Micropipette Technique Study of Natural and Synthetic Lung

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Micropipette Technique Study of Natural and Synthetic Lung Surfactants at the Air−Water Interface: Presence of a SP‑B Analog Peptide Promotes Membrane Aggregation, Formation of Tightly Stacked Lamellae, and Growth of Myelin Figures Elisa Parra,*,† Koji Kinoshita,† and David Needham†,‡ †

Center for Single Particle Science and Engineering (SPSE), Southern Denmark University, Campusvej 55, DK-5230 Odense, Denmark ‡ Department of Mechanical Engineering and Material Science, Duke University, Durham, North Carolina 90300, United States S Supporting Information *

ABSTRACT: The present study is a microscopic interfacial characterization of a series of lung surfactant materials performed with the micropipette technique. The advantages of this technique include the measurement of equilibrium and dynamic surface tensions while acquiring structural and dynamic information at microscopic air−water interfaces in real time and upon compression. Here, we characterized a series of animal-derived and synthetic lung surfactant formulations, including native surfactant obtained from porcine lungs (NS); the commercial Curosurf, Infasurf, and Survanta; and a synthetic Super Mini-B (SMB)-containing formulation. It was observed that the presence of the natural hydrophobic proteins and, more strikingly, the peptide SMB, promoted vesicle condensation as thick membrane stacks beneath the interface. Only in the presence of SMB, these stacks underwent spontaneous structural transformations, consisting of the nucleation and growth of microtubes and in some cases their subsequent coiling into helices. The dimensions of these tubes (2−15 μm diameter) and their linear (2−3 μm/s) and volumetric growth rates (20−30 μm3/s) were quantified, and no specific effects were found on them for increasing SMB concentrations from 0.1 to 4%. Nevertheless, a direct correlation between the number of tubes and SMB contents was found, suggesting that SMB molecules are the promoters of tube nucleation in these membranes. A detailed analysis of the tube formation process was performed following previous models for the growth of myelin figures, proposing a combined mechanism between dehydration− rehydration of the lipid bilayers and induction of mechanical defects by SMB that would act as nucleation sites for the tubes. The formation of tubes was also observed in Infasurf, and in NS only after subsequent expansion and compression but neither in the other clinical surfactants nor in protein-free preparations. Finally, the connection between this data and the observations from the lung surfactant literature concerning the widely reported “near-zero surface tension” for lung surfactant films and intact alveolar surfaces is also discussed.

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reduction.8 The remaining 10% is comprised of specific proteins, the hydrophilic SP-A and SP-D, primarily related to the innate immune response in the alveolar barrier; and, more important for the present work, the hydrophobic SP-B and SPC, which are essential for an efficient surfactant performance.8 Particularly, SP-B is a relatively small peptide whose monomeric form consists of 79 amino acid residues, it is mostly found as 17-kDa dimers and belongs to the saposin protein superfamily.9 Recently, it has been proposed to assemble in its native environment as ringlike, oligomeric complexes formed by multimers of dimers.10 The absence of SP-B expression in animal lungs disrupts trafficking, storage,

INTRODUCTION Fundamental concepts of respiratory mechanics, including the retraction force of the lungs and how it depends on the surface tension in alveoli, were first introduced in 1929.1 However, direct evidence for the surface-active material at the air−fluid interface of the lung was only first reported in the 1950s.2,3 It is now known that alveolar cells produce and secrete a membranous material, the so-called lung surfactant, which ultimately forms oriented films and significantly reduces surface free energy, minimizing the energy expenditure for breathing and avoiding alveolar collapse 4−6 (and see also the comprehensive review by Borden7). Compositionally, natural lung surfactant is a rather complex mixture of lipids and proteins, consisting of around 90% lipids by mass (including phosphatidylcholines, phosphatidylglycerol, and cholesterol), which are mainly responsible for the surface free energy © XXXX American Chemical Society

Received: April 13, 2016 Revised: September 2, 2016

A

DOI: 10.1021/acs.langmuir.6b01420 Langmuir XXXX, XXX, XXX−XXX

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formulations have been extensively evaluated for their ability to reduce the air−water interfacial tension in vitro, the SMBbased formulation has mostly been studied by Captive Bubble Surfactometer.29 Focusing on this new synthetic formulation is important since it provides fundamental information about a compositionally simpler system that retains the lung surfactant activity, which could also bring new and important information regarding its potential activity in treating lung disease. In order to evaluate lung surfactant properties and performance, several surface chemical methods have been developed and optimized over the years. The most widely employed are the Langmuir-Wilhelmy balance (LWB) and the Pulsating and the Captive Bubble Surfactometers (PBS and CBS). 5,6 These three devices present advantages and disadvantages for assessing the surface activity of surfactants: they are all commercially available and, in the case of PBS and CBS, can closely simulate cyclical breathing mechanics of the alveolar environment. Nevertheless, the commercial setups are costly, require relatively large amounts of sample materials, and lack flexibility in surfactant evaluation. The LWB, despite its versatility and the possibility to combine it with many different microscopic and spectrometric methods, is not appropriate for adsorption measurements due to its large surface area (∼cm2) and subphase volume (∼mL). In addition, it does not mimic closely enough the physiological conditions inside alveoli, being not so useful for clinical evaluation of surfactant preparations. On the other hand, the micropipette technique has already been demonstrated as being extremely useful for measuring interfacial tensions of clean, detergent, and lipid-adsorbed monolayers at microscopic air−water interfaces.34,35 It consists of one or more micron-sized glass pipettes operated by micromanipulators, providing a highly versatile experimental platform for performing a range of static and dynamic surface tension measurements. In addition, the imaging system allows acquisition of structural information on the sample at the microscopic level simultaneously with these interfacial measurements. Another advantage of this technique in comparison to the existing surfactometers is that the technique can be adapted to use extremely small amounts of surfactant material in the assays (∼μg), by using a very small delivery pipette that can deposit the material to positions close to the air−water interface. This microinjection technique could be very important for studies involving situations with a very limited sample amount, like the evaluation of surfactant samples obtained from patients with respiratory diseases. The present study presents a microscopic interfacial characterization of a series of lung surfactant materials performed with the micropipette technique, including animal and synthetic formulations. The goal was to report the equilibrium and dynamic surface tensions of these surfactants upon adsorption to microscopic interfaces, and to characterize the microscopic structures formed over time and during area compression at the air−water interface inside the micropipette. Thick membrane multilayers and other lipidic structures were observed beneath the interfaces coated by those surfactant materials with higher protein contents, particularly promoted by the presence of SMB, which highlight the strong membrane activity of this cationic peptide. This paper also attempts to relate this data to the observations already made in the lung surfactant literature concerning the widely reported “near-zero surface tension” for lung surfactant films and intact alveolar surfaces.

and function of surfactant lipids and causes respiratory failure at birth.11,12 Thus, it is strictly required for the assembly of pulmonary surfactant and the formation of stable surface-active films at the air−liquid alveolar interface, making the lack of SPB incompatible with life.9 In vitro, this protein is known to induce aggregation, fusion, and lysis of lipid vesicles;13 to increase the permeability of phospholipid membranes, possibly due to the formation of toroidal or proteolipid pores;14,15 and to promote the creation of multilayer deposits through lateral interactions between opposing membranes.16,17 These in vitro observations for SP-B have been associated with in vivo lung surfactant activities such as the formation and stabilization of the multilayer reservoir, the bilayer-to-monolayer lipid transfer, and other structural transformations occurring in lung surfactant membranes like the formation of lamellar bodies and tubular myelin. Deficiencies or inactivations of this system can produce severe respiratory disorders, such as the neonatal respiratory distress syndrome (NRDS) in premature babies,18 the meconium aspiration syndrome (MAS) in newborns,19 or the acute respiratory distress syndrome related to lung injury (ALIARDS).20 The main clinical treatment for NRDS and MAS involves the administration of exogenous surfactants to replace the absent or damaged surfactant by fully functional preparations, while ALI-ARDS is a far more complex pathology; this treatment still shows low efficacy and the mortality remains high.21 These clinical surfactants can be divided into animalderived preparations, reconstituted from organic extracts of natural surfactants, and synthetic formulations consisting of lipids and simple peptides or surfactant protein analogs.22 While effective, natural surfactant extracts still have some drawbacks associated with pathogen transmission risk, batch-tobatch variation, and low resistance to inhibition, which are the main reasons why developing synthetic versions of clinical surfactants is crucial.21 Among the new synthetic formulations recently under study and development are those containing peptides with sequences and structure based on functional motifs of the native proteins SP-B and SP-C, such as Surfaxin, based on the peptide KL4 and approved by FDA for the prevention of NRDS;23−26 CHF5633, containing synthetic analogs of both SP-B and SP-C, with comparable surface activity to the natural surfactants and superior resistance to inhibition;27 and the new formulations based on the SP-B analogs Mini-B (MB) and Super Mini-B (SMB), which have been demonstrated to reproduce to some extent the in vitro and in vivo activities of full-length SP-B.28 These two SP-B analog peptides consist of the amphiphilic N- and C-terminal helical domains of human SP-B (residues 8−25 and 63−78) joint by an apolar loop domain, adopting a hairpinlike conformation.29 SMB also incorporates the initial N-terminal region (residues 1−7), the so-called insertion sequence, which is rich in prolines so it preferentially positions close to the polar/apolar interface and facilitates a rapid insertion of the peptide into the bilayer.30,31 In addition, SMB appears to form stable dimers and adopt a similar conformation to the saposin fold,29 found also for native SP-B and other saposins, and known to actively bind to and perturb lipid membranes, ranging from local disordering to total membrane permeabilization depending on the specific protein.9 The new synthetic formulations containing SMB, introduced and tested by Walther, Waring, and co-workers, have shown promising results in in vitro and in vivo studies.28,29,32,33 While other natural and commercial animal-derived lung surfactant B


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Figure 1. (A) Micrograph of an air−water interface inside a micropipette, showing the horizontal (X) and vertical (Y) distances used to calculate the radius of curvature of the interface. (B) Plot showing the equilibrium surface tensions between air and pure water (●) and between air and 30 mM PBS containing 150 mM NaCl (○), both at 37 °C, obtained from the slope of the linear regression between ΔP and 2/Rc according to Young− Laplace Law (slope ± SE).

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EXPERIMENTAL SECTION

Rc =

Materials. Clinically approved and commercialized lung surfactants, Curosurf, Infasurf, and Survanta, were obtained from Chiesi Farmaceutici (Parma, Italy), ONY (Amherst, NY), and AbbVie (North Chicago, IL), respectively. These clinically approved formulations are all animal-derived and so will be referred to as such. Survanta and Curosurf are obtained by organic extraction of minced lung tissue (bovine and porcine, respectively).36 During their processing, they undergo a complete removal of cholesterol, and Survanta is further supplemented with extra DPPC, palmitic acid, and tripalmitin.36 Infasurf is obtained from calf lung lavage, and it mostly maintains the original lung surfactant composition except the hydrophilic proteins SP-A and SP-D, which are lost during the organic extraction.36 Native lung surfactant complexes (NS) were obtained from bronchoalveolar lavages of porcine lungs as described previously37 by obtaining stock suspensions in 5 mM Tris buffer (pH 7) containing 150 mM NaCl. The bronchoalveolar lavages were a generous donation from Prof. Jesús Pérez-Gil, and NS was obtained at the facilities of Complutense University (Madrid, Spain). The total phospholipid concentration was determined by phosphorus analysis upon phospholipid mineralization.38 Synthetic lung surfactant formulations, SMB+PL, consisting of the phospholipid mixture DPPC:POPC:POPG 50:30:20 (molar ratio) plus increasing concentrations of the synthetic peptide SMB (0−4% by mass of phospholipids), were provided by Molecular Express Inc. Rancho Dominguez, CA, as multilamellar suspensions. The linear sequence of the synthetic peptide SMB peptide was FPIPLPYCWLCRALIKRIQAMIPKGGRMLPQLVCRLVLRCS. All animal and synthetic surfactants were used as received or after simple dilution in 30 mM phosphate buffer (pH 7) containing 150 mM NaCl. Hexamethyldisilazane (HMDS), buffers, organic solvents (HPLC grade), and other common reagents were purchased from Sigma (St. Louis, MO). Methods. Methodology for Surface Tension Measurements. The micropipette technique consists of one or more micron-sized glass micropipettes operated by micromanipulators and mounted on an inverted microscope stage, providing a highly versatile experimental platform for performing a range of static and dynamic surface tension measurements of clean, detergent- and lipid-adsorbed monolayers at microscopic air−water interfaces.34,35 The method is based on the Young−Laplace equation: ΔP = 2γ /R c

(Y /2)2 + X2 2X

(2)

Before studying the influence of the lung surfactant formulations, measurements were made for the clean air−aqueous interface at the temperature to be used for the surfactants, namely 37 °C. Figure 1B shows the data for measurement of equilibrium surface tension for two different interfaces at 37 °C: between air and Milli-Q water and between air and a 30 mM phosphate buffer solution containing 150 mM NaCl. The measured values were consistent with tabulated values,39 and with the well-known slight increase in air−water surface tension in the presence of electrolytes,40 respectively. This technique was also used to measure the dynamic adsorption of the different surfactants to air−buffer interfaces. Surface tension changes in real time were determined by the creation of a new interface as described previously by Lee et al.34 Briefly, with the micropipette immersed in the surrounding lung surfactant suspension, the meniscus in the pipette was brought to the tip of the pipette by applying the necessary pipette pressure ΔP to overcome the interfacial tension of the meniscus. Then, an air bubble was expelled from the micropipette tip, ΔP was dropped to near zero in ∼1 s, and the interface expanded as it moves rapidly by capillary suction. This technique created a virtually clean interface because of the massive expansion in area of ∼150 times, from the initial meniscus at the pipette tip (∼1600 μm2) to the new meniscus further down the pipette taper (∼240000 μm2). Additional methodological details are given in Supporting Information, including the hydrophobic pretreatment of micropipettes with HMDS and instrumentation details. Methodology for Surface Compression and Structural Studies. The different surfactant samples were tested under progressive surface compression inside glass micropipettes. This technique simultaneously allows for surface tension measurements and direct visualization of the membrane structures associated with, at, or beneath the interface and any potential formation of new structures. Specific hydrophobization treatment applied to the glass pipettes avoids lipid sticking and spreading onto the pipette walls during a compression experiment. Both silanization treatment and an initial exposure of the pipette to the lipid suspension are needed to obtain the best measuring conditions, as explained in the Figure S1. A common protocol for surface compression was used for all tested samples: the pipette was inserted into the tested sample suspensions with a low ΔP of around 4 cmH2O (400 Pa) and kept there while the temperature was equilibrated to 37 °C for 5 min, such that the suspension gradually moved into the pipette and coated the pipette walls. The maximum Rc achieved during this equilibration was around 100 μm (equivalent to an interfacial area A of ∼6 × 104 μm2), and an equilibrium monolayer was allowed to form, measuring the surface tension from the Laplace equation as mentioned above. Then, an applied (positive pipette pressure) ΔP was set to higher values in order

(1)

Where the radius of curvature of the interface Rc is adjusted by the balance between the applied pressure inside the micropipette ΔP and the interfacial tension γ. As represented in Figure 1A, Rc is calculated from the vertical (Y) and the horizontal (X) distances of the central segment cap of the interface, and the following relation is obtained from simple geometry: C


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Figure 2. (A) Equilibrium surface tensions at microscopic air−buffer interfaces in the absence and presence of different surfactant suspensions tested at 1 mg/mL and 37 °C, including SMB peptide, control liposomes (PL, DPPC:POPC:POPG 50:30:20), SMB+PL (4% SMB), and the animalderived formulations Curosurf, Survanta, Infasurf, and Native Surfactant (NS). They were obtained from a series of measurements of the interfacial radii of curvature Rc for increasing applied pressures ΔP according to Young−Laplace Law, whose numerical values are indicated in the legends (mean ± SD, in mN/m). (B) Dynamic adsorption of the different surfactants to air−buffer interfaces, which all achieved the equilibrium values within 100−300 s. The data for time 0 are shown as a reference in brackets to represent that they were taken in independent measurements. to subject the interface to subsequent area compressions. Particularly, three compression stages (C1, C2, and C3) were tested as a series of applied pipette pressures (ΔP) and corresponding meniscus radii of curvature, Rc: C1, ΔP = 0.7 kPa, Rc ∼ 70 μm; C2, ΔP = 1 kPa, Rc ∼ 50 μm; and C3, ΔP = 2 kPa, Rc ∼ 25 μm. The interface was recorded and allowed to equilibrate for 10 min at each applied pipette pressure before the next compression was done. All the processes were continuously monitored and digitally recorded in order to register complete static and dynamic structural information about the sample and any potential transformations occurring next to the interface. This compression assay was done three times for each tested sample, and the number and size of the different structures that appeared beneath the interface was quantified and measured over time in order to determine growth rates and obtain statistical information about each sample. All the image analysis was performed using ImageJ.41

a future publication. As shown by the starred symbols in Figure 2A, the SMB peptide alone in solution at 1 mg/mL also exhibited a certain surface activity, reducing the surface tension down to 39 mN/m. So, despite its low water solubility and the possible existence of peptide aggregates, it reduces the air− water interfacial tension to values comparable to those achieved by amphiphilic proteins such as BSA,42 although the surface monolayer formed by each type of protein would be in principle different in nature due to their different water solubilities (insoluble versus soluble monolayer, respectively). As shown in Figure 2B, after the rapid (1 s) pipette pressure change, the clean air−water meniscus interface was exposed to each of the lung surfactant formulations under diffusioncontrolled conditions. There was an immediate and rapid drop in the measured surface tension from 71.8 mN/m for the clean air−buffer interface to values around 25 mN/m within the first 20 s of exposure, measuring initial adsorption rates of about 1− 4 mN/m/s for the first 30 s. The rate of reduction in surface tension then slowed, and the surface tensions all approached the equilibrium values within 200−300 s of surfactant adsorption to these microscopic interfaces. These results show that an equilibrium surface layer was formed in all cases after 5 min incubation in the presence of the surfactant suspension. This is the reason why incubations of 5 min were initially established prior to any further compression experiments. Interestingly, the peptide also showed a dynamic surfactant-like behavior in the absence of any lipids, although slightly slower than that of the lipid-based materials. Its rate of surface tension reduction was in the order of ∼1 mN/m/s, reflecting its diffusion and incorporation into a monolayer at the air−water interface and reaching its equilibrium value of 39 mN/m in ∼200 s. Formation of Interfacial Microstructures during Compression of the Equilibrated Interface. Results will now be presented for each of the animal-derived and synthetic surfactant formulations that compare the membrane structures present in each of the different samples, especially the effect of SMB concentration on formation and morphology of lipidic structures at the microscopic air−water interfaces inside the tapered micropipette. A series of representative videomicrographs are shown in Figure 3 for each of the animal-derived and synthetic surfactant formulations, showing representative membrane microstructures inside the micropipette at varying compression levels of ∼80% in area. From top to bottom, the

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RESULTS AND DISCUSSION Equilibrium and Dynamic Surface Tensions of the Animal-Derived, Native, And Synthetic-Surfactant Formulations. As can be seen in Figure 2, each of the animalderived, native, and synthetic-surfactant formulations reduced the equilibrium surface tension as compared to the clean air− water or air−buffer interfaces. As shown in Table 1 and in Table 1. Equilibrium Surface Tensions Measured for the Different Surfactant Materials (Mean ± SD) surfactant sample SMB PL SMB+PL Curosurf Survanta Infasurf NS

γEQ (mN/m) 39.4 24.2 23.5 24.5 23.3 21.1 23.0

± ± ± ± ± ± ±

0.2 0.1 0.3 0.1 0.2 0.1 0.3

The materials were tested at 1 mg/mL and 37 °C, and they were obtained from the series of measurements of Rc for increasing ΔP shown in Figure 1A, following the Young−Laplace equation.

a

Figure 2, all the equilibrium values achieved by the different surfactant formulations at these microscopic interfaces were in the range between 21 and 25 mN/m. We measured small but significant differences (±1−5 mN/m) between these equilibrium values that can be attributable to compositional and/or structural differences, whose detailed study will be addressed in D


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tested systems were control liposomes (PL); the animal-derived surfactants Survanta, Curosurf, Infasurf, NS; and the synthetic SMB+PL formulation. All these materials were tested at their original concentrations, without further dilution (between 25 and 75 mg/mL of phospholipid). Starting clean air−buffer interfaces were exposed to the various formulations for 5 min to allow adsorption of the materials (under diffusion-controlled conditions); these are shown in the right column of Figure 3 (panels A, C, E, G, I, and K). The images show that, for these short incubation times of 5 min after initial adsorption, little if any interfacial structures were seen in the initial meniscus (right column). An increase in the micropipette applied pressure then moved the meniscus down the taper to a new smaller radius, thereby subjecting the equilibrated monolayers to surface compression of around 80% with respect to the starting area, as shown in the left column of Figure 3 (panels B, D, F, H, J, and L). Because of their optical scattering, the surfactant suspensions form a somewhat opaque region to the left of the refractory air−water interface. Depending on the composition of the formulation, three types of behavior were found to occur: (i) A complete absence of interfacial accumulation and no formation of multilamellar structures were observed for protein-free pure phospholipid suspensions (PL) and for the low SP-B concentration formulations of Survanta (0.2% SP-B) and Curosurf (0.4% SP-B), even after compression of the surface shown in Figure 3 (panels B, D, and F, respectively). (ii) Irreversible adsorption of surface-active material to the air−buffer interface, seen as the spontaneous diffusion to, and formation of, micron-sized, multilayered membrane structures at the surface leading to the formation of multilayers upon compression of the interface. This process was observed inside the micropipette for compositions that had relatively high concentrations of SP-B or SMB peptide: Infasurf (0.9% SP-B) (Figure 3H), NS (1.3% SP-B) (Figure 3J), and the SMB+PL formulation (4% SMB) (Figure 3L). For these higher protein-containing materials, then, there was a distinct and clear thickening of the surface layer over compression and formation of membrane multilayers more or less parallel to the surface. This was the first indication in our system that SP-B and especially SMB could induce vesicle aggregation and condensation into multilayered structures. (iii) Upon 80% surface compression for Infasurf and SMB+PL, there was a growth of long, tubular structures from the surface-adsorbed material inside the pipette back into the aqueous phase; in some Infasurf samples, a few microtubes were observed with diameters of ∼1 μm (see Figure 3H). For the SMB+PL formulation, a much more extensive tubular growth was observed, with larger lengths and diameters ∼5−10 μm (see Figure 3L). The real-time growth of several microtubes

Figure 3. Microscopic images of the interfacial compression of different surfactant samples. Initial state on the right, final state on the left, arranged from top to bottom by increasing SP-B or SMB contents: the pure-lipid liposomes PL, DPPC:POPC:POPG 50:30:20 (A and B); Survanta (C and D); Curosurf (E and F); Infasurf (G and H); Native Surfactant (NS) (I and J); and SMB+PL formulation (K and L). The images represent the initial stage of the compression (right column), where no or less interfacial structures were visible, and ∼80% surface compression (left column), where a larger accumulation of material led in some samples to the formation of 3D structures beneath or from the interface. All the experiments were done at 37 °C. A video showing the tube formation seen in L is available.

Figure 4. Sequence of micrographs representing different stages on the compression of a NS-coated surface inside the pipette: (A) an initially compressed surface with a high material accumulation and multilayer stacks beneath; (B) surface expansion (180%) that produces elongated membrane structures; (C and D) membrane condensation by compression (75% of the area in B), forming some tubes and coiled structures; and (E) further compression (75%) that led to densely packed membrane layers beneath the surface, from which micrometer-sized tubular structures emerged toward the water side. E


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Langmuir from the surface layers can be visualized for one SMB+PL formulation in Video 1. Interestingly, then, the fact that the protein-free liposomes, Curosurf (0.4% SP-B, 1.4% SP-C), and Survanta (0.2% SP-B, 2.2% SP-C) (Figure 3, panels B, D, and F) did not produce tubes or any other visible structure seems to correlate with the absence of protein or low hydrophobic protein levels in these formulations. Curiously, while Infasurf and especially the SMB+PL formulation readily formed tubes upon area compression, the behavior of NS was more complex, requiring a nucleating expansion to generate tubes, as presented next. Native Surfactant Behavior. As shown in Figure 3 (panels I and J), when the NS−coated interface inside the micropipette received a ∼80% surface compression, some material accumulation and formation of membrane multilayers at the surface occurred, but no significant microtube growth was observed. However, as shown next in Figure 4, when the initial interface in contact with the NS suspension (Figure 4A) was subjected to a surface expansion of ∼180%, new structural conformations were triggered during a subsequent 5 min incubation at this new, larger, expanded radius and area. There was elongation of membranous material and the formation of relatively thick (though not very condensed) membranous layers, as can be seen in the expanded interface of Figure 4B. Then, when this interface was compressed, as shown in Figure 4 (panels C, D, and E), these membranes exhibited a high tendency to accumulate next to the water surface and created thicker multilayers. In this case, the material accumulation was also accompanied by significant tubular growth (diameters ∼10 μm) from the interfacial layer toward the water side, as shown in Figure 4 (panels D and E). These steps corresponded to a surface area reduction of ∼75% between panels B and C; 5 min of equilibration and slight area reduction; and another surface area reduction of ∼75% between panels D and E. Thus, NS did show formation and growth of dense microtubes from the membranes accumulated at the surface but only when triggered by a surface expansion followed by an interface compression. Therefore, in those cases where the hydrophobic peptides (SP-B and SP-C in NS and Infasurf, and the synthetic SMB in the SMB+PL formulation) were present, multilayers and dense lipid microtubes were formed over time, triggered by interfacial compression, and for NS they were triggered by area expansion followed by compression. For NS surface layers, it would appear that the expansion of the initial membrane-coated interface induced new and additional adsorption and likely revealed new material condensation. Although clearly speculation at this point, it could potentially be consistent with a tailored materials-response in vivo to area expansion− compression breathing cycles. Synthetic SMB+PL Formulation. The most complex membrane structures were observed in the SMB+PL formulation, including multilayer formation, tube nucleation, and growth and subsequent transformations. Some representative examples are shown in Figure 5. Two distinguishable aspects of these interfaces were (1) thick multilayers accompanied by the appearance of “zero-tension in the surface” and (2) the nucleation and growth of membrane tubes and its subsequent transformation into membrane helices. One of the most dramatic observations of this whole study was the spontaneous appearance of densely packed multilayered materials formed when a clean air−water interface was simply exposed to the SMB+PL suspension for 5 min or longer. As shown in Figure 5 (panels A and B), multilayers on the

Figure 5. Micrographs showing (A and B) the massively condensed material that had accumulated over a period of 30 min at the interface and (C) the kinds of membrane structures observed in SMB+PL formulations. Long membrane layers beneath the interface, protrusions, and tubules growing from the surface layers were seen inside the pipette, together with helical structures resulting from the coiling of membrane microtubes. The images correspond to three different samples prepared and tested independently from different lipid and peptide stocks, with 4% SMB in all cases. A video showing the realtime formation of the helix formed along the pipette wall in (B) is available.

order of 20−50 μm in thickness built up over an incubation time of 35−40 min, causing a flattening of the interface. This flattening is reminiscent of the flattened air-bubble seen in the captive bubble test upon surfactant adsorption and compression that yield a calculated, near-zero surface tension.43 This was also presumably the case for the MB and SMB samples (although images were not shown in the articles) during repeated compressions of their interface.29,32 The higher optical resolution micropipette images in Figure 5 are direct evidence of the loss of a Laplace pressure effect in the presence of significant adsorption and multilayers formed by the condensation of the SMB+PL material. The second interesting observation was that, as shown in Figure 5 (panels B and C), long multilayered and tubular aggregates were also visualized under the microscope in the presence of these synthetic SMB+PL formulations, in some cases evolving into helical structures. These transformations necessarily involved a 30−40 min incubation and exposure time to the SMB+PL suspension, but the tube growth and further transformations occurred in seconds to minutes. The helices were formed by multiple turns of U-shaped membrane tubes: in Figure 5B, a 7.5 μm a diameter tube coiled to ∼14 μm diameter helix; and in Figure 5C, a 5 μm diameter tube coiled to a helix diameter of ∼15 μm. The real-time formation of the helix formed along the pipette wall in Figure 5B can be seen in Video 2. These morphological changes signify a series of very active processes that likely involve membrane adhesion, fusion, induced curvature, and condensation from individual liposomes in suspension to closely stacked, lipid multilayers and finally to myelinlike figures. Effect of SMB on Structural Transformations and Tube Growth. In order to gain further insight into the specific effects of this synthetic peptide on the complex structures found in SMB+PL formulation, a series of additional observations were made over a range of SMB concentrations (from 0.1 to 4%) and quantified in terms of tube dimensions and growth rates. Three compression stages were done, corresponding to total area compressions ΔA = (Ainitial − Ai)/Ainitial) × 100 of 50, 75, and 93%, respectively. Selected videomicrographs representative for each one of these compression stages are presented in Figure 6 for increasing SMB concentrations, from top to bottom. Each image was taken at the middle of the corresponding 10 min compression stage, being representative of the observed behavior in each case. F


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Table 2. Diameters and Linear and Volumetric Growth Rates of the Tubes in SMB+PL Samples % SMB

# analyzed tubes

0.1 1 2 4

5 15 20 21

diameter (μm) 4.46 3.41 4.73 3.48

± ± ± ±

1.85 1.07 2.19 1.24

linear growth rate (μm/s) 1.76 2.68 2.23 2.73

± ± ± ±

1.24 3.84 2.34 1.70

volumetric growth rate (μm3/s) 28.37 22.38 26.65 22.14

± ± ± ±

28.37 25.11 14.10 12.95

a

They were measured from several tubular structures whose growth from the interfacial membrane layers was visualized in real time inside the micropipette and are listed as a function of SMB contents into DPPC:POPC:POPG (50:30:20) liposomes. All the results are shown as the mean ± SD, calculated from all the tubes analyzed for each composition.

Finally, the growth of these tubular structures, in terms of diameter, length, and volume, was analyzed kinetically and plotted in Figure 7. All the tube growth processes that were visible in the video recordings and grew within the same focal plane from three independent experiments performed for each composition were considered here for analysis. The diameters were observed to remain constant with growth in most of the analyzed cases, as can be seen in Figure 7A. The tube volumes were estimated by multiplying the instantaneous length by the apparent cross-sectional area, calculated from the measured diameters at the equatorial plane of the structure. The increase in tube length and volume versus time are shown in Figure 7 (panels B and C, respectively). Length and volumetric growth rates were then calculated from the averaged slopes in Figure 7 for each SMB concentration and are presented in Table 2. The length and volume growth rates were all positive and relatively consistent for different SMB concentrations, being approximately 2−3 μm/s in length and 20−30 μm3/s in volume. They did, however, display relatively large standard deviations on the same order as their average sizes. Tube measurements were only made out to 60−120 s because after this time they tended to retract back and aggregate as more spherical structures. Interpretation of the Adherent Multilayers, Tubular Structures, and Helices. On the basis of the results in Figures 6 and 7, the presence of SMB peptide is clearly an essential requirement for tube nucleation, since the number of tubes increased with higher SMB contents. However, no specific effect of SMB concentration on the tube growth rate was observed above 0.1% (i.e., there is no clear correlation between the growth rate and the peptide concentration). This tube formation induced by SMB can be related to the well-known membrane activities of SP-B regarding aggregation, fusion, and membrane stacking.16,17,44,45 These activities are likely promoted by the N-terminal sequence,13 also contained in this analog peptide, which has been reported to interact with the bilayer core and deeply affect lipid organization and morphology, promoting the formation of a lipid isotropic phase, which can be the origin of the membrane fusion, bilayerto-monolayer transfer, and multilayer stabilization.46 Nevertheless, the absence of certain segments of the full-length protein in SMB could make a difference in terms of the formation of quaternary, supramolecular structures, since native SP-B has been recently demonstrated to assemble as ringlike, oligomeric complexes formed by multimers of dimers, which could have an impact on any potential activity related to these structures.10 Similarly, SP-B and its N-terminal have already been shown to induce collapse-structures as protrusions from surfactant monolayers, observed by AFM and other imaging

Figure 6. Videomicrographs showing the progressive interfacial compression of SMB+PL-coated surfaces, arranged from top to bottom by increasing peptide contents, from 0 up to 4%. The samples were subjected to 3 sequential compression stages: C1, corresponding to ΔP = 0.7 kPa, leading to Rc ∼ 70 μm (right column); C2, ΔP = 1 kPa and Rc ∼ 50 μm (middle column); and C3, ΔP = 2 kPa and Rc ∼ 25 μm (left column). All the samples were tested at 25 mg/mL and 37 °C. Scale bars = 50 μm. Additional microscopy images corresponding to three independent experiments performed for each sample are available in the Supporting Information.

The outstanding ability of SMB to promote membrane aggregation, tube formation, and subsequent membrane transformations was clear for all the tested samples. Even for the very low peptide concentration of 0.1%, as shown in Figure 6, while not occurring every time, after only 5 min incubation, a tube had formed and, upon compression of the interface, transformed into a more vesicular structure. At concentrations of 1, 2, and 4%, SMB, there was a higher tendency for tube formation activity and dynamics as the SMB content was increased. Initial incubation generated more tubes, which again transformed into more dense vesicular and helical structures upon compression and further incubation times. This was a very complex and structurally rich process, and some variability was observed; additional microscopy images corresponding to three independent experiments performed for each SMB concentration are available in Figures S2 to S5 that correspond to SMB concentrations of 0.1, 1, 2, and 4%, respectively. Size measurements done from these experiments showed a quite large dispersion of the tube diameters, and the most significant feature obtained from the statistical analysis was that a major tube population appeared centered around 2.5−3 μm at the initial compression stage, C1, and increased in number as the SMB concentration was increased. Subsequent compression stages caused the size population to shift slightly to larger diameters while the total number of tubes was reduced, resulting from the fusion of the initially formed tubes and a certain growth of new ones. The average size values are presented in Table 2, and the complete analysis is available in the Figure S6. G


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Figure 7. Tube diameters, lengths, and volumes versus time of the microtubes growing from the surface lipid layers containing increasing SMB concentrations, from 0.1 to 4%, including (A) diameter, (B) length, and (C) volume. The compression stages corresponded to C1, ΔP = 0.7 kPa, Rc ∼70 μm (circles); C2, ΔP = 1 kPa, Rc ∼ 50 μm (squares); and C3, ΔP = 2 kPa, Rc ∼ 25 μm (triangles). All the measurable tubes formed during the course of three independent compression experiments performed for each sample are represented.

and structural techniques.44,45,47,48 Those studies generally described the 3D structures as disklike, flat bicelles attached to the monolayer, and hypothesized a lateral interaction that would lead to stacking and the formation of cylindrical “nanosilos” of a measured height up to 20 nm.48 While similar cylindrical structures were formed in our experiments presented here, the tubes in the pipette grew to much larger dimensions in the presence of SMB (3−5 μm diameter and tens of micrometers in length). Clearly, the two techniques operate on different scales: we cannot confirm the presence of submicroscopic tubes due to our resolution limit of 0.25 μm, while micron-sized structures could be difficult to identify by AFM. In any event, the size ranges found here for the SMB peptide are significantly higher than those described for SP-B and its Nterminal analog.48 As occurs with other nucleation processes, this is expected to be a stochastic process, which is triggered by the presence of an initiator, in this case presumably the SMB peptide. As has been extensively described previously by Lipowsky,49 the nucleation of tubular structures from flat membranes is directly related to changes in the membrane curvature, which can be due to the adsorption or accumulation of any type of particle, molecule, or ion to the membrane surface that will induce a spontaneous curvature and an asymmetric tension. After this nucleation occurs, the tube growth and subsequent transformations seem to follow a universal behavior: the structures observed here are clearly reminiscent of classical myelin figures, which were first described in the 19th century by Virchow.50 These figures, micron-sized cylindrical structures formed by concentric

bilayers of poorly soluble surfactants that in some cases evolve to more complex and coiled conformations, are known to form when water is added to a dense, dry multilayered mass of phospholipids well above their main acyl chain melting transition temperature, Tm. It is thought to be a response to the appearance of a hydration gradient or an external mechanical stress, such as puncturing the sample or applying a strong fluid flow.51 Traditionally, it has been considered that the growth process of these structures in length (L) is governed by diffusion, either of the surfactant in suspension, the lipid molecules within the membrane stacks, or water through the tube base, thus following a diffusive law in the form of L α t1/2.52,53 A more recent model proposes that myelin formation is due to mechanical instabilities, and that the power law for tube growth might have different time dependences due to differences in water hydration.51,54 For instance, by mechanically damaging the smooth surface of a multilayered lipid sample with a needle tip, the time dependence was closer to a power law L α t than to the diffusive model. Our results are indeed consistent with this scenario: by plotting all the measured tube lengths versus time in a double logarithmic plot as shown in Figure 8, the slopes p correspond to the exponent of the time power law. We obtained diverse time dependences for each individual microtube ranging from 0.5 to 1.2, extracted from a quite complex scenario in which some of the tubes followed a diffusive model, with an exponent p ∼ 0.5 (white symbols); the majority of them had p ∼ 1 (black symbols) or even p > 1 (green symbols) consistent with superdiffusive growth; and the rest exhibited a mixed behavior H


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samples: an indirect effect of the SMB peptide, in which its ability to promote fusion and interbilayer adhesion would induce the transformation between individual liposomes into long-stacked multilayers and create a highly dehydrated membrane phase. Then, the nucleation of tubes could occur at fusogenic sites, packing or mechanical defects induced by the peptides, which would result in the gradual rehydration of this adhered phase, leading to the formation of myelin figures. The massive adhesion and multilayer formation could lead to asymmetry of bending moments and induced curvature, which could ultimately also contribute to the evolution from cylinders to more complex geometries like the observed helixes. From Monolayers to Multilamellar Bilayers and “Near-Zero Surface Tensions”. Given the nature of the multilamellar materials that we have now seen can be formed at the air−water interface coated by NS, SMB+PL and, to a lesser extent, Infasurf, and that were perhaps not seen in the CBS experiments because of lower optical resolution, these systems cannot be treated as a single lipid monolayer, as other authors have pointed out as well.57−60 We would certainly expect these multilamellar bilayers to be much more resistant to viscoelastic deformation than their single monolayers. Even if there is a thermodynamic surface tension at the actual air−lipid interface, these thick multilayers would mechanically support the pipette (Laplace) pressure that drops to zero because of a high material shear viscoelasticity. Thus, we propose that these materials, when stacked as strongly adherent multilayers, form viscoelastic materials with zero tension-in-the-surface. This interfacial monolayer will still exhibit a surface tension, which is widely known to be ∼19−24 mN/m for spread monolayer of liquidcrystalline (Lα) phase lipids and can be even higher if any gelphase lipid separation occurs.34 However, it has been widely reported that both natural lung surfactant43 and synthetic peptide formulations29,32 can develop “zero” or “near-zero surface tensions” ( 1 (green); p ∼ 0.5 (white); and nonconstant p (red). Averaged values (mean ± SD) for each SMB concentration are shown in the graphs. Dashed and dotted lines are the L α t1/2 and L α t power laws, respectively.

with a nonconstant p over time (red symbols). No clear correlation was found based on the peptide concentration, since we obtained average time dependences p ∼ 0.9−1 for all SMB contents (see values in Figure 8). Interestingly, we found a decreasing tendency over the compression stages: the time dependences p measured for the tubes formed in the first compression stage, C1, were on average larger than those measured for subsequent compression stages, when the surface is becoming more crowded and the surface layers more dense. These results are consistent with previous observations in which isolated, well-hydrated, myelin figures grew in length linearly over time (p = 1), while the growth from very dense and dehydrated bundles followed a diffusive-like model with time dependence p = 1/2.54,55 Therefore, while the peptide is essential for tube formation, the growth rate of these tubes, once formed, does not appear to be governed or affected by the presence of the peptide. Rather, it is consistent with the universal behavior already observed and studied for simple phospholipid multilayer stacks becoming hydrated. But a clear effect of SMB on tube nucleation was obtained, presumably related to a mechanical impact on the bilayers or the creation of defects. Its presence has been also proven to be crucial for the formation of the initial, multilayer stacks at the interfaces since they were not formed in PL samples. This is consistent again with the proposed fusogenic activities of the SMB peptide on lipid membranes. Hypothetically, the absence of such spontaneous growth of myelin figures in other samples that did form multilayered structures, like NS, could correspond to more complex compositional and structural differences within the lamellar phase: it has been demonstrated that the existence of a continuous, powder phase is required for myelin growth, while no tube growth occurs from phases consisting of stacked multilamellar vesicles in hours.56 On the basis of this framework, we propose the following mechanism for the formation of myelin figures in SMB+PL I


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tubes and other myelin figures. These structures were only seen in Infasurf and more strikingly in SMB-containing formulations, while protein-free liposomes, Survanta and Curosurf did not generate such structures. Interestingly, the behavior of NS was more complex: NS showed microtube formation and growth but only when triggered by a surface expansion followed by compression. Even though some multilayers were formed in these relatively short exposure times, the interface in the tapered pipette remained curved, and a measurable surface tension of 23 mN/m was still recorded. Therefore, even for these initially adsorbed interfaces, the surface tension was still dominated by the Lα phase lipid at the air−hydrocarbon interface. In contrast, 30−40 min exposures to the SMB-containing formulations during slow compression in the micropipette resulted in a massive condensation of the suspension to form 20−30 μm thick multilayers. This was accompanied by nucleation and growth of multiple, micrometer-long, membranous tubular aggregates and helical structures, reminiscent of the classical myelin figures and whose growth is consistent with previous models based on hydration of multilayer stacks. These changes suggest very profound membrane-perturbing activities of SMB that would induce membrane−membrane adhesion, condensation, curvature and fusion, producing new morphological structures from closely stacked lipid multilayers. These results suggest that the same kind of multilayers and myelins could also be present at those millimeter-sized interfaces of the bubble-based surfactometers in the presence of NS or SMB+PL suspensions, although not visible at their optical magnification and resolution. Increasing compression of the bubble would initiate a reduction of the measured mechanical tension to values near zero, and hence, the calculated “near-zero surface tension” would be really a “nearzero mechanical tension-in-the-surface”. That is, there is possibly no need for invoking a “squeeze-out hypothesis”. Rather, it would be the highly viscous multilayered material that supports any Laplace pressure simply by viscoelastic shear of the tightly stacked, multilamellar membranes. These features could then provide the outstanding mechanical performance observed in vivo for lung surfactant along the demanding breathing cycles.

pressure supported by a thermodynamic surface tension at an infinite radius is therefore misleading. Micropipette Technique versus Other Techniques. Here is just a brief comment on adsorption−formation times and structure observation that have been determined by two other techniques compared to what we observe with the micropipette technique. Captive Bubble Surfactometer. Regarding the times it takes to make this multilayered material, we note that, in our experiments, adsorption is diffusion controlled. This is in contrast to the 5 min preincubation time that was used in CBS experiments for example of Walther et al. and Schürch et al., where the chamber was convectively mixed by using a magnetic stirrer.29,43 Therefore, it is conceivable that such convective transport could generate a similarly relatively thick 20−30 μm interfacial material that becomes a soft, viscoelastic bulk material with zero tension-in-the surface. With the Laplace pressure now dropped across a highly viscous multilayered material that would respond to any compression by deforming in a viscoelastic way, these flattened captive bubble interfaces could simply represent similarly adsorbed, stacked interfaces potentially with microscopic tubes and helices that have not been directly visualized until now. High-Resolution Imaging Normal to the Surface. In the context of previous lung surfactant research, our present technique is comparable to the high-resolution imaging technique developed by Ravasio et al.61 This technique allowed real-time visualization of the adsorption of lung surfactant complexes under physiological conditions to microscopic air− water interfaces (200 μm diameter), in a similar range as, although slightly larger than, the present technique. The observations done in that work, however, were normal to the surface plane (i.e., from below using an inverted microscope) and can only get an approximate estimation for the final surface tension values. In contrast, our micropipette technique allows visualization parallel to the axi-symmetric surface plane of the air−water meniscus and can observe directly, in real time, any formation of surface-associated membrane structures growing normal to the interface, while allowing us to make simultaneous measurements of the surface tension and so correlate these with such structural information. It also allows changing the size of the aqueous surface, leading to progressive compression or expansion of the microscopic interfacial film and the real-time visualization of how potential interfacial structures form or change under these conditions.

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ASSOCIATED CONTENT

S Supporting Information *

SUMMARY AND CONCLUSIONS To summarize, we have proposed here a new in vitro technique for lung surfactant characterization that allows for the simultaneous measuring of equilibrium and dynamic surface tensions while acquiring structural information at the microscale. The adsorption of different lung surfactant formulations to air−buffer microscopic interfaces was characterized, and the membrane structures formed during measurement times of 5− 30 min in diffusion-controlled conditions and under progressive surface compression were reported. Equilibrium surface tensions of around 21−24 mN/m were achieved by adsorption of all the lipid-based formulations within 100−200 s. Regarding the microscopic membrane structures formed by lung surfactants over time and under compression, we observed that, in some cases, the lipid-coated surface films underwent large condensation, forming multilayers of the peptide−lipid complexes and even membrane

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.6b01420. Further description of experimental methodology, including instrumentation details and the silanization treatment done to the micropipettes; four supplementary figures of the behavior under compression of SMB+PL samples; detailed analysis of tube numbers and dimensions for increasing SMB concentrations (PDF) Microscopy video of a SMB+PL multilayer with growth and development of thin membrane tubes from the air interface (MPG) Microscopy video of a SMB+PL multilayer with growth and real-time formation of a membrane helix from 5−10 μm thick microtubes (MPG) J


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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +45 65 50 47 67. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank the Danish National Research Foundation for financial support and Molecular Express Inc. for providing the lung surfactant materials and for helpful discussions regarding the new data and future applications of the formulation. This work was supported by Grant 232149 from the Danish National Research Foundation, Niels Bohr Professorship, awarded to D.N. and by a 3-month duration contract service from Molecular Express, Inc.

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ABBREVIATIONS SP-A/B/C/D, surfactant protein A/B/C/D; NS, native surfactant; LB, lamellar bodies; TM, tubular myelin; LWB, Langmuir-Wilhelmy balance; PBS, pulsating bubble surfactometer; CBS, captive bubble surfactometer; MB, Mini-B peptide; SMB, Super Mini-B peptide; DPPC, dipalmitoylphosphatidylcholine; POPC, palmitoyloleyl phosphatidylcholine; POPG, palmitoyloleyl phosphatidylglycerol; HMDS, hexamethyldisilazane; AFM, atomic force microscopy

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REFERENCES

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