Giant Phospholipid Folds on Air-Water Surface: Structure Details

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B: Biomaterials and Membranes

Giant Phospholipid Folds on Air-Water Surface: Structure Details, Formation Pathway and Possible Recycle Mechanism Huiqiong Wu, Jun Zheng, Qiang Li, Rujuan Shen, Ting He, Zhifang Sun, Lunzhao Yi, and Yi Zhang J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.9b01970 • Publication Date (Web): 14 May 2019 Downloaded from http://pubs.acs.org on May 17, 2019

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The Journal of Physical Chemistry

Giant Phospholipid Folds on Air-Water Surface: Structure Details, Formation Pathway and Possible Recycle Mechanism Huiqiong Wu, † Jun Zheng, † Qiang Li, † Rujuan Shen, ‡ Ting He, † Zhifang Sun, † Lunzhao Yi § and Yi Zhang* † †

Key Laboratory of Hunan Province for Water Environment and Agriculture Product

Safety, College of Chemistry and Chemical Engineering, Central South University, Changsha 410083, P. R. China ‡

State Key Laboratory of Powder Metallurgy, Central South University, Changsha

410083, P. R. China §

Research Institute of Food Safety, Kunming University of Science and Technology,

Kunming 650500, P. R. China *Corresponding author: Yi Zhang Address: College of Chemistry and Chemical Engineering, Central South University, Changsha 410083, China Tel: +86-(731) 883-6954, Fax: +86-(731) 883-6964 E-mail: [email protected]

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ABSTRACT: In vitro mimics recognized that the propensity of a negatively charged phospholipid, DPPS, monolayers to self-aggregate to three dimensional (3D) giant folds under over-compression at air/water interface. Time elapsing microscopical observations confirmed that such giant folds were able to float stably on the air/water interface for weeks or even longer. Ex situ Atomic Force Microscopy (AFM) and Transmission Electronic Microscopy (TEM) characterizations pointed out such giant folds were composed of compactly stacked lipid layers. Phospholipase A2 (PLA2), a principal bactericide in human and animal tear secretion was chosen to drive the in situ lipid giant folds disassembly on water and supported substrate surfaces, respectively. Our experimental results confirmed the layer-by-layer structures of the giant folds. Noteworthily, the defect-rich areas of the giant lipid folds were eliminated quickly by PLA2 while defect-free lipid zones were left untouched, suggesting that PLA2 may serve as a high effective and selective regenerator/cleaner of lipid aggregates in physiological circumstance of certain organs .

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INTRODUCTION: Phospholipid monolayer plays critical roles at many organs’ surfaces including but not limited to the lung alveoli,1 ear2 and eye surfaces.3-4 It is well known that the biological functions of lipid monolayers highly rely on their interfacial phase behaviors, especially collapsing behavior.3, 5-8 Monolayer collapse, which refers to a two-dimensional (2D) to three-dimensional (3D) phase transformation upon continual compression at the air/water interface,9-10 is often characterized by a “critical surface pressure” in the pressure-area isotherm.5 Depending on the monolayer elasticity and solubility,11-13 phospholipid monolayer collapse may result in reversible folds,5, 7 buckles,14 vesicles15 and irreversible giant folds,16 respectively. Monolayer collapse behaviors of lipid films at cornea surfaces have attracted widespread attentions from both ophthalmologists and chemists.17 McDonald and colleagues reported that flat lipid layers could be “bunched-up” to “pleated drape” during eyelids closing.18 Similar phenomenon in a dry-eye surface was also recorded.1920

King-smith et al. caught the snap-shots of the lipid layer development on the cornea

surface; inhomogeneous patterns such as thick spots, strip and plateaus were found floating on top of the flatten lipid film.21-22 Korb et al. confirmed that lipid layers which stacked on the cornea surface could quickly spread away from the center of cornea after the eyelid’s opening.23 Both in vivo and in vitro researches have confirmed the appearance of multilayered lipid films on ocular tear films during eyelids blinking, in a typical thickness range from 30 to 100 nm.24 Despite a growing number of inspirational achievements on recognizing the phase behavior of varies lipid aggregating systems that induced by the over-compression of 3



utmost lipid layers during the eyelids blinking process,3,

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23, 25

insights into the

aggregation and disaggregation pathway, as well as the recycle processes of those large lipid aggregates remain insufficient. Inspired by the innovative effort of ophthalmologists, we have set up a real time observation system to monitor the in situ phase behavior of DPPS monolayer upon excessive over-compression, confirming the presence of visible-sized fibril aggregates of lipids.16 Lacking in situ analyzing techniques lead to a knowledge gap between the macro morphology and the structure details of as-formed DPPS giant folds. The self-assembly pathway has been a labyrinth for more than 15 years. As a continuous work of the previous study, we utilized an in situ Atomic Force Microscopy (AFM) system to monitor the time elapsing disassembly process of DPPS giant folds. Phospholipase A2 (PLA2) was chosen to catalyze the hydrolysis of giant folds in both the in situ Brewster Angle Microscope (BAM) and in situ AFM studies; Owing much to the pioneering discoveries that PLA2 has strong expression in eye secretions26-29 and functioned as the principal bactericide,26, 30 the important ocular homeostasis31 and the free fat acid provider32 in eyes. Our observation results disclosed that fibrillar DPPS aggregates were composed of cascading stacks of well-organized lipid monolayers. Time elapsing observation on the in situ disassembly process of DPPS folds indicated that the hydrolysis reaction driven by PLA2 initiated from the defect-rich zone of DPPS fibrils at both liquid-air and liquid-solid surfaces, while the penetration of PLA2 into the DPPS monolayer was the prerequisite step for the enzymatic hydrolysis. This AFM observation coincides with previous reports as well as our own BAM observations that PLA2 can only digest 4


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DPPS monolayer at low surface pressure while it can digest DPPS giant folds at high surface pressure. Although this over-simplified in vitro model may not be comprehensive enough to mirror the whole in vivo situations in eyes, we are inclined to recognize that PLA2 may also serve as a highly effective and selective regenerator/cleaner toward the metabolism of unnecessary large lipid aggerates in tear film, without touching those useful homogenous lipid thin films, in addition to the principal bactericide in tear.

EXPERIMEATAL SECTION Materials. 1,2-dipalmitoyl-sn- Glycero-3-[phosphor-L-serine] (sodium salt) (DPPS), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (sodium salt) (DPPE), 1,2dipalmitoyl-sn-glycero-3-phosphate (sodium salt) (DPPA), 1,2-Dimyristoyl-sn-glycero3-phosphoserine (sodium salt) (DMPS), 1,2-Distearoyl-sn-glycero-3-phosphate (sodium salt) (DSPS), 1,2-Dipalmitoyl-sn-glycero-3-phosphate (sodium salt) (DPPA), (1,2Dipalmitoyl-sn-glycero-3-phosphocholine) (sodium salt) (DPPC), 1,2-Dipalmitoyl-snglycero-3-phosphoethanolamine (sodium salt) (DPPE) and 1,2-Dipalmitoyl-sn-glycero3[Phospho-rac-(1-glycerol)] (sodium salt) (DPPG) were purchased from Avanti Polarlipid Inc. and used as received (99+% purity). PLA2 from Naja mossambica mossambic (which was purchased from Sigma with 90% purity) was used to substitute the PLA2 from human. Chloroform with (99+%) was purchased from Sigma. Deionized ultrapure water, (MilliQ system, 18.2 MΩ) was used as aqueous sub-phase solution in order to avoid salt crystallization. Enzymatic hydrolysis reaction was performed in the Tris-HCl 5



buffer, pH= 8.9, with 150 mM NaCl and 10 mM CaCl2. All experiments were performed at room temperature (19-23 °C). The concentration of PLA2 is 20 units/100 mL, as the final concentration for all experiments. Method.

A home-built Brewster Angle Microscope (BAM) was mounted on a

Nima Langmuir trough (Nima Technique Model 312D), using a CCD camera (FastCam super 10K with Kodak Motion corder analyzer Ps-220, Video Kommunikation GMBH) as the video recorder with the frequency of 30 frames/s. The laser beam (wavelength of 514 nm, Innova 90 C, Choherent) for the BAM was operated at a power of 60 mW to give sufficient intensity for recording. To obtain well-organized phospholipid aggregates, DPPS solution was first spread on the ultrapure water surface by Hamilton syringe. The monolayer was then compressed by “Isotherm Mode” with a slow and constant barrier speed controlled by the computer 30 minutes later. After waiting for another 30 minutes, the concentrated buffer solution was carefully injected into the bulk from the outside edge of barriers, reaching a final buffer pH at 8.9, containing 150 mM NaCl, 10 mM CaCl2, and 5 mM Tris. The barriers were slowly expanded to the pre-decided surface pressure (30 mN/m) by pressure controlling process. Then, we can choose to either transfer the surface aggregates to a pre-treated silicon wafer or study the degradation reaction locally by BAM. For the BAM measurements, we normally waited for 60 minutes to make sure that the surface pressure reached the equilibrium value and then the PLA2 was injected in the same way as pHsalt buffer.

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In situ Atomic Force Microscopy（AFM）experiments were performed with the MFP3D (Asylum Research) in a non-contact mode (tapping mode) to minimize the force driven by the AFM tip. The scan rate was set at 0.5 Hz. To avoid the structural reorganization of DPPS folds and monolayer during the in situ AFM characterization, the chips were firstly treated with piranha solution (H2SO4: H2O2=3:1) for 30 minutes at 338 K and then incubated within 10 mM Ni2+ solution for 10 minutes at room temperature. Finally, the chips were dried with N2 and washed with ultra-pure water for three times. The as-prepared Chips were incubated in ultra-pure water solution before LB transfer. The silicon wafers were pre-immersed into the pure water solution before the spreading of DPPS monolayer. We usually wait for 30-60 minutes after the formation of DPPS giant folds before LB transfer. The LB transfer speed was 2 mm/min under a constant pressure mode. The same LB transfer method was also used to obtain TEM samples: chipping the commercial Cu grid with the pincers of the LB through, immersing the grid before spreading the DPPS monolayer, incubating 30-60 minutes after the formation of DPPS giant fold and then performing LB transfer at constant pressure mode, the lifting speed was 2 mm/minutes. TEM images were acquired in HRTEM mode at 200 kV via a commercial TEM (JEM-2100F). Surface Plasmon Resonance (SPR, Bicore) was utilized to study the dynamic behavior of the enzyme catalytic degradation. The commercial CM5 chips were treated with the standard operation process provided Bicore company to make sure the chips’ surfaces were highly hydrophilic. Besides, our experimental results confirmed that the naked Au chip form Bicore could also be used to transfer lipid films according to the 7



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same treatment that we treated silicon wafers: the chips first treated with piranha solution (H2SO4: H2O2=3:1) for 30 minutes at 338 K and then incubated with 10 mM Ni2+ solution for 10 minutes at room temperature. Finally, the chips were dried with N2 and washed with ultra-pure water for three time. The as-prepared Chips were incubated in ultra-pure water solution before LB transfer. The lipid monolayers, bilayers and the giant folds containing complex were transferred to the as-prepared chips through LangmuirBlodgett technique exactly the same as the preparation of AFM samples, with a 2 mm/min transfer speed under a constant pressure mode.

RESULTS In vitro Observation of the Giant Folds Formation Process. Modified Langmuir trough systems have been used by some ophthalmologists to investigate the phase behavior of lipid layer during tear film breaking up process.27,

33-37

In these mimic

systems, the outmost lipid layer on the eye surface was represented by a spread phospholipid thin film in a Langmuir trough; and the eyelids was represented by the coupled trough barriers. Inspired by this smart design, we combined a home-built Brewster angle microscopy (BAM) system with the Langmuir trough to build a real time observation system. The pressure−area isotherms of a pre-spread DPPS monolayer is shown in Figure S1a. The monolayer collapse of DPPS appeared above surface pressure 55 mN/m, while the area per molecular of DPPS was below 40 Å2, by over-compressing a monolayer beyond its solid phase. Notably, the surface pressure kept on increasing smoothly. A 8


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short movie which recorded the monolayer collapse process via a high-speed CCD camera was attached in the supplementary section (Movie S1b.mp4). According to this movie, a typical monolayer collapse usually accomplishes in 0.3 second. Morphologically, the homogenous lipid layer collapse appears like the breaking of a toughened safety glass. The images of our giant folds are similar to the snap shots caught by King-smith and colleagues38. Time elapsing BAM observations indicate that those DPPS folds can stably stay at the air/water interface for weeks, an abrupt movement of Langmuir trough barrier has no effect on their morphological structures.

Figure 1. A (Scale bar = 10 µm) and B (Scale bar = 500 nm; the red line indicates the section analysis area and the result is shown in Figure S2 in Supporting Information) AFM and C (Scale bar = 100 nm) and D (Scale bar = 5 nm) TEM images of DPPS giant folds.

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Ex situ Structure Characterization and Analysis of The Giant Folds. The ex situ characterizations of the giant folds were performed with AFM and transmission electron microscopy (TEM), corresponding results are illustrated in Figure 1. The AFM image suggests that the giant aggregates are around 100-200 nm-high and 20 nm-wide, the length of each giant fibril can reach 5 mm or even longer (Fig. 1A). The image of the digested giant folds by PLA2 is shown in Fig. 1B and the corresponding section analysis results is shown in Figure S2 to provide a better view. As shown by the results, the remains of the lipid steps are around 3-nm high, which are coinciding with the thickness of a single monolayer of DPPS. Parallel TEM image reveals that the DPPS giant folds are made of step-shaped monolayers (Fig. 1C). The HRTEM result reveals the essential building blocks of DPPS were very well packed at the plateau zones of the giant folds. There were no significant defects appeared in each layers, excluding the structural reorganization induced by LB transfer (Fig. 1D). In situ Disassembly of Giant Folds at Air/Water Surface Driven by PLA2. PLA2 is capable of catalyzing the hydrolysis of an L-phospholipid into a sn-1-acyl-lysophospholipid and a fat acid, effectively tuning the metabolism reactions of lipid.39-43 Actions of PLA2 on phospholipid membranes are extensively studied in all kinds of phospholipids dispersions, including micelles, vesicles, support bilayers, Langmuir monolayers and others.41, 44-47 It has been proven that the catalytic activity of PLA2 varied significantly with the interfacial properties of phospholipid aggregates such as membrane curvature, phase behaviors, and packing properties.42, 48-50 Previous studies also revealed that the hydrolysis reactions always initiate from the phase coexisting 10


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region between liquid condensed (LC) and liquid expended (LE) phase, and PLA2 preferred to attack LC domains rather than the LE domains.48-49 Some researchers proposed that there existed a special recognition process in this kind of reactions.44, 50 Here PLA2 was chosen as the disassembly reagent to further reveal the structural details of the giant folds. BAM was applied to monitor the time elapsing structural changes of the local giant fibrils during the hydrolysis reactions. Simultaneously, the dynamic surface tension changes were recorded via a microbalance pressure sensor. Fig. 2A exhibits the morphology of giant folds one minute prior to the injection of PLA2. Giant irreversible lipid folds are presented as bright uniaxial fibers in BAM images. Fig. 2B was caught at 120 minutes after enzyme injection, at this time point，several defects appeared on the folds-occupied surface. Fig. 2C yields the subsequent morphology change in which significant disfigurements of the bright fibrils were visualized at this time window. Time elapsing observations confirmed that the size of the slit was enlarged along the inner edge circle, indicating that PLA2 was inclined to attack pre-existed defects. It took 8-12 hours for PLA2 to digest most of the giant folds at the air/water surface (Initial surface pressure was 12 mN/m and final surface pressure was 13.6 mN/m) at room temperature, as shown in Fig. 2D-2E.

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Figure 2. Real-time BAM images picked up from the enzymatic digestion evolution process corresponding to Fig. 1C. (A-F): T= 0, 120, 180, 300, 360, 600 minutes, respectively. Scale bars in all figures are 100 μm. It is notable that our parallel observations confirmed that at an initial surface pressure of 30 mN/m, this homogeneous monolayer was able to stay on the air/water interface without apparent structural change during a 48-hours incubation. When the trough barrier was slowly expanded to reduce the surface pressure and kept at 5 mN/m, the structure changed little in the beginning, while distinguishable black zones emerged at a guise-homogeneous monolayer in 300 minutes, confirming that PLA2 was still reactive after a 2-day incubation. This result points out that high surface pressure can inhibit the reactivity of PLA2, coinciding with previous reports by Li et al.48 and Danmen-levison et al.49 12


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To further disclose the relationship between surface pressure of lipids and digestion activity of PLA2, we measured a series of real time dynamic surface tension changes for a series of digestion reactions corresponding to the BAM observations. Usually, PLA2 cleavages an L-phospholipid into two parts at the sn-2 ester linkage: a sn-1-acyl-lyso-phospholipid residue and a fat acid residue. It needs to be pointed out that the digested remains of DPPS, both the sn-1-acyl-lyso-phospholipid and the fat acid are surfactants. The sn-1-acyl-lyso-phospholipid residue of DPPS is soluble, which may return to the buffer solution. The insoluble fat acid residues tend to stay at the surface and may penetrate into the lipid monolayer at lower surface pressure, playing a significant role on the surface pressure changes in the hydrolysis reaction. Since head group of fat acid residue is smaller than that of DPPS, the hydrolysis of DPPS always leads to the decrease of surface pressure. As shown in Fig. 3, curve a provides the dynamic surface pressure isotherm (π-t curve) of a system containing PLA2 and DPPS monolayer (the initial surface pressure was 12.0 mN/m, control experiment 1). The surface pressure increased instantly right after the injection of PLA2, indicating the successful binding and penetration of PLA2 into the lipid monolayer. While the subsequent surface tension decrease was corresponding to the hydrolysis of DPPS monolayer. This result agrees well with previous investigations on DPPC monolayer.42, 49-51

Curve b in Fig. 3 refers to the control experiment 2, confirming that PLA2 showed no catalytic activity towards DPPS monolayer as long as the initial surface pressure was higher than 30 mN/m, showing a similar behavior to that of the DPPC monolayer. 48-49 13



Figure 3. Surface pressure as a functional of time in constant surface area mode with different initial pressure: (a) Fold-free Monolayer, initial pressure was 12 mN/m; (b) Fold-free Monolayer, initial pressure was 36.5 mN/m; (c) Fold-rich mixed monolayer, initial pressure was 12 mN/m; (d) Fold-rich mixed monolayer, initial pressure was 37 mN/m; (e) Fold-occupied complex monolayer which had adsorbed PAH for 10 minutes before PLA2 was added, initial pressure was 12 mN/m. For all curves, the corresponding concentrations of PLA2 are 20 units/100 mL. Curve c in Fig. 3 yields the π-t curve of the above-mentioned system (shown in Fig. 2) which contains DPPS monolayer, DPPS giant folds and PLA2. This curve has the same shape as curve a, the surface pressure increased first and then decreased, suggesting that the mixture contains DPPS monolayer and giant folds exhibits similar hydrolysis behavior to the monolayer at low initial surface pressure. It needs to be mentioned that in the climbing stage, the surface pressure value in curve c reached a higher value than it did in curve a, after which it declined faster, revealing that the 14


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mixture system contained more biting and attacking sites (defects) than a pure monolayer. It is notable that the surface pressure in curve c changed little during another 3-hours incubation (from 540 min to 720 min as indicated in Fig. 3), indicating the end of hydrolysis since PLA2 keeps its activity more than 48 hours. This further indicates that at low surface tension, PLA2 exhibits better catalytic activity against giant folds than that of lipid monolayer. Furthermore, the disassembly of DPPS giant folds lead to those surface “inactive” DPPS molecules (localized inside the giant folds) returning to the surface, therefore resulting the increase of surface tension. The surface pressure peak of curve a appeared at around 30 minutes after the injection of PLA2, while the surface pressure peak of curve c ended at around 70 minutes. In view that we used the same amount of PLA2 in the two reactions, the surface pressure gap between these two peaks should be attributed to the return of inactive DPPS. PLA2 exhibited better catalytic activity at high surface pressure in the mixture system than the monolayer system. Comparing curve d with curve b, we can see that the initial surface pressure increasing step in curve d is caused by the adsorption and penetration of PLA2 (0-40 minutes). The surface pressure drops a little after the first peak, indicating that PLA2 starts to digest the floating folds of DPPS. However, after this short decrease, the surface pressure kept increasing (with several fluctuations) while the surface area was constant in 400 min. We supposed that the second increase is due to the release of highly packed phospholipid molecules that stocked inside the giant folds. Since the PLA2 molecules exist in the water phase and they could only work at the solid/water interfaces, the disassembly started from the interface areas and the 15



untouched DPPS molecules stocked in the ice-mountain-like zone fell down to the water surface after the outside layers were destroyed, therefore led to the increase of surface tension. Obviously, the differences between curve b and curve d were caused by the existence of DPPS giant folds that contain more defects compared with DPPS monolayer. Polyallylamine hydrochloride (PAH) is a positively charged polymer that can effectively bond to the negatively charged head group of DDPS via electrostatic interaction. To further identify if the PLA2 catalyzed hydrolysis is a defect-induced process or the defects are just more reactive compared with non-defect areas, we injected a PAH into the buffer solution right after giant folds were generated in another contrast experiment to isolate the head groups of DPPS aggregates. PLA2 was injected into the buffer 10 minutes later. The corresponding π-t curve e (Fig. 3) shows no surface pressure drop before and after the injection of PLA2 even when the initial surface pressure was set around 12 mN/m. This result substantiates that the penetration of PLA2 into the defects of the lipid aggregates is a prerequisite for it to catalyze the hydrolysis of the giant fibrils and the monolayers. The presence of defects is the key to induced he hydrolysis reaction. In situ Observation on The Structural Change Details during Giant Folds Disassembly. Above mentioned dynamic surface tension investigation suggests that the hydrolysis reaction of free-standing giant folds could last a very long time, indicating that giant folds should have a stuffed structure instead of a hollow one. Hence, we conducted the in situ AFM characterizations to detect their detailed structures. First, 16


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the free-standing giant folds were transferred onto a silicon substrate through the LB technique, then the silicon substrate was glued onto the bottom of the liquid cell of AFM. Tris-buffer and PLA2 solution were sequentially injected into the liquid cell right before the AFM test. Fig. 4 exhibits the time elapsing observations on the PLA2−driving DPPS giant folds disassembly process at the solid/water interface. Structure changes emerged at the peaks several minutes after PLA2’s injection, indicating that the peaks were the earliest sufferers during the reaction. Consequent hydrolysis developed along the parallel direction of the pre-existing channels and notable structure etching appeared (Fig. 4C). This agrees with the well accepted conclusion that PLA2 starts the hydrolysis of phospholipids monolayer and bilayers from the defects at phase boundary.52 In such ice-mountain like lipid folds, the most significant degradation took place around the gap between two peaks, where the sharp promontories were quickly smoothed during the first several scans. Along with the height deterioration of the promontories, more details of structure changes can be disinterred. Several hole and channel like defects appeared later as shown in Fig. 4E, indicating that PLA2 tended to hydrolyze the fibrils along those existed defects. In other words, the enzymatic degradation on tableland always evolved along the pre-existing defects which induced by the disassembly of lipids at mountain-like areas, as shown in Fig. 4F-4G. The tableland was the latest attacked area by PLA2 probably because there were least defects around. Similarly, there was no significant structure degradation on the plateau areas of the folds, even 1800 minutes after the injection of PLA2. These results further confirmed that the 17



original defects are the key reactive sites to initiate the digestion on giant lipid folds. Insight into the degradation process of a pre-existing cave clearly confirms that the mountain-like aggregates were made of well packed “stairs” (Supplementary S3).

Figure 4. Time elapsing in situ AFM images for DPPS fibrils degradation by PLA2. Scans started at (A) 30 minutes, (B) 60 minutes, (C) 100 minutes, (D) 300 minutes, (E) 600 minutes, (F) 900 minutes, (G) 1200 minutes, (H) 1800 minutes. Scale bars in all Figures are 500 nm. All images are raw data and only corrected by the first order flatten. Surface Plasmon Resonance (SPR) and fluid injection techniques were combined to quantitively investigate the dynamic mass changes of giant DPPS folds during the PLA2 driven digestion. The mixed DPPS film that contained giant folds (Fig. 1A) were transferred form water surface to SPR chips through Langmuir-Blodgett technique. 18


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When buffer solution was injected onto the chip surface, the SPR signal changed little (the plateau before 1st injection). We input 6 injections of PLA2 solution one by one, each was flowing through the giant folds while the SPR signal changes were recorded, respectively (Fig. 5). Each injection leaded to a short SPR signal jump period which was assigned to the binding of PLA2, while the subsequent SPR signal decrease was caused by the degradation and removal of lipid aggregates by fluid buffer. We need to emphasize that we had set a 10-hours degradation-break between the end of injection 4 and injection 5. During this time, the DPPS complexes (the mixture of all partially attacked segments) were continuously rinsed by Tris-EDTA buffer. SPR signal drops little during such a long period, indicating that the giant folds still own excellent stability to resist the flooding of buffer even when they had been partially degraded. We calculated the mass loss of several samples and the layer thickness was estimated to be around twenty times of monolayer (detail discussion can be found in supplementary S4).

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Figure 5. The dynamic SPR curve for the degradation of DPPS fibrils. The injection rate for the first two steps is 5 μl/min, and it’s 1 μl/min for the next 4 steps. Each injection contains 80 μl enzyme with a PLA2 concentration of 0.05 mg/ml. T=310 K

DISCUSSION Collapse Pathway of Lipid Monolayer to Giant Folds. Our experimental data indicates that the formation of giant DPPS fibrils is a multi-step process, including at least one relatively faster folding step and one slower elongation step (see Movie S1b.mp4). To date, it is almost impossible to directly obtain the monolayer collapse mechanism of DPPS since the resolution of ultra-fast CCD video is not high enough to identify the detailed structure changes. Fortunately, we caught a series of intermediate states during the phase transition process, which are useful to disclose the collapse mechanism of DPPS monolayer. The most important intermediate image is shown in Fig. S5a, the corresponding section analysis result is shown in Fig. S5b. The results confirmed a monolayer overlapping leaded bilayer of DPPS at first. According to previous innovative efforts,53-54 and the structural details of intermediates caught by in situ BAM and AFM observations, we here propose a possible collapse pathway to the formation of giant folds (Schemes 1). When a maximum-compressed monolayer was continually compressed, a bulking deformation break out at the weakest point of the monolayer then protruded to an evagination (the contrary way to form a caveolae). Ries et al.21 and Zasadzinski et al.’s pioneering research1 were particularly inspirational for this step. Subsequently, the evaginated 20


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monolayer was spreading along the perpendicular direction of the compression force, leading to folds that contained with bilayers. Our high-speed CCD results pointed out that the spread speed of this perpendicular elongation is a fast development process. Such folded bilayers tend to form a tile bending structure upon continuous overcompression and finally expelled from the continuous monolayer to form a collapsed bilayer on top of the highly packed lipid monolayer, as shown in Fig. S4a. According to our high-speed CCD results, this bending and collapsing process is a relatively slow step compared to the folding process. Duplications of above development(s) constructed a giant fold that contains tens of well packed multi-layers of DPPS.

Schemes 1. A possible formation pathway of giant folds. Our experimental results confirmed that lipid family which contains serine headgroup (-PS group, compose both –NH3 and –COOH in their head-groups) exhibits the strongest tendency to form giant folds. The monolayer collapse speed of DPPS, DMPS and DSPS were about the same level. On the other hand, DPPA, which is another negatively charged lipid owns same alkyl-tails as DPPS, exhibited much lower ability to form giant folds while other lipid such as DPPC, DPPE, and DPPG were not able to form giant folds unless mixed with DPPS. Our previous results showed that the increase of DPPC ratio in a DPPS/DPPC mixture would slow down the spreading of monolayer collapse linearly and the giant fold would never be generated when DPPS/DPPC ratio 21



was lower than 1.0.16 These results strongly support that the intermolecular hydrogen bond plays the key role in stabilizing the folded bilayer structure and consequently maintaining the giant fold structure.55

CONCLUSION In this work, the real time BAM observations, ex situ AFM/TEM characterizations were combined to reveal the disassembly process of phospholipid giants. By using PLA2 and characterizing the real time process with both BAM observation and AFM characterization, the layer-by-layer structures of the giant folds proved. Based on this evidence, we proposed a possible development pathway of DPPS at air/water interface. It is notable to point out that PLA2 prefers to attack defect-rich lipid aggregates such as DPPS giant folds, rather than a homogeneous lipid monolayer. This observation is not only helpful in revealing the fold structures but also providing an archetypical example for researching the selective recycling way of the phospholipid aggregates in organs surfaces. We hope that such interesting catalytic advantages of PLA2 may be developed in the future to selectively remove unnecessary irreversible lipid aggerates and consequently generate useful free fat acids as natural lubricants in diseased eyes. ASSOCIATED CONTENT AUTHOR INFORMATION *Corresponding Author：Yi Zhang Tel: +86-(731) 883-6954 22


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E-mail: [email protected] Notes The authors declare no competing financial interests. ACKNOWLEDGMENT The authors thank Prof. T.M. Fischer and Prof. Lars E. Helseth for insight discussion and Dr. Lanping Huang for using of the HRTEM. This work was supported financially by National Natural Science Foundation of China (No. 21773311 and 21473257), and the Hunan Provincial Science and Technology Plan Project of China (2018TP1003). SUPPORTING INFORMATION The Supporting Information is available free of charge on the ACS Publications website http://pubs.acs.org. Supporting information contains a PDF file about Figure S1a (Surface pressure-area relationship during the formation of DPPS folds), Figure S2 (The section analysis of Figure 1b), Figure S3 (Real time observation of structure changes of defective hole during an in situ digestion process), S4 (The semi-quantitative estimation of the thickness of giant folds and related Figure, Figure S4), S5 (The methods of studying the intermediate patterns, related analysis and figure, Figure S5), and S6 (results of in situ observation of the enzymatic digestion of new born giant folds at the silicon surface and related Figure, Figure S6), as well as an MP4 file about the formation of giant folds captured by the real time BAM.

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Giant Phospholipid Folds on Air-Water Surface: Structure Details

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