) (27). Owing to system miniaturiza-

tion, CDS enables high-fidelity simulation of physiological conditions, i.e.,

the core body temperature of 37�C and a relative humidity close to 100%,

using an environmental control chamber.

Specifically, a 30-mL or 1 mg/mL Infasurf or Curosurf droplet was

dispensed on a 5-mmCDS pedestal via a pipette. Immediately after forming

the droplet, the surface tension was continuously recorded and found to be

quickly (within seconds) reduced to an equilibrium value around 22–

25 mN/m, indicating the rapid formation of the adsorbed PS film at the

air-water surface of the droplet (1). To mimic respiration, the adsorbed

PS film was compressed and expanded at 20 cycles per minute, correspond-

ing to a compression rate of 13.3% of the area per second.

Subphase replacement and LB transfer

To transfer the adsorbed PS film for AFM imaging, as shown in Fig. 2, the

subphase replacement was implemented using a coaxial CDS pedestal con-

nected with two motorized syringes, with one withdrawing the phospho-

lipid-vesicle-containing subphase from the droplet at a rate of 1 mL/s and

the other one simultaneously injecting buffer into the droplet at the same

rate. Consequently, phospholipid vesicles in the aqueous subphase (i.e.,

the droplet) were washed away without disturbing the adsorbed PS film

at the air-water surface. After the subphase replacement, LB transfer of

the adsorbed PS film was performed by first quickly inserting a freshly

peeled mica sheet into the droplet followed by slowly lifting the mica across

the air-water interface of the droplet at a rate of 1 mm/min. During the LB

transfer process, the PS film was maintained at a constant surface pressure

(51.5 mN/m) for a prolonged period using CL-ADSA (27,28). All mea-

surements were conducted at 37�C for at least three times. The deposition

ratio of the LB transfer, defined as the ratio between the lost area of the sur-

factant film during the LB transfer and the total surface area of the mica

sheet (29), was estimated to be 1.24 5 0.18, which indicates a complete

transfer of the surfactant film from the air-water surface to the mica surface.

AFM

The lateral structure and topography of the adsorbed PS films were imaged

using an Innova AFM (Bruker, Santa Barbara, CA). Samples were scanned

in the air using the tapping mode with a silicon cantilever of the spring con-

stant 42 N/m and a resonance frequency of 300 kHz. Images were taken at

multiple locations to ensure the reproducibility. Lateral structures and

topography of the samples were analyzed using NanoScope Analysis

(version 1.5).

Surface dilational rheology

Detailed experimental protocols for determining the surface dilational

modulus E ¼ ðdg =dlnAÞ of the adsorbed PS film using CDS can be found

elsewhere (30,31). Briefly, the surface area of the de novo adsorbed PS film

at equilibrium surface tension (ge) after subphase replacement was oscil-

lated in a sinusoidal waveform, with frequencies of 0.01, 0.1, and 1 Hz

and an amplitude of 10% of the initial surface area, using CL-ADSA.

The surface tension response to the surface area oscillation was recorded

as the output and was compared against the surface area oscillation wave-

form as the input. The elastic (Er) and viscous (Ei) components of the sur-

face dilational modulus were determined from the phase shaft (4) between

the input and output waveforms and from the oscillation amplitudes of the

surface area and the surface tension. The loss tangent angle (tan4) was

calculated as the ratio between the viscous modulus and the elastic modulus

(Ei/Er). Spread dipalmitoylphosphatidylcholine (DPPC) monolayers were

also studied as a reference. All measurements were carried out at 37�Cfor at least three times.

RESULTS

Subphase replacement of a dye: proof offeasibility

We first demonstrate the feasibility of the subphase replace-ment technique integrated into the CDS. Fig. S1 shows thewashing and replacement of 1 mg/mL royal blue dye froma 45 mL droplet by simultaneously withdrawing the dye so-lution and replacing it with an equal amount of pure water atthe rate of 1 mL/s. It can be seen that the blue dye in thedroplet was washed away with pure water fourfold (4�)of its original volume (i.e., a total of 180 mL) after 180 s.During the entire subphase replacement process, the surfacetension, surface area, and volume of the droplet remainedconstant. A video clip of the subphase replacement processcan be found in the Supporting Materials and Methods.

Subphase replacement of Infasurf

We then performed the subphase replacement on a 30-mLdroplet of 1 mg/mL Infasurf, using one-, two-, three-,four-, seven-, and 10-fold replacement volumes, respec-tively. As shown in Fig. 3, surface tension, surface area,and volume of the Infasurf droplet remained constant forall subphase replacement volumes, indicating the integrityof the interfacial surfactant monolayer at the air-water sur-face. However, as shown in Fig. 4, dynamic surface activityof Infasurf after 10-fold subphase replacement shows signif-icant deterioration in comparison with the de novo adsorbedInfasurf film. The dynamic surface activity was determinedat physiologically relevant conditions, i.e., 37�C, 100% rela-tive humidity, and less than 30% compression ratio at a fre-quency of 20 cycles/min to mimic the exhalation andinhalation cycles during normal tidal breathing. These re-sults, therefore, indicate that the excessive washing at 10-fold volume may impair the SASR, although not the integ-rity of the interfacial monolayer. At all other replacing vol-umes from onefold to up to sevenfold, the Infasurf film afterthe subphase replacement maintains a similar surface activ-ity as that of the de novo adsorbed Infasurf film, indicating arelatively intact SASR. Reproducibility of Figs. 3 and 4 canbe found in Figs. S2 and S3.

Optimization of the subphase replacementtechnique for adsorbed Infasurf film

Fig. 5 shows the lateral structure and topography of the In-fasurf film after subphase replacement with onefold to 10-fold replacing volumes. Reproducibility of these AFM im-ages can be found in Figs. S4–S9. Fig. 5 also shows the


FIGURE 3 Subphase replacement of a 30-mL,

1 mg/mL Infasurf droplet with various replacing

volumes from onefold (1�) to 10-fold (10�) of

the original droplet volume at a rate of 1 mL/s. It

can be seen that during the subphase replacement

process, the surface tension, surface area, and vol-

ume of the droplet remained unchanged, indicating

the integrity of the interfacial surfactant monolayer

at the air-water surface. To see this figure in color,

go online.

Xu et al.

quantified height analysis of the AFM images at various re-placing volumes. It can be seen that at onefold and twofoldwashing, the AFM images show isolated large aggregateshigher than 100 nm, which are most likely surfactant vesic-ular residuals adsorbed to the solid substrate before LBtransfer, thus indicating inadequate washing to remove allsurfactant vesicles from the subphase. At sevenfold and10-fold washing, on the other hand, the AFM imagesshow a rather flat, mostly monolayer structure with isolatedindividual protrusions of 5–14 nm in height. Especially at10-fold washing, only few isolated protrusions can be found,indicating that the excessive washing process significantlyimpairs the SASR. This AFM observation of the lateralstructure and topography is in line with the compromiseddynamic surface activity found at 10-fold washing (Fig. 4).

At threefold and fourfold washing, the AFM images showconsistent lateral structure and topography with uniformlydistributed multilayer protrusions of 20–28 nm in height.


Given the thickness of a fully hydrated phospholipid bila-yers to be around 4 nm (32), these AFM images revealSASR of five to seven stacked bilayers, in good agreementwith previously reported electron microscopy observations(8) and AFM imaging of compressed PS monolayers to itsequilibrium spreading surface pressure of around 50 mN/m (24,25), which is equivalent to the equilibrium surfacetension (ge) of adsorbed natural PSs at around 22 mN/m.These AFM observations, together with the surface activitymeasurements shown in Fig. 4, therefore collectively indi-cate that an optimal washing procedure exists for the sub-phase replacement technique to balance sufficient removalof the surfactant vesicles from the subphase and efficientpreservation of the functional SASR structures. It shouldbe noted that in addition to the volume of the subphasereplacement, the rate of the subphase replacement wasalso studied and found to have a negligible effect on thelateral structure of the adsorbed Infasurf film (Fig. S10).

FIGURE 4 Comparison of the dynamic surface activity of the subphase-

replaced Infasurf film with various replacing volumes from onefold (1�) to

10-fold (10�) of the original droplet volume. With onefold to up to seven-

fold replacing volumes, the subphase-replaced Infasurf film maintained the

surface activity of the de novo adsorbed Infasurf, indicating a relatively

intact SASR. However, with a 10-fold replacement volume, the sub-

phase-replaced Infasurf film showed impaired surface activity, indicating

that the excessive washing damaged the SASR. Dynamic surface activity

was determined with CDS at 20 cycles per minute, 30% compression ratio,

under physiologically relevant conditions, i.e., 37�C and 100% relative hu-

midity. To see this figure in color, go online.


Adsorbed Infasurf and Curosurf films atphysiologically relevant low surface tensions

Fig. 6 shows the lateral structure and topography of ad-sorbed Infasurf (Fig. 6, a–d) and Curosurf (Fig. 6, e–h) filmsat physiologically relevant low surface tensions. Optimiza-tion of the subphase replacement technique for Curosurfcan be found in Fig. S11. The adsorbed PS films at threephysiologically relevant low surface tensions (i.e., 15, 10,and 5 mN/m) were prepared by compressing the sub-phase-replaced PS films to the target surface tensions at aphysiologically relevant rate of 13.3% of the area per sec-ond, followed by LB transfer at the controlled surface ten-sions (51.5 mN/m) using CL-ADSA. Fig. S12 shows atypical result of LB transfer of Curosurf films at these lowsurface tensions. Figs. S13 and S14 include additionalAFM images that show the reproducibility of the lateralstructure and topography.

As shown in Fig. 6 a, right after the de novo adsorption,i.e., at the equilibrium surface tension (ge) of around 22 mN/m, the Infasurf film shows uniformly distributed isolated in-dividual multilayer protrusions as high as 28 nm, corre-sponding to seven stacked phospholipid bilayers. Uponfurther reducing the surface tension to 15 mN/m (Fig. 6b), the multilayer protrusions are enlarged in the lateraldimension, but not significantly in height. At a surface ten-sion of 10 mN/m (Fig. 6 c), the originally isolated individualmultilayer protrusions are compacted into largely contin-

uous multilayer structures covering a large fraction of theInfasurf film. At the surface tension 5 mN/m (Fig. 6 d), thesecontinuous multilayer structures further overlap to formlarge folded structures indicated by steep increments ofaround 20 nm in height. Such large-scale folded structuresare very similar to the multilayered SASR found with theinnate PS film using electron microscopy (8).

The adsorbed Curosurf film shows a lateral structure andtopography distinctly different from those of Infasurf. Asshown in Fig. 6 e, compared with Infasurf, the de novo ad-sorbed Curosurf film shows only a few but significantlylarger multilayer protrusions in the lateral dimension.When the film is compressed to a surface tension of15 mN/m (Fig. 6 f), more multilayer protrusions appear,but their heights remain relatively unchanged. At a surfacetension of 10 mN/m (Fig. 6 g), the multilayer protrusionsmerge into laterally large domains more than 10 mm insize. When the adsorbed Curosurf film is compressed to avery low surface tension of 5 mN/m (Fig. 6 h), large foldingsof the surfactant film, as indicated by steep increments of�16 nm in height, are observed. To better understand thedifferent lateral structures of adsorbed Infasurf and Curosurffilms under physiologically relevant low surface tensions,next we studied the interfacial rheological properties ofthese two adsorbed PS films.

Surface dilational rheology of adsorbed PS films

Fig. 7 shows the elastic and storage moduli (Er), viscous andloss moduli (Ei), and the loss tangent (tan4 ¼ Ei/Er) of thesubphase-replaced Infasurf and Curosurf films. A DPPCmonolayer at ge was also studied as a reference. For eachfilm, the surface dilational rheology was studied at three fre-quencies of 0.01, 0.1, and 1 Hz, respectively. Detailed pro-cedures of determining the interfacial rheological propertiescan be found in Figs. S15 and S16.

It can be seen that the surface dilational rheological prop-erties of the three films are not appreciably different fromeach other at the same frequency of oscillation, thus indi-cating that the interfacial monolayer of the adsorbed Infa-surf and Curosurf may both be enriched in DPPC.Nevertheless, tan4 of adsorbed Curosurf films is signifi-cantly higher than that of Infasurf films, indicating that Cur-osurf forms a more viscous film than Infasurf. In addition,interfacial rheological properties of all three films demon-strate a clear frequency dependence. Although both Er andEi increase with the frequency of film oscillation, tan4 ofthese films decreases especially when the frequency isincreased to the physiologically relevant value of 1 Hz.

DISCUSSION

Because of its experimental simplicity, spread Langmuirmonolayers have long been used as a standard model forstudying the biophysics of PS films. Despite providing


FIGURE 5 Lateral structure and topography of the subphase-replaced Infasurf film with various replacing volumes from onefold (1�) to 10-fold (10�) of

the original droplet volume. Also shown is a quantified height analysis of the subphase-replaced Infasurf film with various replacing volumes. All AFM

images have the same scanning size of 20 � 20 mm. Images in the first two columns have a z range of 100 nm, whereas the rest images have a z range

of 20 nm. It is found that threefold and fourfold replacement volumes are the optimized volumes because they efficiently remove the vesicles from the droplet

and, meanwhile, preserve the SASR of the adsorbed Infasurf film. To see this figure in color, go online.

Xu et al.

valuable biophysical knowledge of the PS film, especiallyon the quasiequilibrium phospholipid phase behavior andthe physical chemistry of lipid-protein interactions, it iswell accepted that the Langmuir monolayer model is inca-pable of mimicking the highly dynamic nonequilibriumbiophysics of the adsorbed PS film under physiologicallyrelevant conditions (3,8,33–35). As illustrated in Fig. 1,the spread Langmuir monolayer model differs from ad-sorbed PS films mainly in the lack of the multilayeredSASR. Although PS films without a SASR (as in the caseof spread monolayers) or with impaired SASR (as in thecase of 10� replacement volume shown in Figs. 3 and 5)can still maintain low surface tensions under static or qua-siequilibrium conditions, a functional SASR activelyattached to the interfacial monolayer at the air-water surfaceappears to be necessary for maintaining the normal biophys-ical properties of the PS film during dynamic cycling underphysiologically relevant conditions (Fig. 4).

Directly imaging an adsorbed PS film with SASR is tech-nically challenging. Here, with a series of technological ad-


vances in conjunction with the development of CDS,including subphase replacement, in situ LB transfer froma droplet, and real-time surface tension measurements andcontrol using CL-ADSA, we have performed the first, toour knowledge, AFM observation on adsorbed PS films.With the study of two animal-derived modified naturalPSs, Infasurf and Curosurf, we have gained novel insightsinto the biophysical mechanisms of PS films.

Our data provide direct evidence about the multilayerstructure of adsorbed PS films. After de novo adsorption,both Infasurf and Curosurf form multilayer structures thatconsist of an interfacial monolayer at the air-water surfaceplus a multilayered SASR. The specific lateral structureand topography of the SASR are different between Infasurfand Curosurf, most likely related to their different unsatu-rated phospholipid profiles as a result of their different ani-mal sources. Another major compositional differencebetween these two surfactant preparations is cholesterol,which is known to affect the thermotropic melting profileof PS membranes (36). Although Infasurf contains 5–8 wt

FIGURE 6 Lateral structure and topography of the adsorbed Infasurf (a–d) and Curosurf (e–h) films within the physiologically relevant low surface tension

range, including the equilibrium surface tension (ge) around 22–25 mN/m and three surface tensions lower than ge, i.e., 15, 10, and 5 mN/m, respectively. All

AFM images shown in the two middle rows have the same scanning area of 20 � 20 mm and a z range of 20 nm. Images in the top and bottom rows show the

three-dimensional renderings of the zoomed-in AFM images. White arrows point to the locations where the height measurements in the AFM images were

determined. To see this figure in color, go online.


% cholesterol, Curosurf is essentially devoid of cholesterol(24,37). Cholesterol lowers the viscosity of phospholipidmonolayers by promoting the formation of nanodomains(38), which enhance the mixing between tilted-condensed(TC) and liquid-expanded (LE) phases (22). Because it ap-pears likely that the multilayer formation initiates at thedomain boundaries (22–25), this difference could explainwhy the SASR of Infasurf appears as uniformly distributed,isolated, laterally small protrusions, whereas the SASR ofCurosurf appears as individual large protrusions (Fig. 6).

Interestingly, it is found that at physiologically relevantlow surface tensions, i.e., surface tensions less than ge, theSASR structures are compacted, merged, and eventuallyfolded at the very low surface tension of 5 mN/m for bothsurfactant preparations (Fig. 6). Although the lateral dimen-sions of the SASR increase with film compression, theheight of the SASR does not change significantly. This

finding is consistent with our previous observations of Infa-surf and Curosurf using the quasistatic compressed Lang-muir monolayer model (25). We found that multilayeredprotrusions of Infasurf and Curosurf grow in height onlyduring the compression-driven plateau region (25). Afterpassing the plateau, multilayers stop growing in height butonly increase packing density in the lateral dimension as aconsequence of surface area reduction. Such a squeeze-outbehavior in a supersaturated Langmuir monolayer can beexplained by a nucleation-growth model that predicts thatthe formation of a compression-driven multilayer from aninterfacial monolayer above a critical surface pressure initi-ates from a single-step nucleation, followed by growth of thethree-dimensional nuclei, and ends with overlapping of thegrowing nuclei, at which point the size of the nuclei be-comes limited in all directions (39). Once the growth ofthe nuclei becomes restricted, the compression rate exceeds


FIGURE 7 Surface dilational rheological properties of adsorbed Infasurf

and Curosurf films. These rheological properties include (a) the elastic and

storage moduli (Er), (b) the viscous and loss moduli (Ei), and (c) the loss

tangent (tan 4 ¼ Ei/Er). All rheological properties were determined at

37�C and the equilibrium surface tension (ge) around 22 mN/m, with three

frequencies of film oscillation, i.e., 0.01, 0.1, and 1 Hz, respectively. The

DPPC monolayer at ge was included as a reference for comparison.

Xu et al.

the relaxation rate of the film, thereby rendering the film ahigh metastability until ultimate film collapse at the collapsepressure. These data suggest that such a nucleation-growthmodel may also apply to adsorbed PS films, in which molec-ular adsorption to the air-water surface is equivalent to filmcompression in the Langmuir film model.

Consequently, our data suggest an adsorption-inducedsqueeze-out of the PS film, which results in an interfacialmonolayer enriched in DPPC with the attached multilayeredSASR made up of mainly fluid non-DPPC components.Although numerous biophysical studies have demonstratedthe compression-induced squeeze-out (24,25), the adsorp-tion-induced squeeze-out does not involve lateral compres-


sion of the PS film but only spontaneous adsorption ofsurfactant vesicles. It was found that after adsorption, anair bubble in a captive bubble surfactometer required lessarea reduction to reach very low surface tensions than esti-mated from the lipid composition of the surfactant. This wasthought to be due to ‘‘selective adsorption’’ of DPPC at theair-liquid interface of the bubble (9,33), a process known torequire surfactant proteins (40). This study suggests thatDPPC is not selectively adsorbed but rather enriched bysqueeze-out unsaturated phospholipids from the air-watersurface during the spontaneous adsorption process. Thisadsorption-induced squeeze-out model is consistent withthe autoradiographic studies of Yu and Possmayer (11),who found that the lipid composition of adsorbed naturalsurfactant films, including the interfacial monolayer plusthe attached SASR, had no difference from that of thebulk surfactant in the aqueous phase.

Direct extrapolation of the in vitro AFM data to under-standing lung physiology, however, should be taken withcaution. The clinical surfactant preparations used here,i.e., Infasurf and Curosurf, lack SP-A, which is known toplay a significant role in stabilizing the SASR in vivo(41). The adsorbed PS films studied here were formed at arelatively low surfactant concentration of 1 mg/mL, whereasthe surfactant concentration in the alveolar fluid was esti-mated to be one or two orders of magnitude higher (1).Therefore, it is not unexpected that the SASR formed in vivois more complex, thicker, and intrinsically more stable athigh compression rates. Moreover, our AFM data do notallow us to distinguish the orientation of the SASR forma-tion because the adsorbed PS film was LB transferredfrom the air-water surface onto a mica surface. It remainscontroversial whether the SASR orients toward the airside or the aqueous liquid side (42,43).

The adsorption-induced squeeze-out model is supportedby the analysis of interfacial rheology. It was found thatthe rheological properties of de novo adsorbed Infasurfand Curosurf films are very close to those of the DPPCmonolayer at ge, especially at 1 Hz (Fig. 7). Our methodo-logical advances allow us to study the surface dilationalrheological properties of de novo adsorbed PS films underphysiologically relevant conditions, i.e., adsorbed films atge of around 25 mN/m, body temperature of 37�C, andthe frequency of oscillation at 0.1–1 Hz, covering the fre-quency range of normal tidal breathing. It is found thatboth the dilational elastic modulus (Er) and viscous modulus(Ei) increase with increasing frequency from 0.01 to 1 Hz,whereas their ratio (tan4 ¼ Ei/Er) decreases with increasingfrequency, thus indicating that the PS film is significantlymore elastic than viscous under physiologically relevantconditions. Such a frequency dependency of the surface di-lational rheology in the range between 0.1 and 1 Hz is ingood agreement with what was recently reported by Bykovet al. (44) but appears to be somewhat different from thatdetermined at a much lower frequency range. Using captive


bubble surfactometry, W€ustneck et al. studied the dilationalrheology of DPPC monolayers with 2 mol % SP-C at roomtemperature in a very low frequency range between 0.006and 0.025 Hz (45). They found that although the elasticmodulus was relatively independent of the frequency, theviscous modulus decreased significantly with increasingthe frequency of oscillation. Therefore, tan4 still decreaseswith increasing frequency, consistent with what we found inthe high frequency range (Fig. 7).

It is interesting to point out that the surface shear rheologyof spread PS films showed a frequency dependency similarto that of the surface dilational rheology determined here. Itwas found that both the shear elastic and viscous moduli ofDPPC-containing monolayers (46) and natural PS prepara-tions (47,48) (including Infasurf and Curosurf) increasewith frequency in the same frequency range as studiedhere. Dilational rheology and shear rheology representdifferent rheological properties of thin films (49). Althoughthe dilational rheology studies changes in an area at a con-stant shape, the shear rheology concerns changes in shape ata constant area. In terms of PS film, the surface dilationalrheology provides information on the composition of thePS film, which is important for understanding phase transi-tions and film collapse during normal tidal breathing. Sur-face shear rheology provides information on the stabilityof PS films, which is crucial to better understand thespreading and Marangoni flow of PS films (46–48,50).The finding that both dilational moduli and shearmoduli of PS films increase with frequency may have impli-cations in better understanding the biophysical stability ofthe PS films, thus contributing to the design of novel, toour knowledge, surfactant preparations for pulmonarydrug delivery.

CONCLUSIONS

We have developed a novel, to our knowledge, experimentalmethodology called CDS. In conjunction with AFM, CDSallowed the study of lateral structure and topography of an-imal-derived clinical PS films at physiologically relevantlow surface tensions. Our data suggested that a nucle-ation-growth model is responsible for adsorption-inducedsqueeze-out at the PS film, which likely results in an inter-facial monolayer enriched in DPPC with an attached multi-layered SASR. These findings were further supported byfrequency-dependent measurements of surface dilationalrheology. Our study has implications in understanding themechanisms by which the PS film attains low surface ten-sions and stabilizes the alveolar surface.

SUPPORTING MATERIAL

Supporting Material can be found online at https://doi.org/10.1016/j.bpj.

2020.06.033.

AUTHOR CONTRIBUTIONS

L.X. and Y.Y. carried out the experiments and data analysis. Y.Y.Z. de-

signed the research and oversaw the experiments and analysis. Y.Y.Z. wrote

the article. All authors discussed the results.

ACKNOWLEDGMENTS

We thank Dr. Fred Possmayer for valuable comments on our manuscript.

We thank Dr. Walter Klein at ONY Biotech and Dr. Alan Roberts at Corner-

stone Therapeutics for the donations of the Infasurf and Curosurf samples,

respectively.

This research was supported by National Science Foundation grant CBET-

1604119 and the Hawai’i Community Foundation (18ADVC-90802).

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