Silica Nanoparticle-Induced Structural Reorganizations in Pulmonary

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Silica Nanoparticle-Induced Structural Re-organizations in Pulmonary Surfactant Films: What Monolayer Compression Isotherms Do Not Say Olga Borozenko, Manon Faral, Shirin Behyan, Abdullah Khan, Jennifer Coulombe, Christine E. DeWolf, and Antonella Badia ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.8b01259 • Publication Date (Web): 30 Aug 2018 Downloaded from http://pubs.acs.org on September 3, 2018

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ACS Applied Nano Materials

Silica Nanoparticle-Induced Structural ReOrganizations in Pulmonary Surfactant Films: What Monolayer Compression Isotherms Do Not Say Olga Borozenko,†,‡ Manon Faral,† Shirin Behyan,†,‡ Abdullah Khan,‡ Jennifer Coulombe,‡ Christine DeWolf,*,‡,§ and Antonella Badia*,†,§ †

Département de chimie, Université de Montréal, C.P. 6128 succursale Centre-ville, Montréal, QC H3C 3J7, Canada

‡

Department of Chemistry and Biochemistry and Centre for NanoScience Research, Concordia University, 7141 Sherbrooke St. West, Montréal, QC H4B 1R6, Canada

§

FRQNT Quebec Centre for Advanced Materials, Université de Montréal, C.P. 6128 succursale Centre-ville, Montréal, QC H3C 3J7, Canada

Keywords: pulmonary surfactant, inhaled nanoparticles, silica, charge, Langmuir monolayer, phase structure, membrane organization

ACS Paragon Plus Environment

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ABSTRACT The interaction of nanoparticles (NPs) with pulmonary surfactant is important for understanding the potential adverse effects of inhaled engineered and incidental nanomaterials. The effects of a low concentration (0.001 wt%) of charged hydrophilic silica NPs of hydrodynamic diameter of ~20 nm on the phase behavior and lateral structure of lipid-only and naturally-derived surfactant monolayers were investigated at the air/water interface using surface pressure–area isotherms and Brewster angle microscopy, respectively. Atomic force microscopy was used to image the morphology of films transferred onto mica substrate with nanometer resolution. We show that the silica NPs can significantly alter the condensed domain size and shape even in the absence of apparent differences in the monolayer compression isotherms. The cationic particles notably induced structural and morphological progressions in a lipid-only model that are similar to those observed for the natural surfactant film that contains the cationic specific proteins. These findings specifically highlight the impact of the NP charge on the phase transformations in pulmonary surfactant, with implications for the engineering of nanomaterials for commercial use and bioapplications.


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Introduction Whether for investigations of the health effects of ambient air ultrafine particulate1-3 or the development of nanomaterial-based drug carriers,4-6 considerable attention has been placed on the pulmonary system as a portal of entry or delivery. With a large surface area (~100 m2 in human adults) and an extremely thin alveolocapillary barrier, the lungs constitute effective adsorption sites for small molecules and particles.7 Inhaled nanoparticles (NPs) preferentially deposit in the alveolar region and peripheral airways,8 and can translocate to various organs by crossing the alveolar epithelium barrier or are retained in the lungs due to slow clearance.3, 9 In the alveolar region, the NPs likely interact with and/or embed into the surfactant monolayer that covers the air/fluid interface and whose main function is to lower the surface tension in the alveolar sacs.

The

reversible

expansion/compression of this

ultrathin

film

during

inhalation/exhalation via a monolayer-to-multilayer transition optimizes the mechanics of breathing and prevents alveolar collapse on exhalation.10 Pulmonary surfactant is comprised of saturated and unsaturated lipids and proteins (both neutral and charged).11 The saturated lipids enable the film to reach near-zero surface tensions in states of high compression, whereas the unsaturated lipids serve to maintain the fluidity required for easy re-spreading upon expansion. As the film is compressed, fluid-like liquid-expanded (LE) and solid-like condensed phases are formed, with the surfactant protein predominantly associated to the LE phase. Further compression to higher surface pressure leaves an interfacial film consisting of monolayer domains enriched in dipalmitoylphosphatidylcholine (DPPC), the main saturated lipid component of pulmonary surfactant, and closely-attached multilayer structures of fluid nonDPPC lipid and surfactant protein (lipid reservoirs).10, 12 The physicochemical characteristics of the NPs, such as size, surface hydrophobicity, and surface charge, regulate their transport across


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and interaction with the surfactant monolayer, including the formation of a lipoprotein corona on the pristine particle.13-18 The latter can adversely impact the surface activity and biophysical function of the surfactant by altering its phase structure, phase transformations, and mechanical properties. These effects and relevant literature are reviewed in ref 2. We previously investigated the changes in the two-dimensional molecular ordering in phaseseparated lipid-only and naturally-derived surfactant monolayers induced by charged amorphous silica NPs (Scheme 1a) of hydrodynamic diameter of ~20 nm at the air/water interface using Xray diffraction (GIXD) and X-ray reflectivity (XRR).19 Silica NPs are one of the three most produced nanomaterials worldwide as well as being of interest for potential biomedical applications.20-22 Contrary to hydrophobic NPs that are retained in the hydrophobic alkyl chain region of the surfactant film (increased inflammation potential), these hydrophilic silica particles are most likely to pass through the film into the lung lining fluid.14, 18, 23 Although many literature reports on the effects of NPs on surfactant membranes and related systems use anionic NPs (both hydrophobic and hydrophilic),14-15,

23-28

we found that the cationic silica NPs induce larger

changes in the alkyl chain packing in the surfactant and anionic lipid-containing monolayers, and at much lower concentration than previously considered. The anionic silica interacted with the monolayers but induced only minor structural changes. These findings are in line with the higher cytotoxicity exhibited by cationic particles that penetrate and disrupt the negatively-charged cell lipid membrane29-31 compared to the anionic analogues, whose interactions are limited to the membrane surface30-32. A review of the recent literature concludes that silica NPs can induce acute toxicity in vitro and in vivo and that correlating specific particle characteristics, such as surface charge, to particular effects is essential to minimize risk in the use of engineered nanomaterials.21


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We focus herein on NP-induced effects on the phase properties and film morphology. We show that the presence of the charged silica NPs at low concentration (0.001 wt%) in the aqueous subphase induces changes in the condensed domain size and/or shape, and that these structural changes do not always lead to an alteration of the monolayer compression isotherm. The NPs were also found to affect the kinetics of the compression-induced structural reorganization and collapse of the films. These findings contribute to a general understanding of the effects of physicochemical interactions at the nano-bio interface on the organization of surfactant membranes which may serve to guide the design of engineered NPs with low pulmonary toxicity.

Results and Discussion Lipid

The

Systems.

model

systems

investigated

are

DPPC,

DPPC/palmitoyloleoylphosphatidylglycerol (POPG), and the calf lung surfactant extract Infasurf. The lipid structures are shown in Scheme 1b. Although DPPC is generally accepted as being the lipid responsible for generating a near-zero surface tension at the interface during compression,10 by itself, it is not a good surfactant mimic. The disaturated zwitterionic DPPC and monounsaturated anionic POPG are commonly used to generate mixed monolayers that mimic the condensed/fluid phase coexistence of natural surfactant.33-35 DPPC/POPG was prepared in a 7:3 molar ratio representative of the relative proportions of saturated and unsaturated

lipids

in

pulmonary

surfactant.

Analogous

monolayers

of

DPPC/dilauroylphosphatidylcholine (DLPC) were also investigated to discriminate between charge-induced and phase-directed NP–lipid interactions. Infasurf contains the complete set of


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surfactant phospholipids, neutral lipids (mainly cholesterol), and the two hydrophobic and positively-charged surfactant-specific proteins SP-B and SP-C.35 Monolayer Compression Isotherms. Ultrapure Water Subphase. The π–A isotherms of DPPC, 7:3 (mol/mol) DPPC/POPG, 7:3 (mol/mol) DPPC/DLPC, and Infasurf on ultrapure water at 22 oC are shown in black in Figure 1. These isotherms resemble those reported in the literature.19, 36 The isotherm of the single-component monolayer of DPPC, whose gel-to-liquid crystalline phase transition (Tm) is 41 oC,37 exhibits the usual plateau associated with a LE-tocondensed phase transition between 7 and 10 mN m-1.38-39 In the case of the binary lipid mixtures, the phase transition is broadened and shifted to higher surface pressures, between 15 and 20 mN m-1. POPG (Tm = -2 oC)40 and DLPC (Tm = -1 oC)37 both remain in the LE phase until their respective collapse pressures. In the case of the more complex Infasurf mixture, the LE-tocondensed phase transition is no longer apparent in the isotherm. The plateau at 40 mN m-1 is associated with a monolayer-to-multilayer transition in which the LE phase forming lipids are selectively removed from the interface into surfactant reservoirs closely associated with the overlying DPPC-enriched film.10, 12, 41 The DPPC, DPPC/POPG, and Infasurf monolayers can be compressed to between 67 and 70 mN m-1 (i.e., near zero surface tension) before collapsing, while DPPC/DLPC collapses between 50 and 55 mN m-1, as previously reported.42 Although 22 °C is below the physiological temperature, literature has shown the phase behaviour of Langmuir monolayers of DPPC and pulmonary surfactant to be similar, but with the phase transitions shifted to higher surface pressures, so that the findings and conclusions should be transferable to physiological conditions.38, 43-45 Aqueous Nanoparticle Subphases. The lipids were subsequently spread and compressed on aqueous dispersions containing 0.001 wt% of cationic (ζ-potential = +43 ± 1 mV;


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hydrodynamic diameter = 24 ± 7 nm) or anionic (ζ-potential = -12 ± 4 mV; hydrodynamic diameter = 17 ± 5 nm) silica NPs. This subphase NP concentration is the lowest for which any of the systems investigated showed differences in the π–A isotherms and corresponds to a lipidto-NP ratio of approximately 150:1 for the lipid-only systems. This ratio is not the lipid-to-NP ratio at the interface. Prior XRR results indicate that the charged particles are present at the aqueous/lipid monolayer interface under the experimental conditions used in this study.19 For the DPPC and DPPC/DLPC monolayers comprised solely of zwitterionic phosphocholine headgroups, the π–A isotherms show virtually no change in the presence of the NPs (Figure 1, blue and red curves). In the case of the DPPC/POPG monolayer, for which the fluid phaseforming lipid comprises of negatively-charged phosphoglycerol headgroups, the isotherms on the NP subphases are shifted to higher molecular areas above the LE-to-condensed phase transition but collapse at similar surface pressures to the film on water. Additionally, in the presence of the anionic NPs, the phase transition is shifted to higher surface pressures, approximately 20 to 30 mN m-1. The driving force for these shifts is not readily explained by either structural (GIXD and XRR) or morphological (BAM and AFM) data, as will be shown, and remains to be resolved. For Infasurf, the isotherm in the presence of the anionic NPs coincides with that on ultrapure water, while in the presence of the cationic NPs, multiple shorter plateaus and transitions are observed between 50 and 55 mN m-1. To our knowledge, the resolution of multiple transitions has not previously been shown for lung surfactant films, and the origin of these is discussed below. Film Morphology. DPPC. The BAM images for DPPC on water (Figure 2a) show the expected LE/condensed phase coexistence between ~6 and ~10 mN m-1, with typical triskelionshaped domains of ~20–45 µm diameter (π = 9 mN m-1),39 followed by full coalescence of the


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film at higher pressures. The onset of condensed phase domain formation is shifted to higher surface pressures on both the cationic and anionic NP subphases (7–8 mN m-1) compared to ultrapure water (5 mN m-1). Despite the lack of differences observed in the compression isotherms and GIXD measurements that show no change in the unit cell parameters for DPPC in the presence of either NP type at 10 mN m-1 (Figure S1 and Tables S1 to S3 of Supporting Information) and at higher pressure (π = 35 mN m-1)19, the BAM images (Figure 2b,c) demonstrate that the NPs have a significant impact on the condensed phase domain size and shape. The domains are smaller, especially in the case of the anionic NPs (~5 µm in diameter at 9 mN m-1). These also exhibit a quasi-circular (vs. triskelion) shape on the anionic NP subphase, highlighting a significant change in line tension.46 The individual domains reflect differently, yet uniformly within a domain (see Figure 2c, π = 9.1 mN m-1), indicative of a tilted phase where the molecules within each domain have the same azimuthal tilt.47 Phase-Separated Mixed Monolayers. Both BAM (Figures 3a-5a) and AFM (Figures 6a-7a) imaging show that upon compression on ultrapure water, the lipid-only mixtures and Infasurf laterally phase separate into thicker DPPC-rich condensed domains and a thinner LE phase enriched in the low Tm saturated and/or unsaturated lipid(s), and surfactant proteins in the case of Infasurf. AFM analyses reveal apparent height differences ranging from 0.5 to 1 nm (depending on the force applied to the sample) between the condensed and LE monolayer phases that are a convolution of the true monolayer thickness and the phase compliance.48 The AFM tip-sample adhesion is larger over the LE versus condensed phase (Figures S2 and S3), reflecting a difference in the mechanical properties, and possibly the charge (electrostatic forces) in the case of DPPC/POPG and Infasurf.49


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Binary Lipid Mixtures. DPPC/DLPC (Figure 3a) was chosen to induce a similar phase separation as exists within natural surfactant but using a fluid phase-forming lipid that is chemically homologous to DPPC. The onset of domain formation is shifted to higher surface pressure (~12 mN m-1) compared to DPPC, in agreement with the compression isotherm which exhibits a higher phase transition pressure. Phase separation persists until film collapse. Closer examination by AFM of the mica-supported film at 15 mN m-1 (Figure S4) reveals that the fluid phase also contains submicron-sized condensed phase domains that are below the limit of lateral resolution of BAM. The condensed phase domains show internal optical anisotropy,39 which is accentuated on the cationic NP subphase (Figure 3a,b). The anionic particle-induced changes to the domain shape, size, and texture are very similar to those observed for the DPPC film (Figure 3c). A study on the interaction of DPPC with hydrophilic anionic silica NPs shows that in polar medium, the positively-charged choline headgroup mediates the adsorption of DPPC to the anionic particle surface.50 We19 and others51 have reported a small change in the orientation of the phosphocholine headgroup upon interaction with anionic NPs, which in the case of DLPC liposomes, results in localized lipid condensation51. Electrostatic repulsion between the adsorbed NPs has been proposed to prevent the line tension coalescence of the domains,51 leading to the smaller circular domains observed here in the DPPC and DPPC/DLPC monolayers. The negative charge in the DPPC/POPG monolayer yields a similar effect (i.e. smaller condensed domains, Figure 4a). An important difference between the single-component DPPC and binary DPPC/DLPC mixture is that at high pressures, the condensed DPPC-rich domains break up to form branched structures (Figure S5), attributable to the partial miscibility of the DPPC and DLPC.42, 52 This accounts for the decrease in optical contrast between the condensed and fluid phases and the corresponding decrease in domain size, which is again accentuated on the NP


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subphases. The loss of contrast as the lipids re-organize or re-mix followed by BAM over time at higher magnification (Figure S6) reveals the formation of a corona around a brighter core. While complete loss of contrast occurs after ~60 min on a water subphase at a constant pressure of 35 mN m-1, domains are still clearly observable after 2 h on the NP subphases. Clearly, the NP–lipid interactions influence the kinetics of this re-organization. Although DPPC/DLPC turns out to be a poor choice of a phase-separated zwitterionic lipid model because of the compression-induced re-organization of lipid, this mixture allowed us to capture the effect of charged NPs on lipid remixing. In order to determine how the charge of the LE phase influences its interaction with the particles, a film comprised of DPPC and POPG in the same proportion as DPPC/DLPC was selected. Even in the absence of NPs, a change in the headgroup charge from zwitterionic to anionic of the fluid phase-forming lipid impacts the line tension, leading to a noticeable reduction of the domain size on water (Figure 4a versus Figure 3a). Unlike DPPC/DLPC, the domains do not appear to grow significantly in size upon compression. Additionally, in contrast to DPPC/DLPC, at the moderate pressure of 20 mN m-1, AFM imaging indicates the absence of submicron domains within the fluid phase (Figure 6a). Between 30 and 40 mN m-1, the film shows a loss of contrast in the BAM images; the pressure range over which this occurs was found to vary with the batch of POPG, for which no explanation is yet available. AFM imaging for a transferred film at 35 mN m-1 (Figure 6a) shows that this loss of contrast is attributable to the presence of smaller, submicron condensed phase domains that are not resolvable by BAM but contribute to the optical signal. There is no difference in the heights of the micron- and submicron-size domains, suggesting they are in a similar condensation state and have similar compositions. Figure 6c-d summarizes the monolayer surface coverages of the micron- and


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submicron-size domains and micron domain size as a function of the surface pressure. Compression of the DPPC/POPG films seems to destabilize the micron-size domains in favor of the submicron domains, as was observed for the clinical pulmonary surfactant BLES.53 As the film is further compressed to pressures above 40 mN m-1, the condensed phase domains are no longer discernible by BAM or AFM. Changing the headgroup already changes the line tension and the domain size, as noted above, and so no further change is observed in the presence of the anionic NPs (Figure 4c), as might be expected given the potential for electrostatic repulsion between the negatively-charged POPG and particles. AFM confirms a similar break-up of the large condensed-phase domains into nanoscale structures at high surface pressures on the anionic NP subphase (Figure 6b-d). On the other hand, the cationic NPs induce two notable effects in the BAM images (Figure 4b). The first is a decrease in contrast between the LE and condensed phases at all pressures, suggesting a greater adsorption of the cationic silica NPs at the air/water interface which induces a change in the refractive index and resulting Brewster angle. Secondly, after the contrast is completely lost at 30–35 mN m-1 (as is also observed on the water subphase), further compression yields an inversion of the contrast, namely the appearance of dark spots within a continuous bright phase (see images at 53.4 and 59.8 mN m-1). There are two possible causes for this contrast inversion – either there is the appearance of a new phase with different optical properties or the fluid phase thickens due to collapse of the LE phase components12, 35 Such a contrast inversion was previously reported by Ding et al. upon the addition of SP-B to a ternary mixture of DPPC/POPG/palmitic acid and attributed to the acquisition of local order in the fluid phase.54 To identify which of these two possibilities is operative, films were deposited by both Langmuir-Schaefer and Langmuir-Blodgett for AFM imaging. However, because of the


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electrostatic attraction between the positively-charged particles and the negatively-charged mica, AFM images of the transferred films only showed the much larger NPs (data not shown). Transfer from a cationic NP subphase in the absence of a lipid film produced a surface densely covered with NPs confirming that the particles are deposited independently of their adsorption to the lipids. We tried other substrates (i.e., self-assembled monolayer modified gold, unmodified silicon wafers, and silicon wafers treated with Al(NO3)3 to introduce a positive surface charge). These substrates unfortunately changed the morphology of the deposited lipid films in the absence of NPs. Interestingly, no anionic NPs were observed by AFM in the transferred films indicating that these NPs drain with the subphase, reflecting a low strength of interaction with the lipid layer. This contrasts with hydrophobic NPs (co-spread with lipid or surfactant) that are reported to be found in the transferred films.14, 55-56 We have previously shown by GIXD that at 35 mN m-1, the cationic NPs induce a significant reduction of the molecular tilt angle in the condensed phase of DPPC/POPG due to NP-induced condensation of the POPG.19 Wang and coworkers reported a similar effect – binding of anionic NPs to the fluid areas of phosphatidylcholine-based liposomes caused gelation (molecular condensation), and conversely, cationic NPs induced gelled membranes to fluidize locally.51 Similarly, Prenner and coworkers observed POPG–NP clusters by BAM for much larger cationic gelatin particles (~107 nm) and 10-fold higher particle-to-lipid ratio.57 Figure 8a and Tables S4S6 (summary of the GIXD data) show that at 45 mN m-1, within the pressure range that the phase contrast inversion occurs, the cationic NPs induce a change in the lattice of the condensed monolayer phase from an oblique unit cell with the alkyl chains tilted between nearest neighbour and next-nearest neighbour to a distorted hexagonal unit cell with untilted chains. This lattice change on its own is not sufficient to induce the observed inversion in the BAM contrast.


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Notably these changes in unit cell and tilt are also observed for the cationic silica NPs in the presence of Tris-buffered saline, but not on the buffered saline subphase alone (Figure S7), indicating that the reduction in tilt angle is not the result of adsorbed counterions nor are the electrostatic particle–lipid interactions fully screened at physiological salt concentrations. We return to the origin of the contrast inversion in the discussion of Infasurf. Infasurf. Two populations of condensed phase domains are observed in BAM (Figure 5a) and AFM (Figure 7a) for Infasurf on water. The larger domains are approximately 15–20 µm in diameter while the smaller are 2–5 µm. The area fraction of these doubles on compressing from 20 to 33 mN m-1 as the lateral phase separation proceeds (Figure 7c). Moreover, AFM scans of the larger domains reveal a domain-in-domain structure that has previously been attributed to DPPC-rich tilted condensed (TC) cores (1.0 nm higher than the LE phase) within a cholesterolrich liquid-ordered (LO) phase (0.2 nm lower than the TC phase) that originate from the higher cholesterol content of Infasurf (up to 5-8 wt%) compared to other clinical surfactant preparations.35 This domain-in-domain structure is observed even well below the onset of the monolayer-to-multilayer transition plateau at 40 mN m-1. High magnification AFM images (Figure S8) reveal that the TC cores are uniformly flat, but that the surrounding phase is in fact heterogeneous and appears to be comprised of nanoscale serpentine TC domains in a LO matrix that is 0.1–0.2 nm lower in height and holes corresponding to residual trapped LE phase that are 0.8–1 nm lower than the TC phase. Infasurf undergoes the same contrast inversion in BAM (Figure 5a) as observed with DPPC/POPG on the cationic NP subphase upon compression across the monolayer-to-multilayer plateau from 40 to 44 mN m-1. By the end of the plateau, at 45 (Figure S9) or 50 mN m-1 (Figure 7a), the average condensed domain size increases due to the loss of the smaller (2–5 µm) domains which also causes a reduction in the area fraction of the


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condensed phase (Figure 7c and d). The AFM images (Figure S9) show that there is very little height contrast between the condensed domains and the surrounding matrix (excluding the 2-10 nm high protrusions appearing in white in the images). This results from multilayer formation from the LE and/or LO phases.35 Adhesion mapping more clearly shows the different material properties of the condensed domains and surrounding phase. It has been suggested that these residual domains derive from the TC core of the domain-in-domain structures.35 Upon further compression to 60 mN m-1, the multilayer protrusions increase laterally in size (800 ± 200 nm) but not in height (average of 2.3 nm), as previously reported.12, 35 BAM imaging confirms that these multilayer protrusions do not encompass the TC cores which remain visible up to 60 mN m-1 (Figure 9a). In the presence of the anionic NPs, both BAM (Figure 5c) and AFM (Figure 7b) confirm that the bimodal distribution of domain sizes and domain-in-domain structure (Figure S8) are retained. The population of the larger domains visible by BAM, appear to decrease in size above 40 mN m-1 and the contrast in BAM begins to be lost. By 49.5 mN m-1, there is little optical contrast observed, in line with the AFM images at 50 mN m-1 (Figure 7b) that show no distinct LE/condensed phase coexistence. By 60 mN m-1, the AFM imaging shows the re-appearance of the TC cores surrounded by multilayer protrusions. Notably, the residual condensed cores (Figure 7d) and surrounding multilayer structures (reservoirs) on the anionic NP-laden subphase are smaller than those on water. The multilayer protrusions extend 2–40 nm from the surface monolayer. The cationic SP-B and SP-C can be strongly attracted to the anionic silica surface via ion pairing of the protonated N-termini, arginine (1 in SP-C and 5 in SP-B), lysine (2 in SP-B), and histidine (1 in SP-B) side chains with the siloxide groups.58-59 Computational studies suggest that anionic hydrophilic NPs pull out the more positively-charged surfactant protein SP-B, but


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not SP-C, from the interfacial surfactant layer, an effect not observed with cationic or neutral NPs.14 Given that one of the functions of the essential SP-B is to enable membrane folding and multilayer formation,60 this may account for the differences in film morphology observed at high surface pressures. In contrast, the cationic NPs again have a direct impact on the lateral film structure even at low surface pressures where the condensed domain size is reduced to the limit of resolution of the microscope (Figure 5b). Additionally, the presence of silica NPs bound to the monolayer/aqueous interface increases the reflectivity and decreases the contrast.19 By 37 mN m1

, the domains are no longer visible, and upon further compression to above 40 mN m-1, they

reappear with inverted contrast. Figure 8b shows the GIXD data for Infasurf on the three subphases. The tilt angle is not significantly altered (all peaks extend to Qz ≈ 0.3 Å-1) in the presence of the particles. It was not possible to definitively fit the GIXD data for Infasurf due to the low intensity and strongly overlapping peak positions. However, it appears that the Infasurf diffraction pattern on water combines the same two peaks observed for DPPC/POPG on the cationic NP subphase (Figure 8a) with an additional broad peak at a Qxy value between 1.40 and 1.47. Such a peak was also observed for Survanta, another clinical surfactant formulation, and attributed to the formation of a second crystalline phase of unknown origin.45, 61 In the case of Infasurf, this could be due to the presence of the cholesterol-induced LO phase that has been reported previously and observed here.35 The additional intensity observed for Infasurf on a cationic NP subphase can be attributed to a NP-mediated condensation that increases the proportion of molecules in the condensed phase, thus increasing the signal. Figure 9b indicates that the multiple plateaus between approximately 40 and 52 mN m-1 observed in the Infasurf isotherm (Figure 1) on the cationic NP-laden subphase are due to the co-


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existence of different film morphologies, one with the residual condensed phase cores and one continuous (i.e., π = 52.4 mN m-1), that collapse at distinct surface pressures. On the other hand, the Infasurf on water (Figure 9a) maintains a similar morphology (dark condensed cores surround by a brighter area comprising multilayer structures), at surface pressures past the plateau up to film collapse. In this pressure regime, the only observable changes are an increase in brightness of the surrounding phase as more reservoirs are formed. As already noted, Zuo et al. have previously shown that the multilayer reservoirs which encompass the LE and LO phases do not significantly increase in height.35 The large increase in collapsed material on the cationic NP subphase accounts for the shift in the compression isotherm to smaller areas at pressures between 52 and 70 mN m-1. Summary of the Findings. Considerable emphasis has been placed on the NP-induced inhibition of the surface activity of pulmonary surfactant and related systems, with the conclusion that anionic hydrophobic NPs have a much greater inhibitory effect on surfactant function.14,

23

In focusing on NP-induced changes in the lateral film structure and phase

transformations at surface pressures spanning the first appearance of LE/C phase coexistence and film collapse, we find that for hydrophilic, amorphous silica NPs, at the low concentration used, the cationic have a greater effect on the structural re-organization of the films than the anionic, and at subphase concentrations much lower than typically considered. We observe differential effects of the silica particles according to charge. For zwitterionic lipid only containing monolayers (DPPC, DPPC/DLPC), the anionic NPs are found to drastically alter the condensed domain size and shape while the cationic NPs have a much less significant effect. Neither particle type appreciably alters the molecular organization within the condensed phase (GIXD). Both the cationic and anionic silica retard the re-mixing of DPPC and DLPC. When the fluid


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phase is composed of the more biologically relevant and anionic POPG, the impact is inversed, whereby the cationic particles have a significant impact on morphology and phase transformation that is not observed with the anionic particles. Namely, the cationic silica induces the local condensation of the POPG via attractive electrostatic interactions. This condensation is correlated with changes to the molecular organization of the condensed phase (GIXD). Secondly, the presence of the cationic NPs induces a similar monolayer-to-multilayer phase transformation as occurs for Infasurf on water to generate condensed domain cores surrounded by a multilayered fluid phase at high pressures. For Infasurf, which contains a complex mixture of zwitterionic and anionic lipids as well as cationic surfactant proteins, the more significant changes in morphology and molecular organization are still observed for the cationic NPs. Moreover, the morphological changes observed for DPPC/POPG and Infasurf on the cationic NP subphase are more significant than those observed on buffered saline, indicating that the observed differences are not simply due to the presence of ions.62 While the in-vivo implications of such structural changes are not yet known, these findings highlight that surface activity alone is insufficient to fully evaluate the impact of NP–surfactant interactions on pulmonary function.

Conclusions What monolayer compression isotherms do and do not say. Surface pressure-area isotherms are frequently used for preliminary screening of the effects of NPs on the phase behavior of lipid and lipid-protein films to identify the NP concentration(s) for further investigation. We show herein that these isotherms are not necessarily sensitive indicators of NPinduced changes in the lateral film organization and phase transformations. Monolayer


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compression isotherms respond to significant changes in the molecular orientation and packing as well as alterations in the relative proportions of the different phases (i.e., LE versus C or monolayer versus multilayer). In complex systems, some of these changes can counter each other, thereby producing minimal shifts in the isotherm. Moreover, changes in the condensed domain shape do not necessarily result in changes in the average molecular area. In the current case, at 0.001 wt% NP concentration in the subphase, significant changes in the isotherms are only observed for DPPC/POPG (anionic and cationic NPs) and Infasurf (cationic NPs only), even though BAM and AFM imaging show that the NPs impact the condensed domain size and/or shape to some extent for all the systems. There is no evident correlation between changes in the isotherm and the resulting film morphology. Studies have shown that the physicochemical interaction of NPs with pulmonary surfactant can lead to adsorption of the lipid and protein constituents onto the particle surface as well as their selective removal from the surfactant membrane.14-15, 25, 34 The surface pressure-area isotherms in the presence of low concentration of the silica NPs do not indicate the significant lipid loss from the air/water interface that would be required for the formation of a lipid coating on the particle (where loss of lipid would be expected to shift the entire isotherm to smaller molecular areas). This does not preclude the formation of a lipoprotein corona becoming relevant at higher subphase particle concentrations. For example, Guzmán et al. have demonstrated that at 1 wt%, anionic silica NPs become partially coated with lipid and embed into a DPPC monolayer, leading to an isotherm expansion.63 Charged hydrophilic silica NPs affect the structural re-organization and phase transformations of lipid-only and surfactant films. Both the cationic and anionic NPs alter the kinetics of lipid re-mixing in DPPC/DLPC, which may have pertinent implications given the


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complex dynamics and composition of natural pulmonary surfactant. This may be due to NPinduced changes in the lipid mobility and film viscosity. The cationic NPs were found to induce the condensation of the LE phases of DPPC/POPG and Infasurf. Additionally, the cationic NPs impact the collapse into multilayer reservoirs of Infasurf. Origin of the phase contrast inversion observed in BAM. While residual condensed phase cores have been visualized by AFM imaging of transferred films of surfactant at surface pressures above the monolayer-to-multilayer transition plateau,35 these structures have not been previously reported at the air/water interface for natural pulmonary surfactant extracts. Herein, we demonstrate that these structures can be observed as domains of inverted contrast in BAM. The initial loss of contrast observed in BAM before the contrast inversion is due to the average thickness of the surrounding fluid phase being equivalent to the thickness of the condensed cores. The increased thickness of the fluid phase is due to the presence of localized collapsed patches and/or reservoirs. As these multilayer patches grow in size and number, the average thickness of the fluid phase eventually becomes greater than that of the condensed domains, leading to the observed contrast inversion. Role of cationic species in pulmonary surfactant. It is noteworthy that the GIXD contour plot of DPPC/POPG on the cationic NP subphase is very similar to that of Infasurf on water. In fact, the similarities in the structural and morphological progressions determined by both GIXD and BAM imaging for these two systems, containing cationic NPs and cationic SP-B protein, respectively, highlight the roles of charged species, which may include the condensation of the negatively-charged lipids and facilitation of reservoir formation. Similarly, synthetic cationic peptides have also been shown to induce reservoirs and additionally aid membrane folding.64 However, it is clear that the amount of cationic species must be tightly regulated, both in natural


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surfactant and in any synthetic formulation. This is evident from the impact of the cationic NPs on Infasurf whereby the presence of additional cationic species induces changes to the collapse mechanism and accompanying reservoir formation. The presence of both the cationic proteins and NPs appears to be cumulative and induces multiple phase transitions. Such changes can impact the proper functioning of pulmonary surfactant, in particular its re-spreadability.

ASSOCIATED CONTENT Supporting Information. Experimental details, tabulated data summarizing the fitted parameters for the grazing incidence diffraction plots presented in the main text, additional AFM images (at higher resolution, additional surface pressures and different imaging modes) and additional BAM images (at higher magnifications and as a function of time) (pdf)

AUTHOR INFORMATION Corresponding Author *Email: [email protected] and [email protected] Author Contributions OB and MF recorded the surface pressure-area isotherms and performed the BAM imaging. OB carried out the film transfers and AFM imaging. AK performed the GIXD measurements. SB and JC carried out the GIXD analysis. CD and AB interpreted the data and co-wrote the manuscript.


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ACKNOWLEDGMENT AB and CD acknowledge funding from the FRQNT team research project program (grant 2015-PR-183946). OB thanks the FRQNT for a postdoctoral research fellowship. MF thanks the Groupe de recherche en physique et technologie des couches minces for an Arthur Yelon-John Low Brebner summer scholarship. ChemMatCARS Sector 15 is supported by the National Science Foundation under grant number NSF/CHE-1346572. This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357.

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Scheme 1. Structures of the (a) Ion-Stabilized Colloidal Silica Types and (b) Lipids Used in This Work

(a)

(b)


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70

70

DPPC

60

30

40 30

20

20

10

10

0 20

40

60 80 A (Å 2)

100

0 20

120

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60 80 A (Å 2 )

100

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water cationic NPs anionic NPs

50 40 30

π (mN/m )

DPPC/DLPC (7:3)

60

60

Infasurf

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40 30

20

20

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0 20


50 π (mN/m)

π (mN/m)

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DPPC/POPG (7:3)

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water cationic NPs anio nic NPs

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π (mN/m )

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


0 40

60 80 A (Å 2)

100

120

150

300 450 600 Trough area ( cm 2)

Figure 1. Surface pressure–molecular area (π–A) isotherms of DPPC, DPPC/DLPC (7:3), DPPC/POPG (7:3), and Infasurf on ultrapure water and 0.001 wt% aqueous dispersions of cationic and anionic silica NPs. Temperature = 22 oC. The isotherms for Infasurf are reported as trough areas and not molecular areas since its exact composition is not known. The isotherms of the NP-containing subphases exhibited no discernible surface activity.


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(a)

6.3 mN/m

7.6 mN/m

8.2 mN/m

9.4 mN/m

10.3 mN/m

12.7 mN/m

7.6 mN/m

8.2 mN/m

9.2 mN/m

10.2 mN/m

12.9 mN/m

8.2 mN/m

8.6 mN/m

9.1 mN/m

10.0 mN/m

12.5 mN/m

(b)

7.3 mN/m

(c)

7.7 mN/m

Figure 2. BAM images (430 µm × 538 µm) of DPPC as a function of the surface pressure on the different aqueous subphases: (a) ultrapure water, (b) 0.001 wt% cationic NPs, and (c) 0.001 wt% anionic NPs. Light regions correspond to thicker (condensed) domains.


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(a)

12.2 mN/m

15.2 mN/m

20.0 mN/m

32.0 mN/m

41.2 mN/m

51.0 mN/m

17.6 mN/m

20.1 mN/m

29.7 mN/m

40.5 mN/m

49.6 mN/m

17.4 mN/m

20.1 mN/m

29.9 mN/m

39.8 mN/m

52.0 mN/m

(b)

15.0 mN/m

(c)

15.3 mN/m

Figure 3. BAM images (430 µm × 538 µm) of DPPC/DLPC (7:3) as a function of the surface pressure on the different aqueous subphases: (a) ultrapure water, (b) 0.001 wt% cationic NPs, and (c) 0.001 wt% anionic NPs.


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(a)

16.0 mN/m

19.8 mN/m

26.0 mN/m

30.8 mN/m

40.75 mN/m

55.5 mN/m

18.0 mN/m

25.6 mN/m

35.0 mN/m

53.4 mN/m

59.8 mN/m

20.1 mN/m

25.0 mN/m

29.9 mN/m

40.1 mN/m

62.4 mN/m

(b)

15.0 mN/m

(c)

17.6 mN/m

Figure 4. BAM images (430 µm × 538 µm) of DPPC/POPG (7:3) as a function of the surface pressure on the different aqueous subphases: (a) ultrapure water, (b) 0.001 wt% cationic NPs, and (c) 0.001 wt% anionic NPs.


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(a)

23.8 mN/m

35.6 mN/m

40.1 mN/m

41.6 mN/m

42.4 mN/m

47.7 mN/m

29.5 mN/m

36.7 mN/m

40.5 mN/m

42.5 mN/m

45.6 mN/m

33.3 mN/m

40.1 mN/m

42.1 mN/m

45.1 mN/m

49.5 mN/m

(b)

24.1 mN/m

(c)

22.4 mN/m

Figure 5. BAM images (220 µm × 275 µm) of Infasurf as a function of the surface pressure on the different aqueous subphases: (a) ultrapure water, (b) 0.001 wt% cationic NPs, and (c) 0.001 wt% anionic NPs.


35


35 mN/m

53 mN/m area fraction of condensed phase

20 mN/m (a)

0.40

(c)

0.35

Submicrodomain Microdomain

0.30 0.25 0.20 0.15 0.10 0.05 0.00 20

35 Water

(b)

10 average microdomain size (µm )

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

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π (mN/m)

(d)

20 35 Anionic NPs

water anionic NPs

8 6 4 2 0 20

35

π (mN/m)

Figure 6. AFM height images of the DPPC/POPG (7:3) monolayers deposited onto mica at 20, 35, and 53 mN m-1 from (a) ultrapure water and (b) 0.001 wt% anionic NP subphase. Images are 50 × 50 µm2. Adhesion images are provided in Figure S2 of the Supporting Information. Quantification of (c) the area fraction occupied by the condensed domains and (d) microdomain size on compression. Note that the y-axis is the same for both plots in (c).


36

20 mN/m

33 mN/m

50 mN/m

(a)

60 mN/m

0.5

(c)

0.3 0.2 0.1 0.0 20

(b)

water anionic NPs

0.4

average domain size (µm)

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


area fraction of condensed domains

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15

33

45

50

60

π (mN/m)

(d)

water anionic NPs

10

5

0 20

33

45

50

π (mN/m)

Figure 7. AFM height images of Infasurf monolayers deposited onto mica at 20, 33, 50, and 60 mN m-1 from (a) ultrapure water and (b) 0.001 wt% anionic NP subphase. Images are 50 × 50 µm2. Adhesion images are provided in Figure S3 of the Supporting Information. Higher magnification images of the boxed domains are shown in Figure S8. Quantification of (c) the area fraction occupied by the condensed domains and (d) domain size on compression.


37

60


Page 38 of 40

Figure 8. Contour plots of the X-ray intensities versus in-plane scattering vector components Qxy and Qz for (a) DPPC/POPG (7:3) and (b) Infasurf on subphases of ultrapure water (left), 0.001 wt% anionic silica NPs (middle), and 0.001 wt% cationic silica NPs (right) at π of 45 mN m-1.


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


(a)

47.1 mN/m

52.7 mN/m

58.7 mN/m

52.4 mN/m

64.0 mN/m

(b)

47.2 mN/m

Figure 9. BAM images (220 µm × 275 µm) of Infasurf at pressures above the monolayer– multilayer plateau: (a) ultrapure water and (b) 0.001 wt% cationic NPs.


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TOC Graphic


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Silica Nanoparticle-Induced Structural Reorganizations in Pulmonary

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