UNIVERSITY OF CALGARY. Biophysical Characterization of a Biomimetic from the Tear Film Lipid Layer. Matthew Jack Patterson A THESIS

Transcription

1 UNIVERSITY OF CALGARY Biophysical Characterization of a Biomimetic from the Tear Film Lipid Layer by Matthew Jack Patterson A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF BIOLOGICAL SCIENCES CALGARY, ALBERTA JUNE, 2014 Matthew Jack Patterson 2014

2 Abstract Tear film is a three layered structure that lies adjacent to the cornea. It protects the eye from desiccation, bacterial infections, and injury. The biophysical properties of the polar lipid layer were investigated to further understand the role that these lipids may have in the properly functioning tear film. The investigation was accomplished through the use of Langmuir monolayers, Brewster angle microscopy, and differential scanning calorimetry. These experiments were conducted on a tear film biomimetic that consisted of 16:0/16:0 phosphatidylcholine (DPPC), 16:0/16:0 phosphatidylethanolamine (DPPE), 16:0 glucosylceramide (PGC), and 16:0 sphingomyelin (PSM), reflecting their in vivo molar ratios. All four lipids formed very stable monolayers indicating that the polar lipids stabilize the open eye tear film. Brewster Angle Microscopy indicated multilayer formation as a possible mechanism to prevent premature tear film collapse. Finally, tear lysozyme interaction studies indicated increased insertion when the barriers for hydrophobic interactions were minimal. ii

3 Acknowledgements First I would like to thank my supervisor, Dr Elmar Prenner, for not only giving me the opportunity to work in his lab but more importantly the necessary guidance and mentorship to succeed. Research in the Prenner lab has been a truly remarkable and thoroughly enjoyable experience. I would also like to extend my gratitude towards my committee members Dr Hans Vogel and Dr Vanina Zaremberg for their time investment and insightful suggestions. Thank you to Dr Max Anikovskiy for his role as internal/external examiner (Спасибо, товарищ). And thank you to Dr Michael Hynes for agreeing to act as the neutral chair for my thesis defence. I would also like to extend my sincerest appreciation to all the Prenner lab members, both past and present. Thank you to Patrick Lai for our late night gym and lab sessions. To Mark Mahadeo, thank you for all the lunches and educating me on BAM. Thank you to Mohamad Hassanin for pushing me to apply for scholarships and to write. To Neil Berezowski, thank you for our time in out of the lab together and for teaching me how to deposit and use the DSC. على دائما أنا,لك شكرا ) Thank you to Weiam Daear for being my friend and helping me learn Arabic I would also like to acknowledge Dr Leo Nguyen and Mauricio Arias for their help in the.(!حق Biophysics Facility. Thank you to Dr Ronald McElhaney at the University of Alberta for the use of his VP-DSC. Last but certainly not least, I would like to pledge my utmost appreciation and gratitude to my loving parents, Eva and Jack Patterson; I cannot thank you enough for all that you have done for me. To my darling sister, Sarah Patterson, who inspired me to pursue higher education (one day I will have as many degrees as you do)! And finally, thank you to all my friends who have put up with me all these years. iii

4 Table of Contents Abstract... ii Acknowledgements... iii Table of Contents... iv List of Tables... vii List of Figures... ix List of Abbreviations... xviii CHAPTER ONE: INTRODUCTION Tear Film Structure Mucin Aqueous Subphase Tear Film Lipids The Lipid Layer Polar Lipid Layer Lipid-Protein Interactions Pathology Hypothesis and Goal Hypothesis CHAPTER TWO: MATERIALS AND METHODS Langmuir Monolayers Brewster Angle Microscopy Langmuir Troughs Isotherm and Isocycle Trough BAM Trough Circular Trough Solutions Surface Pressure-Area Isotherms Isotherm Analysis Surface Pressure Area Isocycles iv

5 2.6.1 Isocycle Analysis BAM Isocycles Lysozyme Injections Differential Scanning Calorimetry Differential Scanning Calorimetry Procedure CHAPTER THREE: SURFACE PRESSURE-AREA ISOTHERMS Individual Lipid Systems Pure Lipid Compression Modulus Binary Lipid Systems Binary Lipid Compression Modulus Tertiary and Quaternary Lipid Systems Tertiary and Quaternary Lipid Compression Modulus Compression Isotherm Summary CHAPTER FOUR: FILM RESPONSE TO MULTIPLE COMPRESSIONS Surface Pressure-Area Isocycles Individual Lipid Systems Binary Lipid Systems Tertiary and Quaternary Lipid Systems Isocycle BAM Images Individual Lipid Systems Binary Lipid Systems Tertiary and Quaternary Lipid Systems Compression Isocycle Summary CHAPTER FIVE: LYSOZYME INJECTION STUDIES Individual Lipid Systems Binary Lipid Systems Tertiary and Quaternary Lipid Systems Monolayer Rigidity and Lysozyme Adsorption Comparisons v

6 CHAPTER SIX: DIFFERENTIAL SCANNING CALORIMETRY RESULTS Individual Lipid Systems Binary Lipid Systems Tertiary and Quaternary Lipid Systems DSC Results Summary CHAPTER SEVEN: CONCLUSIONS AND FUTURE DIRECTIONS Conclusions Future Directions REFERENCES APPENDIX vi

7 List of Tables Table 1. Composition of the polar lipid layer from tear film, represented in percentage of total polar lipids [30, 32]. The ± is the standard deviation Table 2. The 15 different lipid systems used in the Langmuir trough experiments with their corresponding average molecular weights and ratios. Ratios listed are molar ratios based on the tear film polar lipid layer composition [30]. The average molecular weight was necessary for the computer program to allow plotting of the molecular area for the isotherm experiments Table 3. Results from the compression isotherms of the pure lipid systems. Isotherms were completed on a 1x PBS buffered subphase at room temperature. The error is represented by the standard deviation; n 3. The fourth column contains the literature values for the collapse pressures over a pure water subphase Table 4. The average compression modulus (β, mn/m) at different surface pressures (mn/m) and the average maximum compression modulus value (mn/m) for the individual lipid systems. The compression modulus was obtained from the compression isotherms using the equation in section Error was recorded as the standard deviation (n 3) Table 5. Results from the compression isotherms of the binary lipid systems. Ratios represent the molar fractions of each lipid. Isotherms were completed on a 1x PBS buffered subphase at room temperature. Error is represented by the standard deviation; n Table 6. The average compression modulus (β, mn/m) at different surface pressures (mn/m) and the average maximum compression modulus value (mn/m) for the binary lipid systems. The compression modulus was obtained from the compression isotherms using the equation in section Error is reported as the standard deviation (n 3) Table 7. Results from the compression isotherms of the tertiary and quaternary lipid systems. Ratios represent the molar fractions of each lipid. Isotherms were completed on a 1X PBS buffered subphase at room temperature. Error is represented by the standard deviation; n vii

8 Table 8. The average compression modulus (β, mn/m) at different surface pressures (mn/m) and the average maximum compression modulus value (mn/m) for the tertiary and quaternary lipid systems. The compression modulus was obtained from the compression isotherms using the equation in section Error was recorded as the standard deviation (n 3) Table 9. The RMSD values from the pure lipid systems. The 1 st Cycle RMSD column describes the 1 st compression and expansion. The 5 th Cycle RMSD column describes the 5 th compression and expansion. The Inter-Cycle RMSD column is the overall difference between the 1 st and 5 th cycles. n Table 10. The RMSD values from the binary lipid systems. The 1 st Cycle RMSD column describes the 1 st compression and expansion. The 5 th Cycle RMSD column describes the 5 th compression and expansion. The Inter-Cycle RMSD column is the overall difference between the 1 st and 5 th cycles. n Table 11. The RMSD values from the tertiary and quaternary lipid systems. The 1 st Cycle RMSD column describes the 1 st compression and expansion. The 5 th Cycle RMSD column describes the 5 th compression and expansion. The Inter- Cycle RMSD column is the overall difference between the 1 st and 5 th cycles. n Table 12. The average RMSD of each lipid studied taken from all systems that had that lipid present, excluding the quaternary system. The bolded values were significantly different from each other (p<0.05). n= Table 13. DSC data for the enthalpy, T m, and T 1/2 of the pure lipid MLVs Table 14. DSC data for the enthalpy, T m, and T 1/2 of the binary lipid MLVs. The ratios listed are the molar ratios viii

9 List of Figures Figure 1. Cartoon of the pre-corneal tear film showcasing the three layers and their relative widths in µm [10] Figure 2. (A) In the absence of the polar lipid layer, the non-polar lipids would aggregate and not spread evenly across the aqueous subphase of the tear film. (B) The polar lipid facilitates rapid and even spreading of the nonpolar layer by making favourable interactions with both the aqueous subphase and non-polar lipids Figure 3. Isotherm cycle of human meibomian extract [39]. The graph is read from right to left to represent the film being compressed to smaller areas followed by expansion to larger areas. When looking at a single trace the compression isotherm is the above curve (arrows pointing leftwards) while the expansion isotherm is the bottom curve (arrows pointing rightwards). Permission obtained from Investigative Ophthalmology on 14/05/ Figure 4. Images from molecular simulations illustrating the formation of TG- and COrich protrusions to the air side of the interface at high surface pressures [41]. The area per molecule was (A,D) 64 A 2, (B,E) 53.1 A 2, and (C,F) 42.1 A 2. PC headgroups are shown in orange, TG in pink mesh, CO in green, and FFA (palmitate) in red. Simulations were between 2000 and 3200 ns long Figure 5. Chemical structures for the major polar lipids found in tear film [30, 32]. (A) 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), (B) 1,2-dipalmitoylsn-glycero-3-phosphoethanolamine (DPPE), (C) D-glucosyl-ß-1,1' N- palmitoyl-d-erythro-sphingosine (PGC), (D) N-palmitoyl-D-erythrosphingosylphosphocholine (PSM) Figure 6. Proposed three layered organization of the tear film. The green rectangles represent transmembrane mucin proteins found in the glycocalyx layer with attached sugars represented by purple hexagons. Shown in the aqueous phase are the crystal structures of tear proteins lactoferrin (pdb 1FCK), lysozyme (pdb 2LYZ), and lipocalin (pdb 1XKI), with dissolved salts. The inner polar lipid monolayer is represented by blue lipids and the outer nonpolar layer by black lipids, with inserted tear proteins present. Image is not to scale ix

10 Figure 7. A generalized surface pressure-area isotherm. Plateaus are areas of phase coexistence and kinks represent phase transitions. The horizontal slope at the smallest molecular area represents monolayer collapse. G stands for gas, L E for liquid expanded, and L C for liquid condensed Figure 8. Principle behind Brewster Angle Microscopy. When p-polarized light is incident on a subphase at the Brewster angle in the absence of a film no light will be reflected. In the presence of a lipid film, however, the interfacial properties will change, causing some of the light to be reflected Figure 9. Schematic of the Langmuir trough used for both the isotherm and isocycle experiments. Lipid was deposited on to the subphase and then compressed to a selected area using the electronically controlled barrier. A Wilhelmy plate immersed 1 mm into the subphase was used to detect surface pressure changes Figure 10. Schematic of the BAM microscope and BAM trough. The two moveable barriers can be seen at their open positions. Lipid was deposited near the right barrier. Missing from the photo is the pressure sensor Figure 11. Image of the circular trough used for lysozyme injection studies. 1x PBS buffer was added to the inner compartment. The syringe used to inject lysozyme solution can be seen inside the injection port. It was placed into position prior to lipid deposition to prevent monolayer perturbation during the experiment. Additionally, all components were secured in place using lab tape Figure 12. (A) Hypothetical example of a single compression expansion isocycle. The film is first compressed to a given surface pressure then expanded, resulting in a loop like curve. The blue trace is the compression isotherm; the red trace is the expansion isotherm. (B) Hypothetical example of two complete isocycles. The first cycle in blue and the 5 th cycle in green were compared to how well they overlapped with each other Figure 13. Hypothetical surface pressure (Π) vs. time graph. Once the surface pressure had equilibrated, lysozyme was injected into the subphase at the time indicated by the arrow. The trials were then allowed to run for an additional 1600 seconds. The change in surface pressure, Π, was recorded based on the average surface pressure from the last 100 seconds minus the baseline x

11 Figure 14. An example DSC thermogram of the gel to liquid-crystalline phase transition. Illustrated are the thermodynamic properties directly reported from the DSC where T m is the melting temperature, T 1/2 is the peak width at half-height, and H cal is the enthalpy corresponding to the area under the peak Figure 15. Average compression isotherm (n 3) for the four different pure lipid systems on a 1x PBS subphase. DPPC (red), DPPE (green), PGC (purple), and PSM (blue). Arrows highlight points of interest mentioned such as kinks or regions over isothermal overlap Figure 16. A PGC glucose head group hydrogen bonding to surrounding water molecules. A kosmotropic anion is shown on the left, polarizing one of the water molecules Figure 17. Representative compression modulus curves for the four different pure lipid systems. DPPC (red), DPPE (green), PGC (purple), and PSM (blue). The black line represents the compression modulus value (100 mn/m) in which monolayers transition from the L E to the L C phase. Arrows highlight specific points of interest such as local minima Figure 18. Schematic illustrating the increased distance between DPPE head groups following anion adsorption (red circle) Figure 19. (A) Average compression isotherm (n 3) for the different binary lipid systems. The ratios listed are the molar ratios. 3DPPC:2DPPE (red), 1DPPC:1PGC (green), 3DPPC:2PSM (purple), 2DPPE:3PGC (light blue), 1DPPE:1PSM (orange), and 3PGC:2PSM (dark blue). (B) Enlarged display of the high surface pressure region of the binary compression isotherms. Double sided arrow emphasizes the near isothermal overlap at higher pressures Figure 20. A cartoon illustrating the packing differences between the binary systems of DPPC (red disks) and PGC (purple disks), and PSM (blue disks) and PGC, at 5 mn/m. The scale used to draw the circles was 5 Å 2 : 1 cm 2. The box has an area of 350 Å 2. (A) has 20 disks of 10 DPPC and 10 PGC representing the 1DPPC:1PGC molar ratio; (B) has 20 disks of 12 PGC and 8 PSM representing the 3PGC:2PSM molar ratio. The dark grey behind the lipid disks represents the free surface area. Panel B has more free surface than A illustrating how greater amounts of larger lipids can lead to film expansion xi

12 Figure 21. Representative compression modulus curves for the six different binary lipid systems. 3DPPC:2DPPE (red), 1DPPC:1PGC (green), 3DPPC:2PSM (purple), 2DPPE:3PGC (light blue), 1DPPE:1PSM (orange), and 3PGC:2PSM (dark blue). The black line represents the compression modulus value (100 mn/m) in which monolayers transition from the L E to the L C phase. Arrows highlight areas where the slope changes, kinks are present, or other points of interest Figure 22. Average compression isotherm (n 3) for the four different tertiary and quaternary (3DPPC:2DPPE:3PGC:2PSM) mixtures on a 1x PBS subphase. 3DPPC:2DPPE:3PGC (red), 3DPPC:2DPPE:2PSM (green), 3DPPC:3PGC:2PSM (purple), 2DPPE:3PGC:2PSM (orange), and quaternary mixture (blue). Arrows highlight plateau regions Figure 23. Representative compression modulus curves for the different tertiary and quaternary lipid systems. 3DPPC:2DPPE:3PGC (red), 3DPPC:2DPPE:2PSM (green), 3DPPC:3PGC:2PSM (purple), 2DPPE:3PGC:2PSM (orange), and quaternary (blue). The black line represents the compression modulus value (100 mn/m) in which monolayers transition from the L E to the L C phase. Arrows highlight specific points of interest such as local minima Figure 24. Examples illustrating which part of the isocycle belongs to the compression or expansion trace (A) and what is meant by the 1 st and 5 th cycles (B). The arrows in panel B illustrate comparisons within a cycle (the longer arrow) and between cycles (the shorter arrow) Figure 25. Selected surface pressure-area isocycles of the single lipid systems. (A) DPPC (B) DPPE (C) PGC (D) PSM. The first cycle is shown in blue and the fifth cycle in red. Each film was compressed until 15 mn/m before collapse and expanded until zero surface pressure was reached. The lipid system is shown above each graph. n Figure 26. Selected surface pressure-area isocycles of the binary lipid systems. (A) 3DPPC:2DPPE (B) 1DPPC:1PGC (C) 3DPPC:2PSM (D) 2DPPE:3PGC (E) 1DPPE:1PSM (F) 3PGC:2PSM.The first cycle is shown in blue; the fifth cycle is in red. Each film was compressed until 15 mn/m before collapse and expanded until zero surface pressure was reached. The lipid system is shown above each graph. n xii

13 Figure 27. Selected surface pressure-area isocycles of the tertiary and quaternary lipid systems. (A) 3DPPC:2DPPE:3PGC (B) 3DPPC:2DPPE:2PSM (C) 3DPPC:3PGC:2PSM (D) 2DPPE:3PGC:2PSM (E) 3DPPC:2DPPE:3PGC:2PSM.The first cycle is shown in blue and the fifth cycle in red. Each film was compressed until 15 mn/m before collapse and expanded until zero surface pressure was reached. The lipid system is labelled above each graph. n Figure 28. BAM images from the DPPC isocycles. The figure is split into columns containing images from the different isocycle regions, labelled at the top of the figure. Each image is 218 x 271 microns; the scale bar is 50 microns Figure 29. BAM images from the DPPE isocycles. The figure is split into columns containing images from the different isocycle regions, labelled at the top of the figure. Each image is 218 x 271 microns; the scale bar is 50 microns. Inset is an enlarged image from the orange box Figure 30. BAM images from the PGC isocycles. The figure is split into columns containing images from the different isocycle regions, labelled at the top of the figure. Each image is 218 x 271 microns; the scale bar is 50 microns. Inset is an enlarged image from the orange box Figure 31. BAM images from the PSM isocycles. The figure is split into columns containing images from the different isocycle regions, labelled at the top of the figure. Each image is 218 x 271 microns; the scale bar is 50 microns. Insets represent enlarged images from the boxes Figure 32. BAM images from the 3DPPC:2DPPE isocycles. The figure is split into columns containing images from the different isocycle regions, labelled at the top of the figure. Each image is 218 x 271 microns; the scale bar is 50 microns Figure 33. BAM images from the 1DPPC:1PGC isocycles. The figure is split into columns containing images from the different isocycle regions, labelled at the top of the figure. Each image is 218 x 271 microns; the scale bar is 50 microns xiii

14 Figure 34. BAM images from the 3DPPC:2PSM isocycles. The figure is split into columns containing images from the different isocycle regions, labelled at the top of the figure. Each image is 218 x 271 microns; the scale bar is 50 microns. Inset is an enlarged image from the orange box Figure 35. BAM images from the 2DPPE:3PGC isocycles. The figure is split into columns containing images from the different isocycle regions, labelled at the top of the figure. Each image is 218 x 271 microns; the scale bar is 50 microns. Insets represent enlarged images from the boxes Figure 36. BAM images from the 1DPPE:1PSM isocycles. The figure is split into columns containing images from the different isocycle regions. Frame C's insert is a magnification of the orange box. Each image is 218 x 271 microns; the scale bar is 50 microns. Inset is an enlarged image from the orange box Figure 37. BAM images from the 3PGC:2PSM isocycles. The figure is split into columns containing images from the different isocycle regions, labelled at the top of the figure. Each image is 218 x 271 microns; the scale bar is 50 microns. Insets represent enlarged images from the boxes Figure 38. BAM images from the 3DPPC:2DPPE:3PGC isocycles. The figure is split into columns containing images from the different isocycle regions, labelled at the top of the figure. Each image is 218 x 271 microns; the scale bar is 50 microns. Insets represent enlarged images from the boxes Figure 39. 3D images of a bright spot domain (A) and of the surrounding L C phase (B) from 3DPPC:2DPPE:3PGC films at 27.1 mn/m during the 1 st compression. (C) Representative compression modulus curve from the 3DPPC:2DPPE:3PGC system. The blue line represents the surface pressure (27 mn/m) where the potential multilayer formation occurs Figure 40. BAM images from the 3DPPC:2DPPE:2PSM isocycles. The figure is split into columns containing images from the different isocycle regions, labelled at the top of the figure. Each image is 218 x 271 microns; the scale bar is 50 microns. Inset represents enlarged image from the green box Figure 41. 3D analysis of the bright circular domain from the 5 th compression of the 3DPPC:2DPPE:2PSM film at 32.2 mn/m xiv

15 Figure 42. BAM images from the 3DPPC:3PGC:2PSM isocycles. The figure is split into columns containing images from the different isocycle regions, labelled at the top of the figure. Each image is 218 x 271 microns; the scale bar is 50 microns Figure 43. BAM images from the 2DPPE:3PGC:2PSM isocycles. The figure is split into columns containing images from the different isocycle regions, labelled at the top of the figure. Each image is 218 x 271 microns; the scale bar is 50 microns. Inset represents enlarged image from the green box Figure 44. 3D analysis of the circular bright domain from the 5 th compression of the 2DPPE:3PGC:2PSM film at 27.5 mn/m Figure 45. BAM images from the 3DPPC:2DPPE:3PGC:2PSM isocycles. The figure is split into columns containing images from the different isocycle regions, labelled at the top of the figure. Each image is 218 x 271 microns; the scale bar is 50 microns. Insets represent enlarged images from the boxes Figure 46. 3D analysis of the circular bright domain from the 3DPPC:2DPPE:3PGC:2PSM (quaternary) film during the 1 st compression at 28.2 mn/m (A) and the 5 th compression at 31.9 mn/m (B) Figure 47. Surface activity of 50 µg/ml of lysozyme determined by an increase in surface pressure following protein injection into a 1x PBS buffer subphase in the absence of a lipid film and measured by a surface pressure sensor Figure 48. Single lipid system lysozyme injections. The x-axis represents the different initial surface pressures prior to lysozyme injection. For each initial surface pressure, the final lysozyme concentration was 0.05 mg/ml. The y-axis is the change in the initial surface pressure following lysozyme injection. (A) DPPC (B) DPPE (C) PGC (D) PSM. Significance was determined in relation to the control, * p<0.05, ** p<0.01. The error bars represent the standard deviation. n Figure 49. Surface pressure change following lysozyme injection at 30 mn/m in a PSM monolayer. The figure is used to emphasize the gradual decrease in surface pressure over 30 minutes xv

16 Figure 50. Binary lipid system lysozyme injections. The x-axis represents the different initial surface pressures prior to lysozyme injection. For each initial surface pressure, the final lysozyme concentration was 0.05 mg/ml. The y-axis is the change in the initial surface pressure following lysozyme injection. (A) 3DPPC:2DPPE (B) 1DPPC:1PGC (C) 3DPPC:2PSM (D) 2DPPE:3PGC (E) 1DPPE:1PSM (F) 3PGC:2PSM. Significance was determined in relation to the control, * p<0.05, ** p<0.01, *** p< The error bars represent the standard deviation Figure 51. Tertiary and quaternary lipid system lysozyme injections. The x-axis represents the different initial surface pressures prior to lysozyme injection. For each initial surface pressure, the final lysozyme concentration was 0.05 mg/ml. The y-axis is the change in the initial surface pressure following lysozyme injection. (A) 3DPPC:2DPPE:3PGC (B) 3DPPC:2DPPE:2PSM (C) 3DPPC:3PGC:2PSM (D) 2DPPE:3PGC:2PSM (E) 3DPPC:2DPPE:3PGC:2PSM. Significance was determined in relation to the control, * p<0.05, ** p<0.01, *** p<0.005.the error bars represent the standard deviation Figure 52. Cartoon showcasing the different head group size and orientations of the lipids used in the polar layer biomimetic. The hydrocarbon tails were drawn at the same distance from each species. A vertical orientation (PGC) or a small head group (DPPE) allows for greater hydrophobic interactions between lysozyme and the lipid hydrocarbon tails Figure 53. Change in initial surface pressure following lysozyme injection ( Π) versus compression modulus (β). The compression modulus was taken from the values at the surface pressure prior to injection. These pressures were; 10 mn/m (A), 20 mn/m (B), and 30 mn/m (C). Each point represents one of the 15 different lipid systems Figure 54. DSC thermograms of the pure lipid systems. DPPC (red), DPPE (green), PGC (purple), and PSM (blue). The final concentration was 1 mg/ml of lipid. The arrows, red for DPPC and purple for PGC, represent observed lamellar gel to ripple gel phase pre-transitions. The red shaded box represents the melting range of the natural tear film xvi

17 Figure 55. DSC thermogram of the six binary systems in their specified molar ratios. 3DPPC:2DPPE (red), 1DPPC:1PGC (green), 3DPPC:2PSM (purple), 2DPPE:3PGC (light blue), 1DPPE:1PSM (orange), and 3PGC:2PSM (dark blue).the final lipid concentration was 1 mg/ml. The red shaded box represents the melting range of the natural tear film Figure 56. DSC thermogram of the tertiary system 3DPPC:2DPPE:2PSM. The vertical line represents the T m of pure DPPC and PSM (green line). The red shaded box represents the melting range of the natural tear film Figure 57. DSC thermogram of the tertiary system 3DPPC:2DPPE:3PGC. The vertical lines represent the T m of pure DPPC (green line) and pure DPPE (red line). The red shaded box represents the melting range of the natural tear film Figure 58. DSC thermogram of the tertiary system 3DPPC:3PGC:2PSM. The vertical line represents the T m of pure DPPC or PSM (green line). The red shaded box represents the melting range of the natural tear film Figure 59. DSC thermogram of the tertiary system 2DPPE:3PGC:2PSM. The vertical lines represent the T m of pure PSM (green line) and pure DPPE (red line). The red shaded box represents the melting range of the natural tear film Figure 60. DSC thermogram of the quaternary system 3DPPC:2DPPE:3PGC:2PSM. The vertical lines represent the T m of pure DPPC or PSM (green line) and pure DPPE (red line). The red shaded box represents the melting range of the natural tear film Figure 61. DSC thermogram of the quaternary system 3DPPC:2DPPE:3PGC:2PSM in the presence of lysozyme at a 30:1 lipid to protein ratio (A). The vertical lines represent the T m of pure DPPC or PSM (green line) and pure DPPE (red line). The red shaded box represents the melting range of the natural tear film. Panel B represents overlay comparison of the lipid pure thermogram onto lysozyme and quaternary mixtures in their specified lipid to protein ratios. Quaternary only (blue), 30:1 lipid to protein (red), and 15:2 lipid to protein (green) xvii

18 List of Abbreviations AMP β BAM CB CE CO Cp C s DES DPPC DPPE DSC FFA G GC L C L E MLV NMR Π PBS PC Antimicrobial peptides Compression modulus Brewster angle microscopy Cerebrosides Cholesterol esters Cholesterol oleate Heat capacity at constant pressure Compressibility Dry eye syndrome 1,2-dipalmitoyl-sn-glycero-3-phosphocholine 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine Differential scanning calorimetry Free fatty acids Gas Glucosylceramide Liquid condensed Liquid expanded Multilamellar vesicle Nuclear magnetic resonance Surface pressure Phosphate buffered saline Phosphatidylcholine xviii

19 PE PG PGC PS PSM RMSD SM TG T m T 1/2 WE Phosphatidylethanolamine Phosphatidylglycerol D-glucosyl-ß-1,1' N-palmitoyl-D-erythro-sphingosine Phosphatidylserine N-palmitoyl-D-erythro-sphingosylphosphocholine Root mean squared deviation Sphingomyelin Triglycerides Transition temperature Temperature at half height Wax esters xix

20 1 Chapter One: Introduction Initial studies of tear film date back to 1946 when Wolff first described the trilaminar, or three layered, structure of tear film comparing it to layers of paint [1]. While adjustments and revisions have since been made regarding tear film organization, this basic idea provides a good starting point for discussion. The multilayered fluid structure not only offers a high quality optical surface necessary for vision but also protects the eye from foreign particles and pathogens, prevents excessive evaporation, and transports nutrients to the corneal epithelium [2]. Despite constant exposure to the elements, it is an amazing feat that the eye does not dry out nor succumb to bacterial infections. The tear film structure is generally between µm thick over the cornea with increasing film thickness along the lid margin, at roughly 270 µm [3, 4]. The thickness affects tear physiology and function, especially tear flow, film breakup, and the tear volume [5-7]. The tear fluid itself also exhibits viscoelastic, non-newtonian properties, where whole tears were shown to be shear thinning [8]. This means that tear film is both viscous because it resists deformation in a time dependent manner and elastic because it is capable of reversible deformation. Tear film also exhibits shear-thinning properties because its viscosity decreases as more shear stress is applied, providing better lubrication for the eyelids during a blink [8]. Additionally, tear film can be classified differently depending on the situation. For example, in the presence of a contact lens, the tear film is divided into the pre- and post-lens tear film [9]. The pre-conjunctival tear film that lines the sclera or white of the eye underneath the lid, is the least studied and thus the least understood because it is not involved in vision [10]. Tear film adjacent to the cornea is referred to as the pre-corneal tear film (fig. 1). The pre-

21 2 corneal tear film is the most studied and will be the focus of this thesis. Any mention of tear film from here on will refer exclusively to the pre-corneal tear film, unless stated otherwise. Figure 1. Cartoon of the pre-corneal tear film showcasing the three layers and their relative widths in µm [10]. 1.1 Tear Film Structure Mucin The classical description of tear film suggests three distinct layers lying adjacent to the epithelial cells of the eye, consisting of a posterior or inner mucus layer, a middle aqueous layer, and an anterior or outer lipid layer [1, 11]. However, in recent years the idea of a distinct mucus

22 3 layer has been questioned, blurring the lines between the aqueous and mucus layers, which is especially evident if the highly hydrophilic nature of the mucus is considered [12]. Mucus, consisting of mostly high molecular weight glycosylated proteins known as mucins is found on the apical surfaces of all wet-surfaced epithelial cells [13]. Within tear film, mucins exist as both soluble and membrane associated proteins. The latter form the glycocalyx, a type of gel found along the corneal epithelium, while the soluble mucins are distributed throughout the aqueous layer [14]. Initial hypotheses suggested that mucins were primarily responsible for the high, non- Newtonian viscosity of tear film, while also providing lid lubrication, wettability to the cornea, and stability to the overall tear film structure [15]. Recently, however, it was determined that the corneal epithelium itself is hydrophilic, thus negating the necessity of mucins to hydrate the surface [16]. As well, Tiffany et al. were unable to detect any significant amount of soluble mucin within tears, suggesting that mucin does not play a major role in the interfacial or rheological properties of tears [17]. All of these findings indicate that the primary role of mucins, within tear film, may be to solely act as a barrier to pathogens and debris along the ocular surface, much like mucin within the respiratory system [18, 19] Aqueous Subphase The aqueous subphase of tear film originates from within the lacrimal glands and contains many different electrolytes, metabolites, and proteins [20]. One important role of this layer is to carry nutrients and oxygen to the cornea, which is avascular. The protein concentration within the aqueous layer is very high at 8 mg/ml, with at least 200 distinct proteins present [21]. Due to the constant exposure to the environment, it is not surprising that many of these proteins are involved in wound healing, inflammatory response, and provide

23 4 protection from pathogens [22]. de Souza et al. have identified lysozyme, lactoferrin, tear lipocalin, secretory immunoglobin A, immunoglobin G, lipophilin, and serum albumin, as the major proteins in the aqueous subphase [22]. These are found at a variety of concentrations, ranging from mg/ml for immunoglobin A at the low end, and up to mg/ml for lysozyme [23]. As well, it has been shown that in addition to the aforementioned properties, many tear proteins are surface active and may play a role in stabilizing the tear film structure [24, 25]. Significant portions of this proteome, however, remain unidentified [26]. This is possibly due to the wide concentration ranges and because the three dominant proteins, lysozyme, lactoferrin, and tear lipocalin, make up 80% of the total, obscuring the results for proteins present at much lower quantities [26] Tear Film Lipids The anterior lipid layer lies at the air-water interface on the eye s surface. The lipids were initially believed to exclusively originate from the meibomian glands (found along the inside of each eye lid). The lipid secretion, often referred to as meibum, is delivered to the eye lid margin either through small aliquots following a blink, or as a steady secretory process [27]. Meibum is rich in non-polar lipids, primarily comprised of cholesterol esters (CE) and wax esters (WE) [28]. The fatty acid chains on these esters are very long, ranging from carbons, and can be both saturated and unsaturated [29]. Of these, oleic acid (18:1) is the predominant species at 31%, followed by linoleic acid (18:2) and stearic acid (18:0), with small amounts of free cholesterol [29]. In terms of the phospholipids present, phosphatidylcholine (PC), phosphatidylethanolamine (PE), and sphingomyelin (SM), make up the major species (table 1) [30, 31]. Other polar lipids such as cerebrosides (CB) have also been detected within the lipid

24 5 layer [30]. The fatty acyl chains of the polar lipids are fully saturated, with 14, 16, or 18 carbons in length [32]. For sphingolipids, the amine linked tail has a chain length that is generally 12, 14, or 16 carbons long. It has to be emphasized that 24% of the phospholipid fraction is still unknown [30, 32]. Recent discoveries have suggested the presence of lyso phospholipids and plasmalogens within the polar lipid layer [33, 34], suggesting that these lipids may make up the remaining unknown fraction. However, for this thesis the investigations regarding the polar lipids of tear film will focus on those lipid species listed in table 1 because they have been validated through multiple sources [28, 30-32]. Table 1. Composition of the polar lipid layer from tear film, represented in percentage of total polar lipids [30, 32]. The ± is the standard deviation. Lipid Mean % Phosphatidylcholine 21.6 ± 5.3 Phosphatidylethanolamine 16.7 ± 4.0 Cerebrosides 23.5 ± 8.5 Sphingomyelin 14.0 ± 8.1 Unknown 24.0 ± 2.7 Controversy has surrounded the lipid composition of tear film, arising in differences regarding both the amount and types of lipids present [28]. Furthermore, the different compositions have been shown to be dependent on the analysis method [28]. For instance, the

25 6 incidence of phospholipids within meibum has varied considerably, with estimates in the range of 0 to 16% by dry weight [28]. Although the entire lipidome of the tear film remains unclear and somewhat disputed, there is an obvious need for the presence of polar lipids. Without polar lipids the very hydrophobic CE and WE would not spread evenly across an aqueous surface, but rather aggregate into droplets (fig. 2A). On the other hand, the polar phospholipids and cerebrosides spread rapidly, due to their amphiphilic properties. The polar head groups make favourable interactions with the aqueous subphase, while the non-polar acyl tails would orient towards the air, forming a monolayer. A polar monolayer provides the necessary framework for the spreading of hydrophobic CE and WE, by minimizing their interaction with the aqueous subphase (fig. 2B). In fact, if there were no phospholipids within meibum, it would suggest that not all tear film lipids are of meibomian origin. Some have postulated that non-meibomian lipids may originate from either conjunctival cells (those that line the inside of your eyelid) or from corneal epithelial cells [35].

26 7 Figure 2. (A) In the absence of the polar lipid layer, the non-polar lipids would aggregate and not spread evenly across the aqueous subphase of the tear film. (B) The polar lipid facilitates rapid and even spreading of the non-polar layer by making favourable interactions with both the aqueous subphase and non-polar lipids. 1.2 The Lipid Layer Due to the initial controversy over the lipid layer composition of tear film, the majority of the literature has focused on meibum extracts or the hydrophobic components exclusively. Many of these experiments involved compressing films of lipid samples at the air-water interface,

27 8 simulating the pre-corneal environment, to infer biophysical properties. These so called surface pressure-area isotherms are reviewed in Chapter 2, section 2.1. One of the earliest experiments on tear film investigated the surface activity of meibomian extracts. These lipids were reported to be surface active, and reached a surface pressure of 12.4 mn/m [36]. However, these experiments were conducted on a water subphase at a ph of 5.5, and a compression rate of 20 mm 2 /min. Both factors have been shown to affect surface pressure measurements which may account for the far smaller surface pressure values compared to recent findings of >20 mn/m [37, 38]. Mudgil and Millar continued the meibomian lipid surface experiments by revisiting surface pressure-area changes but expanded their investigations to include different physical conditions such as temperature, subphase type, nature and concentration of divalent cations, and osmolarity [39]. Changes were evident in the maximum surface pressure between a buffered subphase and a pure water subphase, but not by the osmolarity or buffer type [39]. Interestingly, while heating the meibomian lipids produced only isotherm differences at the largest molecular areas, cooling the meibomian lipids increased the surface pressure at the smallest molecular area [39]. Isotherms were completed at 20, 30, 40, and 50 C, either starting at 20 C and warmed up to 50 C, or starting at 50 C and cooled down to 20 C. The heating results are to be expected, as at higher temperatures the molecules would take up larger areas due to increased movement. However, cooling enhanced the surfactant properties of meibum (fig. 3), exhibited by an increase in the surface pressure at smaller molecular areas [39]. These results suggest that lipid packing and reorganization occurs following the input of thermal energy and made the film more resistant to collapse. Meibum exhibits a melting range of C due to its complex composition [40]. Heating above the upper melting limit may induce different phases upon cooling that would not normally be

28 9 encountered during the initial heating, explaining the dissimilar surface properties seen between heated and cooled meibum samples. Finally, the meibomian lipid isotherms indicated the reversible formation of multilayers, illustrated by multiple compression expansion cycles reaching molecular areas of roughly 10 Å 2 /molecule, with little hysteresis between the cycles [39]. Molecular areas this small are physically impossible without monolayer collapse and/or multilayer formation. In the greater context of tear film, the act of blinking necessitates a film that rapidly compresses and reversibly expands, but also resists collapse at high surface pressures, both conditions observed for meibomian lipids in vitro. As well, hysteresis describes the dependence of the system on both its current and past environments. In the case of isotherm cycles, more hysteresis is present in cases where there are larger differences between compression and expansion of the same film. These differences arise from changes in packing during compression and are dependent on the elasticity of the film. Hysteresis is an indication of the degree of deformation that has occurred. In figure 3, there is less hysteresis present at 41 C than at 35 C.

29 10 Figure 3. Isotherm cycle of human meibomian extract [39]. The graph is read from right to left to represent the film being compressed to smaller areas followed by expansion to larger areas. When looking at a single trace the compression isotherm is the above curve (arrows pointing leftwards) while the expansion isotherm is the bottom curve (arrows pointing rightwards). Permission obtained from Investigative Ophthalmology on 14/05/2014. However, beyond the basic understanding of a non-polar lipid layer resting on a polar lipid monolayer, the detailed organization of tear film remains poorly understood. In vitro molecular organization experiments have combined near atomistic molecular dynamic simulations and different microscopic techniques such as X-ray diffraction, Brewster angle, fluorescence, and atomic force microscopies, in conjunction with Langmuir film isotherms [41].

30 11 Grazing incidence X-ray diffraction and fluorescence microscopy of a bovine meibomian film revealed the co-existence of expanded and condensed phases at surface pressures in the range between 15 and 45 mn/m [42]. Using a tear film model made up of PC, free fatty acids (FFA), cholesterol oleate (CO), and triglycerides (TG), atomic force microscopy showed the formation of a multilayered structure present at surface pressures of 20 and 30 mn/m [41]. It was earlier hypothesized that during a blink tear film reversibly compresses to form multilayered structures, as demonstrated in the aforementioned papers, instead of rupturing or collapsing at each blink. Using coarse grained simulations of the same tear film model, the lipid layer exhibited massive restructuring as the pressure increased from 15 mn/m to 50 mn/m [41]. During compression to smaller molecular areas, phase separation between the PC-FFA layer, at the water surface, and the CO-TG layer become more pronounced (fig. 4). At 42.1 Å/mol full separation between the PC-FFA and the CO-TG populations resulted in multilayer formation (fig. 4C,F) [41]. Even more striking, the non-polar layer formed hemisphere like protrusions towards the air (fig. 4F), while the FFA acted as a mediator between the phospholipids and non-polar lipids [41]. In the presence of only phospholipids, reduction to these molecular areas would generally fold into the water subphase to reduce the free energy of the system at high surface pressures [43]. However, due to the presence of hydrophobic lipids residing on top of the phospholipids, the formation of globular hydrophobic protrusions towards the air would possibly constitute a lesser free energy cost, allowing for reversible folding. On the other hand, when the ratio of polar to hydrophobic lipids is low, the tear film stability is decreased and reversible compression is removed because a continuous planar lipid layer is not formed at the air-water interface when compressed [44]. As well, this suggests that the composition of tear film needs to be investigated further and is most likely not entirely of meibomian origin. Finally, the molecular phenomena of reversible folding

31 12 and organization of the different lipid phases has only been observed in vitro and will need to be confirmed in vivo. Figure 4. Images from molecular simulations illustrating the formation of TG- and CO-rich protrusions to the air side of the interface at high surface pressures [41]. The area per molecule was (A,D) 64 A 2, (B,E) 53.1 A 2, and (C,F) 42.1 A 2. PC headgroups are shown in orange, TG in pink mesh, CO in green, and FFA (palmitate) in red. Simulations were between 2000 and 3200 ns long.

32 Polar Lipid Layer While many surface activity experiments have been conducted with the different phospholipids, sphingomyelins, and ceramides [45], not much work has been done analyzing mixtures of these lipids in the context of tear film, especially outside of biosimulations [41]. Common to the listed polar lipids are high surface activity and stability of these films when compressed to small molecular areas [46-48]. As well some PC, SM, and GC, are very compressible suggesting that they may also play an important role during blinking [48]. Finally, the structure of some of the major polar lipids present in tear film exhibit local charges, but are zwitterionic, and thus overall neutral (fig. 5). These localized charges may form favourable electrostatic interactions with many of the proteins found in tear film, and therefore be a driving force for protein interactions and insertion to the lipid layer. However, more work needs to be completed on the polar lipid species and their impact on the tear film layer to better understand the biophysical behaviour of the tear film.

33 14 Figure 5. Chemical structures for the major polar lipids found in tear film [30, 32]. (A) 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), (B) 1,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine (DPPE), (C) D-glucosyl-ß-1,1' N-palmitoyl-D-erythro-sphingosine (PGC), (D) N-palmitoyl-D-erythro-sphingosylphosphocholine (PSM). 1.3 Lipid-Protein Interactions Figure 6 illustrates the proposed possible molecular organization of tear film. As mentioned earlier, the surface activity of tear film does not only depend on the lipid components. While the tear proteins have been observed to engage in a variety of roles, such as antibacterial protection, many have also been implicated in the stability of tear film. To explore this potential further, surface activity experiments were first conducted on individual tear proteins and then

34 15 compared to meibomian lipid isotherms. The surface activity of individual tearproteins lipocalin,, lactoferrin, lysozyme, secretory IgA, and human serum albumin, was investigated by Tragoulias et al. and compared to that of meibomian lipids and tear fractions [24]. Tear fractions had a maximum surface pressure of 24.0 mn/m, with tear proteins ranging from 16.5 mn/m for lipocalin and 25.7 mn/m for lysozyme, while meibomian films collapsed at 15.0 mn/m [24]. These results indicate that individual tear proteins were most similar to the isotherm profile of the tear fractions, not only with respect to the maximum surface pressure obtained but also the degree of hysteresis and the location of isotherm inflection points [24]. These points or kinks in isotherms represent phase transitions in the monolayer, when changes in lateral packing and density occur. As well, the large hysteresis seen in the protein isotherm data has been attributed to a wide variance in structural domains due to both protein folding/unfolding and the orientation of different domains at the air-water interface [49]. These studies indicate that the surface activity of tear film and therefore its stability, most likely arises from the complex interplay of soluble proteins with the lipid layer at the air-water interface.

35 16 Figure 6. Proposed three layered organization of the tear film. The green rectangles represent transmembrane mucin proteins found in the glycocalyx layer with attached sugars represented by purple hexagons. Shown in the aqueous phase are the crystal structures of tear proteins lactoferrin (pdb 1FCK), lysozyme (pdb 2LYZ), and lipocalin (pdb 1XKI), with dissolved salts. The inner polar lipid monolayer is represented by blue lipids and the outer non-polar layer by black lipids, with inserted tear proteins present. Image is not to scale. Miano et al. simulated a model tear comprised of bovine meibomian lipids on a saline subphase to test for protein integration into the film [50]. Lysozyme, lactoferrin, lipocalin, IgA,

36 17 and serum albumin were shown to insert into the bovine meibomian film at initial surface pressures up to 30 mn/m [50]. As well, tear lipocalin had the greatest affinity for insertion, causing surface pressure increases in films with initial surface pressures in excess of 35 mn/m [50]. Finally, while all of the tear proteins adsorbed to the bovine meibomian film in a time dependent manner, lysozyme did so almost three times slower than the other tear proteins, suggesting that folding changes may occur prior to insertion [50]. Further, interfacial enzymatic activity assays on lysozyme showed that the native activity was retained when adsorbed to the meibomian lipids but was not observed in the absence of lipid [50]. This indicates that meibomian lipids may stabilize or protect the tear proteins against denaturation following their insertion. Insertion of lysozyme to the air-water interface was also investigated in the presence of differently charged phospholipid monolayers. Mudgil et al. demonstrated that at surface pressures below 10 mn/m lysozyme inserted to monolayers of either PC, PE, phosphatidylserine (PS), and phosphatidylglycerol (PG) [51]. PC and PE are zwitterionic, while PG and PS carry a net negative charge. At the experimental ph of 7.4, lysozyme will be positively charged, thus the initial adsorption to these phospholipid monolayers may be electrostatically driven. However, the time dependent insertion into meibomian films, as described above, may be due to the rearrangements of hydrophobic protein residues, leading to interactions with the non-polar acyl tails. Indeed, net negative charge was not a necessity for insertion as PE films had the same maximum surface pressure for lysozyme insertion of 20 mn/m as did PG and PS [51]. These results reaffirm that lysozyme may insert into the lipid layer of tear film, thereby contributing to the stability of the overall structure.

37 18 Originally referred to as tear specific pre-albumin, tear lipocalin is also known to adsorb to various phospholipids (PC, PG-PC mix, and PC-sphingosine mix) and meibomian monolayers [52, 53]. Tear lipocalin absorbed to these films in a time dependent manner, with greater increases in surface pressure and less time to reach maximal surface pressure increases than lysozyme [52]. Additionally, lipocalin interactions with anionic and zwitterionic phospholipid monolayers had similar insertion rates and maximum insertion surface pressures to lysozyme [52]. This suggests that tear lipocalin insertion is independent of the monolayer surface electric charge. Furthermore, lipocalin can exist in both a lipid bound and lipid free form. Experiments with both the apo- and holo-tear lipocalins exhibited adsorption to meibomian lipid films, whereby the apo forms produced more stable films [53]. Adsorption of both forms of tear lipocalin changed the lateral packing of the meibomian films. Studies using fluorescently labeled lipid showed that holo-tear lipocalin produced bright large patches while the apo form had a mix of large and small domains [53]. It was originally believed that apo-tear lipocalin recycled incompatible lipids from the corneal epithelium and transferred them to the interfacial lipid layer [54]. While tear lipocalin can bind a wide range of neutral and charged lipids it exhibits no lipid transfer activity between liposomes or proteoliposomes [52]. This suggests that tear lipocalin s primary role is not to scavenge corneal lipids, but instead to impart stability to the tear film. It is also possible that the binding of lipid is required to ensure proper folding of tear lipocalin. The third most abundant tear protein, lactoferrin, provides additional antibacterial potential while also inhibiting biofilm formation, both important functions of the innate immune system associated with the ocular surface (reviewed in [55]). The antibacterial properties of lactoferrin result from its ability to outcompete bacteria in binding free iron, an essential

38 19 bacterial cofactor [56]. Furthermore, many potent antimicrobial peptides (AMPs) have been synthesized using sequences from lactoferrin [57]. In terms of its role within the lipid layer of tear film, surface tension experiments have shown that lactoferrin was capable of inserting into meibomian lipid extracts [50]. Finally, lactoferrin insertion occurred at a broad range of surface pressures, including those at the upper physiological tear film surface pressure of 30 mn/m [50]. Also present within the aqueous subphase of tears is secretory phospholipase A 2 (PLA 2 ) protein, albeit at much lower concentrations compared to the previously discussed soluble proteins at approximately 0.03 mg/ml [58]. While there currently exists no evidence regarding PLA 2 s direct interaction with the tear film lipid layer either through adsorption or insertion, PLA 2 may instead play an indirect role. For instance, PLA 2 catalyzes the hydrolysis of the sn-2 acyl bond on phospholipids giving rise to free fatty acids and lysophospholipids [59]. Lysophospholipids have unique physical properties when compared to their diacylphospolipid precursors [60], suggesting that the activity of PLA 2 may have far reaching implications regarding the form and function of the tear film lipid layer due to the production of lysophospholipids. For example, because of their structure lysophospholipids have been shown to disrupt biological membranes [60] and alter the structure of PC liposomes [61]. However, more work is needed to determine the impact of PLA 2 on tear film in terms of both direct protein interactions and those associated with lysophospholipids. 1.4 Pathology The cornea is one of the few areas on the human body that is not continually covered by a protective epidermal layer, exposing it both to the elements and to microbial invasion. Yet, due to the ongoing dynamic action of the pre-corneal tear film, the ocular surface does not dry out,

39 20 nor is it easily infected. The aqueous subphase is continuously recycled as tears flow down through the upper lid margin from the lacrimal gland and out through the nasal cavity. At the same time, the lipid layer undergoes compression and expansion with every blink. However, the tear film eventually ruptures and needs to be reformed. The tear break up time, or TBUT, depends on both the age and eye colour of the subject where TBUT decreases with both age and individuals with brown eyes, but is independent of the gender [62, 63]. It has been suggested that the cornea from blue eyed individuals is more sensitive than brown, necessitating a stronger tear film for protection [62]. In healthy individuals, TBUT occurs roughly every 20 to 50 seconds after the initial spreading following a blink, and between 0 and 10 seconds in pathological cases [64]. Deficiencies in the tear film result in severe irritation and pathological conditions such as Dry Eye Syndrome (DES) [65]. DES is quite prevalent, affecting 10 to 20% of the adult population in the United States, leading to corneal ulcerations and scarring, increased bacterial infections, and severe eye irritation and discomfort [65]. There are many deficiencies that can lead to DES but generally they fall into two broad categories: lacrimal or aqueous deficient DES and evaporative DES [66]. Both types lead to tear hyperosmolarity, which is thought to be the primary cause of irritation, ocular damage, and inflammation in DES patients [67, 68]. Related to the topic of this thesis, abnormalities of the lipid layer have been found responsible for many cases of evaporative DES [69]. Furthermore, it has recently been discovered that DES may result not only from tear films deficient in lipid but also arise when the lipid composition is altered [70]. More specifically, a reduction of saturated wax esters was correlated with DES conditions [70], suggesting that an increase in film fluidity may cause early tear break up.

40 21 Finally, as the polar lipid layer is responsible for providing stability to the overall tear film, therefore deficiencies in this layer lead to a shorter TBUT, increased evaporation, hyperosmolarity, and eventually a DES disease state [30]. As the polar lipid fraction of tear film is the least understood, further investigations are needed to determine the interactions within this layer that are responsible for the biophysical characteristics of the normal tear film, such as film stability. This will provide insights into DES and may contribute to better treatments in the future. 1.5 Hypothesis and Goal Discrepancies over the composition and function of the healthy tear film lipid layer have led to a significant dearth in the literature, especially when the polar lipids are concerned. The goal was to biophysically characterize the major components of the polar lipid fraction to better understand their role in the normal functioning tear film. This was accomplished by analyzing the influence of the various lipid species on the different physical properties of tear film including film stability, compressibility, lateral domain organization, and phase behaviour. A biomimetic model system was developed using the lipids from figure 5 in the percentages listed in table 1. The composition of this model was 3 parts 1,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC), 3 parts D-glucosyl-ß-1,1' N-palmitoyl-D-erythro-sphingosine (PGC), 2 parts 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), and 2 parts N-palmitoyl-Derythro-sphingosylphosphocholine (PSM). Additionally, as it has been shown previously, proteins from the aqueous layer of tear film play a major role in the functioning of the lipid layer. To this end, one of the major protein

41 22 fraction components, lysozyme, was tested in terms of its ability to penetrate into the model polar lipid monolayer Hypothesis The physical properties of the tear film lipid layer depend on contributions from different polar lipid species and their interactions with protein, potentially including distinct roles for specific lipid classes and subclasses.

42 23 Chapter Two: Materials and Methods 2.1 Langmuir Monolayers As the polar lipid layer forms a monomolecular film on top of the aqueous layer, a Langmuir trough offers a significant advantage over other techniques when studying these lipids in vitro. This technique involves the spreading of an amphiphilic film, such as lipids, over an aqueous subphase contained inside a Teflon coated trough. Once it equilibrates, the film at the air-water interface can be compressed or expanded by moveable barriers to different molecular areas. This provides a suitable experimental method of blinking that directly reports on physical characteristics such as surface pressure, as well as, on properties that can be derived such as lateral compressibility and phase transitions [71, 72]. Surface pressure is the two-dimensional analogue of pressure and is related to the change in surface tension through the following relationship: where Π is the surface pressure in mn/m, Y 0 is the surface tension in the absence of a film in mn/m, and Y is the surface tension with a film present at the interface in mn/m. This formula not only indicates that the higher the surface pressure the lower the surface tension, but also the surface pressure at which surface tension is equal to zero, which in the case of water is at 72 mn/m. The open eye tear film has a surface tension between mn/m which would translate into a surface pressure between mn/m [17]. For Langmuir trough experiments, a surface pressure sensor detects changes in the forces acting on a so called Wilhelmy plate,

43 24 which is typically a thin rectangular piece of platinum or filter paper that is immersed approximately 1 mm into the subphase. Surface tension pulls the Wilhelmy plate downwards, while increases in surface pressure push the plate upwards. Compression and expansion of Langmuir films is usually recorded as surface pressure versus molecular area (Å 2 /molecule). These so called surface pressure-area isotherms, or isotherms, are conducted at a given constant temperature (fig. 7). Figure 7. A generalized surface pressure-area isotherm. Plateaus are areas of phase coexistence and kinks represent phase transitions. The horizontal slope at the smallest molecular area represents monolayer collapse. G stands for gas, L E for liquid expanded, and L C for liquid condensed.

44 25 At larger molecular areas and surface pressures below 0.1 mn/m, the monolayer behaves like a two dimensional gas. In this so called gas phase (G), individual molecules have very limited interactions with each other. Thus the barriers encounter no resistance to compressing the film and therefore, the surface pressure does not increase [37]. As the barriers compress the monolayer to smaller molecular areas, the lipids come into closer contact. The resulting increase in van der Waal s interactions between the hydrophobic tails of the lipids causes both a rise in surface pressure and a transition into a liquid expanded (L E ) phase [37]. Transitions between phases are seen as kinks or slope changes within the isotherm. The L E is characterized by more ordering of the molecules when compared to the gas phase. Upon further compression, the monolayer will transition from the L E phase to the liquid condensed (L C ) phase. The Lc phase has more ordering and more rigidity when compared to the more fluid and loosely packed L E phase [73]. In the L C phase, the monolayer is at its most ordered, with the orientation of the molecules pointed vertically [37]. In some monolayers, L C -L E phase mixing can occur evident by a plateau region in the isotherm (fig. 7) with regions of L C and L E lipids existing simultaneously. Continued compression of the monolayer will lead to collapse, where multilayers are formed and molecules are ejected into the subphase. Collapse is seen as a horizontal slope or drop in pressure in the isotherm. 2.2 Brewster Angle Microscopy Brewster Angle Microscopy (BAM) is a method used for the in situ visualization of lipid monolayer domain formation in real time. The technique uses a combination of plane-polarized light, refractive differences, and the Brewster angle to image heterogeneities at gas-liquid interfaces [74]. P-polarized light that strikes the interface between two fluids of differing

45 26 refractive indexes will not reflect when this light is incident at the Brewster angle (fig. 8). The Brewster angle is dependent on the interfacial refractive indices, which for the air-water interface is Figure 8. Principle behind Brewster Angle Microscopy. When p-polarized light is incident on a subphase at the Brewster angle in the absence of a film no light will be reflected. In the presence of a lipid film, however, the interfacial properties will change, causing some of the light to be reflected. However, when a lipid film is deposited onto the subphase, a new interface is formed with differing refractive properties [74]. When the p-polarized light is incident at this new interface light is reflected, which can then be detected (fig. 8). The intensity of reflected light is related to the ordering of the deposited film, with regions of higher density reflecting brighter [75]. In films where phase separation occurs, the lateral organization or domain formation can be directly visualized, seen by a contrast in the reflected light intensity [76]. The formation of these

46 27 domains depends on differences between competing attractive and repulsive forces that determine their morphology [77]. Line tension drives the formation of domains through attractive van der Waal s interactions, while electrostatic and aligned intermolecular dipole forces seek to disrupt and break up domains [77]. The combination of these forces determine the size and shape of the domain, and additional factors are the subphase composition and structure of the deposited molecules [78, 79]. Another often used technique for observing the lateral organization of monolayers is fluorescence microscopy. Here, lipids are conjugated to fluorescent probes that have a preference for partitioning into different phases [78]. Similar to differences in refractivity seen with BAM, domains are visualized by contrasts between bright and dark areas depending on the localization of the fluorophores. In difference to the label free BAM, however, fluorescent probes are potential contaminants in the monolayer system. They may have influence on the film`s phase behaviour or domain structure, thus artifacts will have to be considered [80]. Therefore, in the context of imaging the lipid layer from tear film, BAM becomes extremely valuable. Indeed, one of the first uses of BAM was visualizing phase separation from meibomian extracts [81]. However, in regards to the polar lipids from tear film, the lateral domain organization remains unknown. 2.3 Langmuir Troughs Isotherm and Isocycle Trough All experiments involving isotherms and isocycles were completed on a 200 cm 2 Teflon coated Langmuir trough obtained from KSV Nima (Medium Trough model, Espoo, Finland). Figure 9 outlines the trough schematic. This trough has a subphase volume of 100 ml.

47 28 Monolayer`s were compressed by a single moveable Teflon barrier at a speed of 96 cm 2 /minute. The surface pressure was measured using a 20 mm Wilhelmy Plate and a Nima model PS4 surface pressure sensor (Espoo, Finland). All experiments were conducted at room temperature. Figure 9. Schematic of the Langmuir trough used for both the isotherm and isocycle experiments. Lipid was deposited on to the subphase and then compressed to a selected area using the electronically controlled barrier. A Wilhelmy plate immersed 1 mm into the subphase was used to detect surface pressure changes.

48 BAM Trough The Langmuir trough used in the BAM experiments was obtained from Accurion (BAM 601 model, Gӧttingen, Germany). This Teflon trough has an area of 950 cm 2, with a subphase volume of 400 ml. The surface pressure was measured using a 20 mm Wilhelmy Plate and a Nima model PS4 surface pressure sensor (Espoo, Finland). Figure 10 illustrates the microscope set-up. One arm contains the laser, light polarizer, and a compensator. The other arm houses the objective, analyzer, and detector. Both the trough and the microscope are positioned on a Halcyonics anti-vibration system, from Accurion (Gӧttingen, Germany). Images were obtained using the EP3 View software, version 2.30 also from Accurion (Gӧttingen, Germany). Each image taken was a compilation of 60 frames with a resolution of 1 micron. The resulting image had a size of 218 microns x 271 microns. All experiments were conducted at room temperature.

49 30 Figure 10. Schematic of the BAM microscope and BAM trough. The two moveable barriers can be seen at their open positions. Lipid was deposited near the right barrier. Missing from the photo is the pressure sensor Circular Trough For the lysozyme injection experiments a Teflon circular trough with a subphase volume of 25 ml and area of cm 2 (6 cm diameter) was used. As seen in figure 11, the trough has no moveable barriers but is equipped with an injection port (red box). The surface pressure was measured using a 20 mm Wilhelmy Plate and a Nima model PS4 surface pressure sensor (Biolin Espoo, Finland). All experiments were conducted at room temperature. This trough was designed in our laboratory and built at the Faculty of Science Machine Shop (University of Calgary).

50 31 Figure 11. Image of the circular trough used for lysozyme injection studies. 1x PBS buffer was added to the inner compartment. The syringe used to inject lysozyme solution can be seen inside the injection port. It was placed into position prior to lipid deposition to prevent monolayer perturbation during the experiment. Additionally, all components were secured in place using lab tape. 2.4 Solutions As the techniques employed are very sensitive, great care was used throughout each experiment to prevent contamination. To this extent, all tools and equipment first underwent a 4 solvent wash consisting of acetone, methanol, hexane, and chloroform prior to use. The synthetic lipids used were purchased from Avanti Polar Lipids (Alabaster, AL). Lipids were weighed using a Sartorius MC 5 Microbalance (Gӧttingen, Germany). Lipid stock solutions were

51 32 dissolved in a 7:3 (v/v) mixture of chloroform:methanol to a final concentration of 1.0 mg/ml. Lipid mixtures were prepared from these stock solutions into binary, tertiary, and quaternary mixtures according to the ratio of 3DPPC:2DPPE:3PGC:2PSM (mol/mol/mol/mol), based on the tear film polar lipid composition (table 2). These samples were dried under argon and stored in a -20 C freezer until further use, where the samples would then be re-solvated in 7:3 chloroform:methanol to a final concentration of 1.0 mm. HPLC grade chloroform and methanol were used when solvating lipids.

52 33 Table 2. The 15 different lipid systems used in the Langmuir trough experiments with their corresponding average molecular weights and ratios. Ratios listed are molar ratios based on the tear film polar lipid layer composition [30]. The average molecular weight was necessary for the computer program to allow plotting of the molecular area for the isotherm experiments. Lipid System Average Molecular Weight (g/mol) DPPC DPPE PGC PSM DPPC : 2 DPPE DPPC : 3 PGC DPPC : 2 PSM DPPE : 3 PGC DPPE: 2 PSM PGC : 2 PSM DPPC : 2 DPPE : 3 PGC DPPC : 2 DPPE : 2 PSM DPPC : 3 PGC : 2 PSM DPPE : 3 PGC : 2 PSM DPPC : 2 DPPE : 3 PGC : 2 PSM 709.2

53 34 A 1x phosphate buffered saline (PBS) solution was used in all experiments because of its similarity to the aqueous layer from tear film, which had been established previously in the literature and is a common buffer used for the experiments within this thesis [51, 52, 82]. This buffer consisted of mm NaCl, 2.68 mm KCl, 40.6 mm Na 2 HPO 4 7H 2 O, and 7.05 mm KH 2 PO 4. All salts were purchased from Sigma-Aldrich (Oakville, Canada) and weighed out on a Mettler Toledo Excellence Plus analytical balance (Mississauga, Canada). The water used in the buffer was purified by ion exchange using the Synergy 185 Millipore with Simpak2 purifying system (Billerica, USA) to a final resistivity of 18.2 MΩ. The buffer was made to a final ph of 7.4. Human lysozyme, which was purchased from Sigma-Aldrich (Oakville, Canada) was provided by the Vogel lab. Stock solutions were made in the 1x PBS buffer to a final concentration of 25 mg/ml or 17.5 mm. A very high initial concentration was chosen such that small volumes could be used in the protein injection experiments. When injecting underneath monolayers, smaller volumes lead to smaller perturbations. These stock solutions were kept refrigerated at 4 C and used within 3 days of the initial preparation. 2.5 Surface Pressure-Area Isotherms All isotherm experiments were conducted on a 200 cm 2 trough mentioned previously. Before each trial, the trough, the Hamilton gastight syringe, and Wilhelmy plate were all thoroughly cleaned. The trough and the syringe underwent the same 4 organic solvent wash described in section 2.4, while the Wilhelmy plate was cleaned by washing 6 times with boiling water. Once cleaned, 100 ml of the 1x PBS buffer was added to the trough. After a ten minute wait period, to allow for solvent evaporation, a buffer isotherm was conducted to ensure that the

54 35 trough was adequately cleaned. If the resulting isotherm did not show any surface pressure increases, 20 µl of 1 mm lipid sample were added drop wise to the PBS subphase. After another 10 minute waiting period, to allow for solvent evaporation, the lipid film was compressed at a rate of 96 cm 2 /min to a final area of 10 cm 2. For each system, this was completed in at least triplicate (n 3) and at room temperature. The molecular area of each isotherm was calculated by the Nima software using molecular weight of the system (table 2), the concentration of the sample, the amount deposited, and Avogadro s Number. After each isotherm, the subphase and film were removed using vacuum suction and subsequently cleaned with the 4 organic solvent wash again. In between lipid systems, the Wilhelmy would be washed and replaced with a clean one Isotherm Analysis For each of the 15 lipid systems the collapse pressure, collapse area, lift-off area, and compression modulus were determined. Both the collapse pressure and collapse area were found based on the point on the isotherm where the surface pressure decreased rapidly or exhibited a horizontal slope (fig. 7). The lift-off area, or the point where the deposited film begins to exert a measurable surface pressure increase [83], was recorded when surface pressures were in excess of 1 mn/m. The compression modulus of each system was determined by first calculating the compressibility using the following equation:

55 36 where C s is the lateral compressibility (m/mn), A is the area occupied per molecule (Å 2 /molecule), and Π is the surface pressure (mn/m). The derivative was calculated in blocks of 5 data points using the slope function within the Excel 2007 program, from the Microsoft Office Suite (Redmond, USA). The compression modulus (β) is the reciprocal of the compressibility and was chosen because it is more generally used within the literature due the compression modulus values having similar orders of magnitude with surface pressure. 2.6 Surface Pressure Area Isocycles After the initial spreading, tear film remains stable over multiple blinks before collapsing and having to be reformed [84]. Surface pressure-area isocycles help to simulate the compression and expansion that tear film undergoes during blinking. These experiments will shed light on which polar lipids have the greatest impact on film stability over multiple blinks. Isocycles were completed on the same trough as the isotherms, with the same care taken during the experimental set-up. Additionally, the same buffer and deposition volumes were used. For the isocycle experiments, however, each film was compressed and expanded five times; compressing first to a surface pressure 15 mn/m below the collapse pressure of that particular film before expanding back to 0 mn/m. Compression was stopped at this point for two reasons. The first being that collapse nucleation sites may start forming at surface pressures below the collapse plateau seen on the isotherm [85, 86]. Had the limiting surface pressure been higher the isocycle experiments would, therefore, be testing reversible collapse instead of stability over multiple compressions. The second reason for stopping at that surface pressure was due to a delay between the pressure being detected and the barrier stopping from moving. This lag period usually leads to unwanted surface pressures in excess of 5 mn/m. For example, setting the compression limit to 30 mn/m

56 37 will often lead to surface pressures larger than 35 mn/m before the barriers have a chance to stop, especially in cases with rapid surface pressure changes. The cycles were also continuous, having no rest period between compression and expansion Isocycle Analysis The isocycle data was both quantitatively and qualitatively analyzed. The ability of the film to resist deformation is related to the degree of hysteresis exhibited during the isocycle. Hysteresis describes a system where the internal state depends not only on its current environment but also on its past. Within the context of compression and expansion isocycles, the shape of a given cycle will be different depending on whether it is going through expansion or compression if hysteresis is present. The less hysteresis observed during the isocycle, the more stable and resilient the monolayer is to deformation. The extent of hysteresis was determined qualitatively by visualizing the isocycle from each system in a surface pressure vs area graph (fig. 12). Plots were made of the first and final fifth cycle. From figure 12A it is clear that the compression isotherm (blue curve) is different than the expansion isotherm (red curve), seen by a shift to smaller molecular areas. This indicates a hysteresis between compression and expansion of the film. Additionally, we also examined the difference between entire cycles, specifically between the 1 st (purple curve) and 5 th (green curve) cycles (fig. 12B). This was completed by analyzing how well the cycles overlapped with one another. Using figure 12 as an example, the hysteresis appears larger between the compression and expansion isotherms than the difference between entire isocycles. That is, the blue and red curves (fig. 12A) appear more distinct compared to the purple and green ones (fig. 12B).

57 38 Figure 12. (A) Hypothetical example of a single compression expansion isocycle. The film is first compressed to a given surface pressure then expanded, resulting in a loop like curve. The blue trace is the compression isotherm; the red trace is the expansion isotherm. (B) Hypothetical example of two complete isocycles. The first cycle in blue and the 5 th cycle in green were compared to how well they overlapped with each other.

58 39 The second method for analyzing the isocycle data took advantage of the Root Mean Squared Deviation (RMSD) equation, allowing for a more quantitative approach. The RMSD was determined using the following formula: RMSD A A where N is the total number of data points, A is the molecular area at that given data point (Å 2 /molecule), and Π is the surface pressure at that given area (mn/m). Surface pressures were only compared past the lift-off area of the isotherm. For each system three RMSD values were determined. These values were between the compression and expansion isotherms (the red and blue curves from figure 12A) of the 1 st and 5 th cycles, and between the entire first and fifth cycles (the purple and green curves from figure 12B). In terms of the RMSD equation, this means that when comparing compression and expansion isotherms from within the same cycle, the surface pressures of compression would be input at Π 1,A and the surface pressures of expansion would be the input at Π 2,A. When finding the RMSD between cycles, all of the surface pressures past lift off for cycle 1 would be entered into Π 1,A and the entire surface pressures past lift off for cycle 5 would be entered into Π 2,A. Therefore, the RMSD is an indication of how different the compression isotherm is from the expansion isotherm, as well as how different the selected isocycles are from each other. The higher the RMSD value the larger the difference and therefore, the more hysteresis. Using the RMSD equation in this context appears to be the first of its kind as no published data was found in other monolayer isocycle experiments.

59 BAM Isocycles BAM images of the lateral domain organization from each film were obtained as they underwent successive compression-expansion cycles. The cleaning procedure for the trough and the tools used was the same as the one described for the isotherm experiments. In contrast to the smaller trough experiments, 40 µl of 1 mm lipid sample was added drop wise to the PBS subphase instead of 20 µl. Before imaging, an isotherm was completed and compared to data from previous experiments to ensure the quality of the prepared sample. During compression and expansion, at least 3 representative images were taken at approximately every 5 mn/m change in surface pressure. This was done for the compression and expansion isotherms from both the 1 st and 5 th cycles. 2.8 Lysozyme Injections Tear proteins inserted within either the polar or non-polar lipid layers in tear film are believed to contribute to the overall stability of the tear film structure [24, 38]. Here we studied the extent to which one of the most abundant tear proteins, lysozyme, interacts with model polar lipid monolayers. This was accomplished by protein injection studies into the subphase. Using the circular trough described in section 2.3.3, 20 ml of PBS buffer was added after 4 solvent washing the trough and all the tools. Following this, 1 mm of lipid solution was added drop wise until the desired surface pressure was reached. After deposition, there was a 10 minute period wait time to allow for solvent evaporation and the surface pressure to stabilize. All surface pressures recorded after the evaporation period were within 1 mn/m of the desired values of 10 mn/m, 20 mn/m, and 30 mn/m. As previously mentioned, the open eye tear film surface pressure is between mn/m [17]. However, due to the pressure from blinking as well as

60 41 constant respreading, the surface pressure of tear film must transition through a broad range of values. Therefore, the three surface pressures chosen were in an attempt to survey lysozyme adsorption at different tear film conditions. Once the film had stabilized, lysozyme dissolved in 1x PBS was injected into the subphase using a 1 ml Tuberculin Slip Tip syringe from BD (Franklin Lakes, USA) ml of lysozyme was injected into the subphase to a final concentration of 50 µg/ml, which is the concentration of lysozyme used in previous injection studies [51]. Inside the trough was a magnetic stir bar to ensure proper mixing. Trials at the three different surface pressures ran for 1600 seconds and the change in surface pressure ( Π, mn/m) was recorded (fig. 13). Π was determined by subtracting the baseline surface pressure before injection from the average surface pressure of the last 100 seconds. For each of the 15 different lipid systems, a negative control was employed by injecting lysozyme free buffer into the subphase of a monolayer at a surface pressure of 10 mn/m. Additionally, a positive control where lysozyme was injected into the subphase with no monolayer present was completed as well, resulting in a pressure increase of 9.0 ± 0.4 mn/m. Each surface pressure measurement and control were conducted in triplicate and at room temperature.

61 42 Figure 13. Hypothetical surface pressure (Π) vs. time graph. Once the surface pressure had equilibrated, lysozyme was injected into the subphase at the time indicated by the arrow. The trials were then allowed to run for an additional 1600 seconds. The change in surface pressure, Π, was recorded based on the average surface pressure from the last 100 seconds minus the baseline. 2.9 Differential Scanning Calorimetry Differential scanning calorimetry (DSC) is a powerful technique for easy determination of lipid thermodynamic properties during phase transitions [87]. The most commonly studied lipid transition using DSC is the gel to liquid-crystalline phase transition, whereby the lipid acyl tails become more disordered at a sample specific temperature [87]. This temperature is dependent on hydrocarbon chain length, the nature of the polar head group, and properties of the solvent such as ph and ionic strength [88]. DSC directly reports on phase transition temperature (T m ), peak width at half-height (T 1/2 ), change in enthalpy of the transition ( H), and change in

62 43 heat capacity at constant pressure ( C p ), by measuring the difference in heat released or absorbed during a phase change. The T m is the temperature in which half of the lipids will exist in the gel phase and the other half in the liquid-crystalline phase; it is the temperature found at the peak of the thermogram (fig. 14) [87]. T 1/2 reports on the cooperativity of the phase transition, where a small T 1/2 denotes a more cooperative transition and a large T 1/2 a more noncooperative one [87]. Transitions that are cooperative have acyl tails that melt together at the same time, while non-cooperative occurs when there is a broader distribution of chain melting. H reports on whether the transition was endothermic (positive H) or exothermic (negative H) with the magnitude of H corresponding to how much heat was absorbed or released during the transition. The C p is the amount of thermal energy required to increase the temperature of the system by 1 C at a constant pressure. The DSC instrument contains two cells that are simultaneously heated at the same rate over a range of temperatures. Within the sample cell are multilamellar vesicles (MLVs) suspended in buffer. MLVs are liposomes that contain multiple lipid layers. The reference cell of the DSC contains only buffer. To get accurate results, the reference cannot release or absorb heat over the temperatures scanned. Furthermore, these cells are enclosed in an adiabatic jacket such that no heat is transferred into or out of the cells except from that supplied by the experiment. During thermotropic events, the calorimeter will detect any temperature differences between the sample and reference, supplying more or less heat to the sample cell to keep both cells at the same temperature. The result is a thermogram that has C p as a function of temperature (fig. 14). The phase transition is either endothermic if the curve is upwards from the baseline due to heat release or exothermic if it points downwards due to heat uptake.

63 44 Figure 14. An example DSC thermogram of the gel to liquid-crystalline phase transition. Illustrated are the thermodynamic properties directly reported from the DSC where T m is the melting temperature, T 1/2 is the peak width at half-height, and H cal is the enthalpy corresponding to the area under the peak. The phase transition of meibomian extracts is broad, having a melting range between C [40]. The average corneal surface temperature is 34.3 C [89] suggesting the potential for coexisting phases within the tear film. Additionally, the individual tear film polar lipids melt at far higher temperatures, ranging from 41 C to 89 C [88, 90]. However, little data to date has been reported on lipid mixtures, especially so in regards to the biomimetic ratios of the model systems presented in this thesis. The goal, therefore, is to test the impact the different biomimetic combinations have on depressing the melting temperature such that it is more in line with that of

64 45 the meibomian extract values. The T m of the biomimetic system in the presence of lysozyme is important as well because the protein could also affect the melting temperature Differential Scanning Calorimetry Procedure Thermodynamic data using DSC was found on the 15 different systems shown in table 2. MLVs were prepared for the sample cell of the DSC using the following method. First, the lipid sample was dissolved in 7:3 chloroform:methanol (v/v) mixture, which was then dried down under argon and placed into a vacuum oven for a minimum of 4 hours (Isotemp Model 280A from Fisher Scientific, Walthamm USA). The films were then rehydrated using the 1x PBS buffer to a final concentration of 1.0 mg/ml. This solution was then vortexed (Vortex Genie 2 from Scientific Industries, Bohemia, USA) and sonicated (FS110H from Fisher Scientific, Waltham, USA) until all lipid had become suspended in solution. It was then degassed for 15 minutes using a ThermoVac vacuum (General Electric Health Care, Mississauga, Canada). Finally, the MLV lipid sample was injected into the sample cell of the DSC (VP-DSC from Microcal, now part of General Electric Health Care, Mississauga, Canada), with 1x PBS buffer injected into the reference cell. Once loaded into the cells, the DSC was set to scan at a rate of 10 C/hour, with each trial scanning at least 10 C above and below the transition temperature to adequately represent the entire transition. Each trial was comprised of five heating scans. Trials with 1x PBS buffer in both the sample and reference cells were conducted to establish a baseline for data analysis. The same procedure was followed for the addition of lysozyme to quaternary MLVs but included adding lysozyme to the rehydrated vesicles to make a 30:1 or 15:2 lipid to protein molar ratio solution. The final concentration of lipid was 1 mg/ml and the final concentration of lysozyme was or mg/ml.

65 46 Chapter Three: Surface Pressure-Area Isotherms Using a biomimetic model to study the interactions within the polar lipid layer of tear film confers distinct advantages. Firstly, it allows for complete control over the experimental conditions, i.e. the exact composition of the lipid film in terms of the lipid concentration, species, or length and nature of the acyl chains. With good experimental technique the exact amount of lipid can be prepared and deposited every time, ensuring that variability between trials is low. Secondly, model systems with defined compositions allow for straightforward comparisons following the alteration of individual components on biophysical characteristics, such as stability, that are relevant for the structure and function of the natural tear film. With this in mind, the four lipids present in this model were examined individually, and then in binary, tertiary, and quaternary configurations. 3.1 Individual Lipid Systems The compression isotherms of the pure lipid systems, deposited onto a 1x PBS subphase, can be seen in figure 15. The take off from zero pressure of the DPPC isotherm occurred at 98.5 ± 3.1 Å 2 /mol. The film remained in the L E phase until the surface pressure reached approximately 5.9 mn/m. At this point L E -L C phase coexistence occurred, evident by the plateau in the isotherm (fig. 15, red arrow). Following the L E -L C plateau, the surface pressure steadily increased until film collapse at 60.0 ± 1.0 mn/m. This maximum surface pressure for DPPC was lower than the literature value of 72 mn/m [91]. However, 72 mn/m is the ideal collapse pressure of DPPC that depends on many factors such as trough geometry and subphase composition. The collapse of DPPC has been observed over a range of values, some as low as 48

66 47 mn/m with many in between [92-94]. The presence of both chaotropic and kosmotropic ions in the subphase have been shown to interfere with the lateral packing of lipid monolayers [39, 95]. Chaotropes and kosmotropes refer to groups of anions that belong to the Hoffmeister series, a system that ranked the relative influence that these ions had on the physical behaviour of macromolecules within an aqueous environment [96]. Generally speaking, kosmotropes increase the surface tension (or decrease the surface pressure) and decrease the solubility of proteins, while chaotropes decrease the surface tension (increase the surface pressure) and increase the solubility of proteins [97]. Indeed, it was observed that chaotropic anions, in the absence of kosmotropes, stabilized DPPC monolayers exhibited by an increase in collapse pressure [95]. In terms of the PBS subphase used in our experiments, Cl - is a relatively weak chaotrope, while H 2 PO - 4 and HPO 2-4 are considered kosmotropic, with HPO 2-4 being the stronger kosmotrope of the two [97]. With this in mind, it appears that the kosmotropic ions within the subphase are having a greater affect than chaotropic ions because there was a decrease in the observed DPPC collapse pressure when compared to that of its ideal collapse pressure. However, it is important to note that our recorded value of 60.0 ± 1.0 mn/m was within the literature range reported earlier. For DPPE, the monolayer left the gas phase at 73.1 ± 0.2 Å 2 /mol, a much smaller area than DPPC (fig. 15, green line). DPPE lipids differ from DPPC only in regards to their head group. The choline head group of DPPC is bulkier than the ethanolamine head group, making the effective area of each DPPC molecule larger. Steric effects from DPPE molecules would, therefore, occur at smaller molecular areas than that of DPPC. Additionally, hydrogen bonds between DPPE molecules decrease the molecular distances from each DPPE lipid. Following take off, the surface pressure of DPPE increased slowly, existing first in the L E phase (fig. 15,

67 48 green arrow). At approximately 65 Å 2 /mol the film transitioned into the L C phase; more rapid pressure increases followed this transition. The DPPE film collapsed at 46.6 ± 0.7 mn/m (table 3). The ideal collapse pressure of DPPE is 50 mn/m, indicating that our results were lower than the literature values [98]. Again, the published value for DPPE collapse was on a water subphase, which could account for the discrepancy observed here. Figure 15. Average compression isotherm (n 3) for the four different pure lipid systems on a 1x PBS subphase. DPPC (red), DPPE (green), PGC (purple), and PSM (blue). Arrows highlight points of interest mentioned such as kinks or regions over isothermal overlap.

68 49 PGC took off at the smallest molecular area, 57.7 ± 0.2 Å 2 /mol (fig. 15, purple curve). Like DPPE, the rate of surface pressure increase was constant based on the linear slope of the isotherm. Following take off, the film transitioned from gas phase to L C before collapsing at 49.1 ± 3.1 mn/m (table 3). The collapse of PGC monolayers over a water subphase was reported to be 65 mn/m [97]. Of all the model lipids it appears that PGC was, therefore, the most affected by the buffer subphase, evident by the large drop in maximal surface pressure. Compared to the other lipids in this series, PGC has the highest potential for hydrogen bonding due to the four hydroxyls on the glucose head group, allowing it to hydrogen bond not only with itself but also the surrounding water. Kosmotropic anions have been shown to destabilize macromolecules through polarizing the hydrogen bond between the macromolecule and their hydration shell, weakening the bond (fig. 16) [99]. It may be possible then, that if the stability of PGC monolayers over a water subphase is conferred through strong hydrogen bonds, it follows that by weakening these bonds the monolayer would destabilize, leading to lower collapse pressures. Additionally, from start to finish, the PGC isotherm occurred over a shorter range when compared to all the individual model lipids. The PGC monolayers transitioned from gas phase, to L C, to collapse all within 19 Å 2 /mol. This indicates that PGC films must be compressed to smaller areas before steric hindrance between molecules exert an increase in surface pressure. Attractive forces from hydrogen bonding between PGC molecules cause tighter lipid packing without increasing the surface pressure. Finally, being the only lipid in the model series that carries no localized charges may additionally account for these smaller molecular areas, as there would be no electrostatic repulsion between PGC molecules.

69 50 Figure 16. A PGC glucose head group hydrogen bonding to surrounding water molecules. A kosmotropic anion is shown on the left, polarizing one of the water molecules. PSM, the last of the selected lipid system isotherms, shared many similar characteristics with the DPPC isotherm (fig. 15, blue curve). Like DPPC, there was evidence of L E -L C phase coexistence, occurring for PSM at approximately 13 mn/m (fig. 15, blue arrow). Furthermore, once DPPC and PSM monolayers entered the L C phase, there were many apparent points of overlap (highlighted by the black arrow in figure 15) before PSM collapsed at 60.1 ± 0.1 mn/m (table 3). The structures of these lipids share many features, only differing in regards to the backbone. DPPC has a glycerol backbone that links to two palmitate chains, while PSM has a sphingosine backbone that links to one palmitate tail. As well, the amide and hydroxyl groups in sphingosine allow PSM to compete for hydrogen bonds while DPPC has no such potential. PSM films took off at a lower molecular area, occurring at 83.8 ± 3.0 Å 2 /mol (table 3), most likely due to hydrogen bonding bringing the lipids closer together. Additionally, the sphingosine tail of PSM contains one degree of unsaturation at the fourth carbon. This double bond is in the trans confirmation, which has been shown to cause condensation of monolayers in previous studies

70 51 due to the more vertical orientation of the hydrocarbon tail [100]. Table 3 summarizes the important data points observed from the compression isotherms of the pure lipid systems. Table 3. Results from the compression isotherms of the pure lipid systems. Isotherms were completed on a 1x PBS buffered subphase at room temperature. The error is represented by the standard deviation; n 3. The fourth column contains the literature values for the collapse pressures over a pure water subphase. Lipid System Take Off Area Collapse Pressure Collapse Pressure (Å 2 /mol) (mn/m) over H 2 O (mn/m) DPPC 98.5 ± ± [92-94] DPPE 73.1 ± ± [98] PGC 57.7 ± ± [97] PSM 83.8 ± ± [101] Pure Lipid Compression Modulus To assess the interfacial elastic packing interactions of the different model lipids, each isotherm was analyzed in terms of its compression modulus (β). Similar to the in-plane elastic moduli of area compressibility used for characterizing bilayers, the compression modulus reports on the elasticity and rigidity of monolayer films [102, 103]. Additionally, the compression modulus is correlated to the monolayer s phase [72]. Values under 12 mn/m represent the gas phase, between 12 mn/m to 100 mn/m to the liquid expanded, between 100 mn/m and 250

71 52 mn/m to the liquid condensed, and finally above 250 mn/m to the solid phase [104]. This indicates that the higher the compression modulus the lower the lateral elasticity. Finally, local minima in compression modulus values represent monolayer phase coexistence during transitions, characterized by high in plane elasticity [72]. Figure 17. Representative compression modulus curves for the four different pure lipid systems. DPPC (red), DPPE (green), PGC (purple), and PSM (blue). The black line represents the compression modulus value (100 mn/m) in which monolayers transition from the L E to the L C phase. Arrows highlight specific points of interest such as local minima.

72 53 For each of the individual lipid species, the compression modulus was plotted as a function of the surface pressure (fig. 17); keeping in mind that the higher the compression modulus (β) the lower the interfacial elasticity. At the peak β values, the observed elasticity was greatest for DPPE > DPPC > PGC > PSM. These peak values occur when the film is nearest to collapse, which is when it is the most rigid. It appears then, that the glycerol-based lipids have higher in-plane elasticity when compared to the sphingoid-based lipids, in this instance. The apparent inelasticity of the sphingosine lipids was most likely from their hydrogen bonding potential. As mentioned earlier, the amide and hydroxyl of the sphingosine backbone can participate in hydrogen bonds making these films more rigid. For example, from table 4 the max β value of DPPC (158.6 ± 4.85 mn/m) was significantly lower than that of PSM (204.0 ± 5.84 mn/m), at a p-value < On the other hand, the lipids studied can also be classified in terms of their head groups. For instance, they can be separated into those with head groups that can hydrogen bond vs those that cannot, those with choline head groups vs those with other structures, and finally, overall head group size and orientation. All the lipids studied can hydrogen bond with the exception of DPPC. PSM and DPPC both contain choline head groups while DPPE and PGC do not. In terms of size, DPPE would be the smallest. The difference between the choline containing lipids and PGC would depend on the orientation of the glucose head group in PGC. Nuclear Magnetic Resonance (NMR) studies of PGC bilayers exhibited few conformational solutions of the saccharide head group. These solutions all fell within a narrow orientation window in which the glucose head group fully extends itself, maximizing interactions with the bulk water [105]. The vertical orientation of PGC s head group would give it a smaller effective area than if it were angled more parallel to the bilayer. In contrast, the choline head group on DPPC and PSM

73 54 orients itself almost parallel to the bilayer surface, such that the positive charge on the amine can interact with the negative charge of the neighbouring phosphate [106]. Thus, when comparing the lipids within their own backbone group (sphingosine vs glycerol), the larger the effective area of the head group the more inelastic the film was. The max β of DPPC (158.6 ± 4.85 mn/m) was significantly higher than that of DPPE (109.2 ± 9.14), at a p-value < However, the max β value of PGC ( ± 31.1 mn/m), was not significantly different from the max β value of PSM (204.0 ± 5.84 mn/m). This may indicate that the sphingosine backbone had more influence over the fluidity than the lipid head group, based on the smaller difference in β max values between PGC and PSM. The sphingosine tail differs from the two 16:0 glycerol based lipids because it is two carbons longer and has a trans double bond at carbon 4. However, while cis unsaturation plays a major role in the fluidity and interfacial elasticity of monolayers, trans mono-unsaturation behaves similar to saturated lipids, especially when the double bond occurs closer to the backbone [107]. The similarity is due to the comparable orientation of trans monounsaturated tails to saturated ones with similar tail lengths [108]. As well, the first three carbons of sphingosine have been reported to be configurationally related to the glycerol backbone of PC, indicating that the 18:1 length is effectively more alike a 16:0 hydrocarbon acyl tail [103, 109]. On the other hand, within the interface of the sphingo lipids, the hydroxyl group and NH- from the amide link would hydrogen bond with neighbouring molecules, increasing the rigidity. The glycerol based lipids are missing these chemical groups and therefore the ability of the interface to hydrogen bond. With that in mind, the influence of sphingosine on elasticity is most likely from the hydrogen bonding potential within the interface of the lipid. Perhaps even more interesting was the observation of a fairly elastic, fluid DPPE monolayer characterized by low β values throughout the compression isotherm (table 4). DPPE

74 55 generally forms very rigid monolayers exhibited through rapid surface pressure increases when compressed over small molecular areas [98]. This rigidity imparted to PE lipids is due to the presence of a positively charged primary amine that is capable of hydrogen bonding. The hydrogen bond between the amine and neighbouring phosphate of PE molecules is a strong interaction that promotes lipid ordering. However, as mentioned previously, this phenomenon was observed overtop of a water subphase, not a buffered one. Chaotropic anions, such as the Cl - in PBS, have been previously shown to insert into the head group region of phospholipid monolayers, especially during the more fluid L E phase [95]. In the case of DPPE, the positive charge on the primary amine would be more accessible to dissolved anions than the quaternary amine on DPPC or PSM. Anionic insertion through coulombic attraction would lead to an exchange between the ethanolamine-phosphate interaction with an ethanolamine-anion interaction (fig. 18), creating distance between neighbouring lipids and preventing the acyl tails from interacting. This would not only decrease van der Waal`s interactions between acyl tails but also disrupt the hydrogen bonds between the head groups and phosphates of neighbouring PE molecules.

75 56 Figure 18. Schematic illustrating the increased distance between DPPE head groups following anion adsorption (red circle). It also appears that any anions adsorbed to the DPPE films remained until collapse because of the relatively gentle slope in figure 17 (green arrow) suggesting little change in the interfacial elasticity as the surface pressure increased. Had bonded anions been desorbed during any point in the compression there would be an evident increase in the compression modulus from increased rigidity. DPPE molecules would re-establish the hydrogen bonds between them, resulting in tighter packing and decreased elasticity. On the other hand, the bulkier head groups are less susceptible to fluidization through anionic adsorption evidenced by their much higher β max values when compared to DPPE. Figure 17 also confirms the L E -L C phase coexistence in DPPC and PSM monolayers. These phase transitions were characterized by the local minima exhibited at approximately 6 mn/m for DPPC and 13 mn/m for PSM (fig. 17, red and blue arrows respectively). This is in contrast to DPPE and PGC which showed rapid increases in β with no apparent minima, indicating that these films existed at one state until collapse.

76 57 Table 4. The average compression modulus (β, mn/m) at different surface pressures (mn/m) and the average maximum compression modulus value (mn/m) for the individual lipid systems. The compression modulus was obtained from the compression isotherms using the equation in section Error was recorded as the standard deviation (n 3). Lipid System β at 10 mn/m β at 20 mn/m β at 30 mn/m Maximum β (mn/m) (mn/m) (mn/m) (mn/m) DPPC 43.3 ± ± ± ± 4.9 DPPE 85.9 ± ± ± ± 9.14 PGC ± ± ± ± 31.1 PSM 47.6 ± ± ± ± Binary Lipid Systems All of the deposited binary lipids formed very stable films, collapsing at surface pressures near or in excess of 60 mn/m, with the exception of the 2DPPE:3PGC mixture (fig. 19A). This was not surprising given that DPPE and PGC formed the relatively least stable monolayers of the pure lipids tested. The binary system of these two lipids was also the most condensed, having the smallest take off area (55.8 ± 2.0 Å 2 /mol, table 5). This value was comparable to that of an individual PGC film (table 3), indicating that in terms of packing density, PGC may have a stronger impact than DPPE. Both of these lipids have hydrogen bonding groups, which increases the overall strength of the interactions between the molecules. Hydrogen bond interactions make for more rigid films as was discussed earlier, accounting for the more condensed film when compared to the other binary systems present. From the isotherm, it also appears that the

77 58 2DPPE:3PGC film undergoes a transition directly into the liquid condensed phase from the gas phase, before finally collapsing at 51.0 ± 3.5 mn/m (table 5). The next most condensed film, 3PGC:2PSM, took off at a molecular area of 61.9 ± 2.7 Å 2 /mol (table 5). The isotherm of this film had a very similar overall shape to that of 2DPPE:3PGC (fig. 19A). The exceptions were that the 3PGC:2PSM isotherm was shifted to larger areas, and collapsed at a higher surface pressure (58.8 ± 0.89 mn/m). PGC is the common element found in both films and may be the lipid responsible for creating a more condensed or tightly packed monolayer. The bulkier choline head group of PSM most likely accounts for the larger take off area in the 3PGC:2PSM film compared to 2DPPE:3PGC. Increased steric hindrance from choline over ethanolamine would prevent tighter interactions between molecules leading to initial surface pressure increases at larger molecular areas. Absent in this isotherm as well, was the characteristic kink seen during the compression of pure PSM films. This kink, as mentioned earlier, was indicative of the phase transition from the L E to the more ordered L C phase. As compression isotherms report on the overall thermodynamic state of the monolayer, it is safe to assume that based on the 3PGC:2PSM isotherm, the film exists primarily in the L C phase between take off and collapse. Therefore, at least at these molar ratios, PGC appears to have a larger impact on film lateral organization than PSM does. The presence of PGC condenses and orders the PSM film.

78 Figure 19. (A) Average compression isotherm (n 3) for the different binary lipid systems. The ratios listed are the molar ratios. 3DPPC:2DPPE (red), 1DPPC:1PGC (green), 3DPPC:2PSM (purple), 2DPPE:3PGC (light blue), 1DPPE:1PSM (orange), and 3PGC:2PSM (dark blue). (B) Enlarged display of the high surface pressure region of the binary compression isotherms. Double sided arrow emphasizes the near isothermal overlap at higher pressures. 59

79 60 With all this in mind, it`s not surprising that the third most condensed binary film would be 1DPPC:1PGC (fig. 19A). While this film took off at much larger area (71.9 ± 0.72 Å 2 /mol, table 5) than the previous two PGC containing binary films, the lateral packing was very similar to the 3PGC:2PSM monolayer. There was almost complete overlap between these two films from approximately 12 mn/m until just before collapse. 1DPPC:1PGC was slightly more stable, collapsing at 64.4 ± 0.93 mn/m, 6 mn/m higher than 3PGC:2PSM. On the other hand, the 1DPPC:1PGC film differed from the other two PGC films in that there was a noticeable slope change following the initial take off (fig. 19A, green arrow), indicative of a phase change. Therefore, based on the compression isotherm data, the 2DPPE:3PGC and 3PGC:2PSM films undergo a smooth transition from gas phase, to L C, and finally to collapse. Whereas, 1DPPC:1PGC monolayers additionally entered the L E phase before transitioning into the more ordered L C state. The appearance of an L E phase when compressing the DPPC containing PGC binary film that was absent in 3PGC:2PSM may be for two reasons. The first reason could be that there are more L E forming molecules overall in the 1DPPC:1PGC film than in the 3PGC:2PSM film, based on the molar composition of the two systems. While PGC is inhibiting the more fluid state, it stands to reason that when the concentration of PGC is lower, there would be more of a propensity for the monolayer to transition into the L E phase. Additionally, the L E phase of pure DPPC monolayers occurs at larger molecular areas than pure PSM monolayers (fig. 15). While the total lipid concentration was the same for all trials, the abundance of individual lipids was less when in mixtures. Because there is less PSM in the binary mixture, the onset of L E phase would be shifted to smaller areas. However, because PGC is also present, as you compress to smaller areas, instead of seeing a phase transition into the L E state, PGC hydrogen bonding may become significant. This causes the film to become more ordered overall,

80 61 transitioning straight into the L C phase instead. The other reason why 1DPPC:1PGC monolayers transition into an L E phase and 3PGC:2PSM does not, could be the result of packing differences between DPPC and PSM (fig. 20). Figure 20 illustrates how the packing depends on the lipid composition of the monolayer. The area of each disk was determined based on the molecular area from each pure lipid isotherm at a surface pressure of 5 mn/m. This surface pressure was chosen because it was where the greatest difference existed between the isotherms of 1DPPC:1PGC and 3PGC:2PSM. By calculating the total molecular area taken up by the lipid disks and subtracting that from the total area of the box illustrates how much free space is available at that particular surface area. In terms of the example from figure 20, 20 lipid molecules from the 1DPPC:1PGC system would correspond to Å 2 while 20 3PGC:2PSM molecules would be equal to Å 2. Thus the amount of free surface area available in figure 20 is 80.8 Å 2 for 1DPPC:1PGC and Å 2 for 3PGC:2PSM if the area sampled is equal to 350 Å 2. Furthermore, PSM can hydrogen bond and DPPC cannot, making PSM more rigid than DPPC to begin with and thus possibly preventing 3PGC:2PSM from entering the L E phase. 3DPPC:2DPPE monolayers followed next in terms of take off area, moving from smaller molecular areas to larger (fig. 19A). At a slightly larger area than 1DPPC:1PGC, 3DPPC:2DPPE films transitioned out of the gas phase at 77.2 ± 1.1 Å 2 /mol (table 5). Like 1DPPC:1PGC, the inflection point at approximately 60 Å 2 /mol would indicate a phase transition from the L E to L C state. Following this transition, the surface pressure steadily increased until collapse at 61.8 ± 0.58 mn/m (table 5). On the other hand, 1DPPE:1PSM films took off at 94.2 ± 1.3 Å 2 /mol and collapsed at 64.9 ± 0.46 mn/m. At these molar ratios, DPPE had a more condensing effect on DPPC monolayers compared to PSM ones because 1DPPE:1PSM monolayers took off at areas over 15 Å 2 /mol greater than those of 3DPPC:2DPPE. This is in contrast to the films made with

81 62 the other hydrogen bonding lipid present, PGC. As noted earlier, the 1DPPC:1PGC and 3PGC:2PSM isotherms were similar with the exception that 3PGC:2PSM monolayers were more condensed. Based on these results, DPPE and PGC have a larger impact on condensing monolayers depending on the nature of the other lipids. In the case of the binary systems studied, the impact was greatest when both lipids shared the same backbone. For example, because DPPE has a glycerol backbone, it condensed the other glycerol based lipid, DPPC, more so than it did the sphingosine based PSM.

82 63 Figure 20. A cartoon illustrating the packing differences between the binary systems of DPPC (red disks) and PGC (purple disks), and PSM (blue disks) and PGC, at 5 mn/m. The scale used to draw the circles was 5 Å 2 : 1 cm 2. The box has an area of 350 Å 2. (A) has 20 disks of 10 DPPC and 10 PGC representing the 1DPPC:1PGC molar ratio; (B) has 20 disks of 12 PGC and 8 PSM representing the 3PGC:2PSM molar ratio. The dark grey behind the lipid disks represents the free surface area. Panel B has more free surface than A illustrating how greater amounts of larger lipids can lead to film expansion.

83 64 A shoulder or kink was observed in the 1DPPE:1PSM isotherm at approximately 45 mn/m, suggesting a phase transition (fig. 19B, orange arrow). While individual PSM monolayers did exhibit phase mixing, this occurred at 13 mn/m (fig. 15, blue arrow). The kink observed in the 1DPPE:1PSM isotherm was, therefore, most likely not due to phase mixing because it occurred at surface pressures over 30 mn/m greater than that of pure PSM. Furthermore, from the pure DPPE compression isotherm, it was hypothesized that anions were adsorbing to the monolayer, disrupting the hydrogen bond network and fluidizing the film. Anions from the subphase, therefore, could be adsorbing to the binary 1DPPE:1PSM monolayer based on the presence of DPPE. Additionally, it was observed previously that greater lipid ordering in DPPC films prevented anion absorption because the head groups become unavailable for ion binding due to tighter lipid packing [95]. However, these studies focused on DPPC monolayers and not DPPE. As evidenced by the steady continuous slope of the DPPE isotherm (fig. 15, green curve), it appeared that the affinity of adsorbed anions to the DPPE molecules prevented any ions from desorbing. This would suggest that DPPE has a greater affinity for anions possibly due to the accessibility of a primary amine over a quaternary amine. However, in the case of the 1DPPE:1PSM films, the total number of DPPE lipids was lower, decreasing the films overall affinity for ion absorption. As there were fewer DPPE molecules in the 1DPPE:1PSM binary mixture compared to pure DPPE films, the influence from increased lipid ordering found at higher pressures may be greater than the favourable electrostatic interactions between DPPE and the adsorbed anions. It would stand to reason that as the surface pressure increased in the 1DPPE:1PSM film, any bound anions would be released based on previous literature illustrating anion exclusion with greater lipid ordering [95]. Therefore, packing changes from ion desorption at 45 mn/m was most likely the reason for the observed kink in the 1DPPE:1PSM isotherm.

84 65 Of all the binary systems, the least condensed film was 3DPPC:2PSM. This was not surprising as this was the only film in which neither PGC nor DPPE were present. The 3DPPC:2PSM compression isotherm exhibited initial surface pressure increases at 97.9 ± 3.3 Å 2 /mol, which continued at a constant rate until approximately 16.5 mn/m. At this point in the isotherm, the monolayer transitioned from the L E state to L C (fig. 19A, purple arrow). Once in the L C phase, the surface pressure rapidly increased until monolayer collapse at 63.2 ± 0.83 mn/m (table 5). The take off area of 3DPPC:2PSM was almost identical to that of individual DPPC films, which were 98.5 ± 3.1 Å 2 /mol, compared to that of PSM at 83.8 ± 3.0 Å 2 /mol. While the initial lateral organization of 3DPPC:2SM was most impacted by DPPC, as evidenced by the take off area, it appears that the phase behaviour was more in line with PSM. For instance, the characteristic kink of the PSM isotherm was observed near the same location in the 3DPPC:2PSM isotherm, albeit at a slightly larger molecular area (60 vs 65 Å 2 /mol) and a slightly higher surface pressure (13 vs 16 mn/m). Finally, as the films were compressed to smaller molecular areas, the differences between the binary systems became less evident. At surface pressures in excess of 40 mn/m, all of the isotherms, with the exception of 2DPPE:3PGC, had noticeable regions of overlap or were within 2 Å 2 /mol of each other (fig. 19B). These surface pressures, where overlap occurred, were greater than those found in the open eye tear film (30 mn/m [17]), suggesting that at least in the binary combinations, the tear film components behaved similarly during blinking pressures. Most of the variability between systems occurred at surface pressures below this region with the exception of 1DPPC:1PGC and 3PGC:2PSM monolayers. A summary of the binary system compression isotherms results can be found in table 5. All films were very stable, with most collapsing in excess of 60 mn/m. Zero surface tension is equal to a surface pressure of 72 mn/m on an

85 66 aqueous subphase, which these systems clearly approached. The only exceptions were the two PGC containing mixtures, 2DPPE:3PGC and 3PGC:2PSM. DPPE and PGC on their own had the lowest collapse pressure so it was not surprising to see their binary mixture behaving in a similar fashion, collapsing at the lowest surface pressure, 51.0 ± 3.5 mn/m. 3PGC:2PSM collapsed at 58.8 ± 0.89 mn/m which was more comparable to the other binary system collapse pressures than it was to 2DPPE:3PGC. Table 5. Results from the compression isotherms of the binary lipid systems. Ratios represent the molar fractions of each lipid. Isotherms were completed on a 1x PBS buffered subphase at room temperature. Error is represented by the standard deviation; n 3. Lipid System Take Off Area (Å 2 /mol) Collapse Pressure (mn/m) 3DPPC : 2DPPE 77.2 ± ± DPPC : 1PGC 71.9 ± ± DPPC : 2PSM 97.9 ± ± DPPE : 3PGC 55.8 ± ± 3.5 1DPPE : 1PSM 94.2 ± ± PGC : 2PSM 61.9 ± ± Binary Lipid Compression Modulus To determine the impact on interfacial elasticity when the model system lipids were mixed into simple binary systems, we measured the β as a function of surface pressure (fig. 21).

86 67 Based on the peak β values alone, it appears that 1DPPC:1PGC and 3PGC:2SPM films were the most inelastic, reaching values in excess of 190 mn/m (table 6). The remaining four binary lipid systems had very similar β max values, all within 5 mn/m of 160 mn/m (table 6). From these results, all of the binary systems collapsed within the L C phase. However, it is important that we look at the entire data, not focusing exclusively on collapse behaviour alone. The three PGC films (1DPPC:1PGC, 2DPPE:3PGC, and 3PGC:2PSM), were the most rigid at initial surface pressures, evidenced by the rapid increase in β values at low surface pressures when compared to the remaining three systems. This initial β increase was the most rapid for 2DPPE:3PGC monolayers, reaching β values greater than 100 mn/m at surface pressures below 5 mn/m. This indicates that 2DPPE:3PGC films entered the L C phase immediately following the gas phase, while all other binary systems existed in the L E state at these pressures. PGC appears to have a condensing or ordering effect on the lipids studied with the greatest impact on DPPE monolayers. Based on the lipid ratios from the 2DPPE:3PGC system, if the mixing were ideal the take off area would be 63.9 Å 2 /mol, using the pure lipid take off areas of 73.1 Å 2 /mol for DPPE and 57.7 Å 2 /mol for PGC. This value is larger than the observed take off area for 2DPPE:3PGC at 55.8 Å 2 /mol, which was closer to pure PGC. Pure DPPE was shown to be more fluid than the other lipids (table 4), however, when mixed with PGC it formed the most rigid monolayer overall. 2DPPE:3PGC was overall more rigid because even though it collapsed at a lower β value, these monolayers existed exclusively in the more ordered L C while the others did not. The next most inelastic binary film was 3PGC:2PSM (fig. 21). Like 2DPPE:3PGC before it, this film underwent initial ordering seen by a rapid increase in β values at low surface pressures. However, unlike the 2DPPE:3PGC films, there was an evident kink on the β vs

87 68 surface pressure graph during the initial rise in β values, below the L C threshold (fig. 21, blue arrow). One of the differences between PSM and PGC monolayers is that PSM can exist in the L E phase while PGC does not. The kink in the compression modulus graph may then be a result of phase mixing between the PGC and PSM molecules. As phase mixing occurs, the interfacial elasticity increases, evidenced by drops in β values at those surface pressures. At the kink s surface pressure (4 mn/m) the PGC lipids would exist in the L C phase while the PSM ones would be in the less ordered L E state. The kink in figure 21 for 3PGC:2PSM is followed by a short decrease in the slope, confirming the change in film elasticity. The decreased slope is brief, rapidly increasing before another kink was observed at a surface pressure of approximately 20 mn/m. PSM monolayers on their own undergo phase mixing as evidenced in figure 17, occurring at 14 mn/m. Therefore, while the PGC lipids in the binary film will continue in the L C state at 20 mn/m, the PSM lipids may be undergoing a phase transition from the L E to the L C state, with a brief period of phase overlap in between. The initial transition of PSM lipids into the L C phase could account for the kink at 20 mn/m and the completed transition could account for the last kink at 26 mn/m. Additionally, PGC may be influencing the transition of PSM from L E to L C because it occurred at a higher surface pressure than it did in the absence of PGC (20 mn/m vs 14 mn/m).

88 69 Figure 21. Representative compression modulus curves for the six different binary lipid systems. 3DPPC:2DPPE (red), 1DPPC:1PGC (green), 3DPPC:2PSM (purple), 2DPPE:3PGC (light blue), 1DPPE:1PSM (orange), and 3PGC:2PSM (dark blue). The black line represents the compression modulus value (100 mn/m) in which monolayers transition from the L E to the L C phase. Arrows highlight areas where the slope changes, kinks are present, or other points of interest.

89 70 The last of the more rigid PGC binary films was 1DPPC:1PGC (fig. 21). Unlike the two previously discussed monolayers, the lower pressure β increases were initially less rapid. This films elasticity was more similar to the PGC less binary systems at lower surface pressure, evident by substantial overlap in figure 21 (green arrow). The initial rise in β values corresponds to the region on figure 21 where β increased almost vertically at the lower surface pressures. At these pressures, the DPPC containing PGC binary system were more elastic than both the DPPE and PSM containing PGC films. The increased rigidity from PGC binary mixtures with DPPE or PSM was most likely from hydrogen bonding, increasing the lipid ordering. DPPC was the only lipid studied without hydrogen bonding potential, which may account for difference in fluidity between the PGC binary systems. Additionally, pure DPPC monolayers at surface pressures under 5 mn/m were more elastic than any other lipids studied, which may account for the apparent resistance to chain ordering caused by PGC. Indeed, once the surface pressure exceeded 5 mn/m, the rigidity quickly increased in the 1DPPC:1PGC film as it did in the pure DPPC film. The rapid increase in the β values continued until the surface pressure reaches approximately 12 mn/m, corresponding to the kink in figure mn/m also roughly corresponds to the surface pressure at which pure DPPC monolayers transition completely into the L C phase, after L E -L C coexistence, based on the results from figure 17. This kink, therefore, may represent the point at which the DPPC lipids in the 1DPPC:1PGC films become more ordered, represented by the slope change. The binary system containing the two glycerol based lipids, 3DPPC:2DPPE, had unique compression modulus behaviour (fig. 21). The initial compressibility was most likely due to both of the lipids existing in the L E phase based on their individual compressibilities described earlier. The presence of a kink at 10 mn/m can most likely be described by the DPPC lipids

90 71 transitioning from L E to the L C state, causing a change in the interfacial elasticity (fig. 21, red arrow). This kink was more pronounced than the kink from the 1DPPC:1PGC system previously described, most likely due to the relative increase in the transitioning lipid. To be more specific, there are 3 parts DPPC to 2 parts DPPE present here, when previously the mixture was a one to one ratio of DPPC to PGC. The relative increase in DPPC lipids within the overall film may correspond to a relatively more pronounced kink during the transition. Additionally, on the PBS subphase DPPE was quite fluid, most likely enhancing elasticity of this binary system as well. However, the rigidity of pure DPPC monolayers increased substantially at higher surface pressures, which was also seen here by a rapid increase in β values following the kink until reaching a maximum. It was at this point that 3DPPC:2DPPE behaved uniquely. The maximal β for 3DPPC:2DPPE was reached at lower surface pressures than other lipid mixtures tested and was maintained over extended surface pressure increases, exhibited as a maximum plateau instead of a peak. This was in contrast to the other binary systems which become their most rigid immediately prior to collapse, evidenced by the sharper β peak followed by a rapid decline. For 3DPPC:2DPPE, the max β was reached at surface pressures approximately 20 mn/m below collapse, and was maintained eventually until collapse. The plateau indicates that the rate of pressure increase was perfectly maintained during this compression region as β is determined by taking the 1 st derivative from the surface pressure-area isotherm. The natural open eye tear film surface pressure (between mn/m [17]) would fall within this compressibility plateau suggesting that DPPC and DPPE mixtures may have a role in maintaining the lipid packing of the fully formed tear film. If we compare this to the behaviour of individual lipids, this plateau may be due to the action of DPPE. Of all the individual lipids, the changes in β over large

91 72 surface pressures were the smallest for DPPE (fig. 17). DPPE may then be stabilizing the monolayer at specific rigidities, with the extent of its action dependent on the other lipid present. This phenomenon, to a degree, was also observed in the 1DPPE:1PSM film (fig. 21). Between surface pressures of 12 mn/m and 31 mn/m, while the changes to β were not flat they were far smaller than in other non-dppe lipid systems. The other interesting feature of the 1DPPE:1PSM monolayer was the presence of a valley or local minimum at approximately 38 mn/m (fig. 21, orange arrow). As mentioned earlier, local minima in compression modulus vs. surface pressure graphs correspond to increased interfacial elasticity associated with phase coexistence. The presence of this feature on the 1DPPE:1PSM figure appeared at much higher surface pressures than the phase mixing associated with the individual components. More specifically, the valley for 1DPPE:1PSM would be characteristic of L E -L C phase coexistence based on the elasticity change. While PSM individually exhibits this behaviour it takes place at surface pressures 25 mn/m lower than the apparent transition in the 1DPPE:1PSM binary system. As suggested in the previous section, this local minimum was most likely from adsorbed anions being released back into the subphase. Large negative ions will insert into phospholipid head groups driven by the electrostatic interactions and from increased entropy associated with shedding a highly coordinated hydration shell [110] but will be excluded as the fluidity of the film decreases [95]. More specifically, large anions can have highly ordered hydration shells, coordinating up to 30 individual water molecules per anion, which decreases to between waters following adsorption to the interfacial region in lipid monolayers [110]. In the 1DPPE:1PSM curve from figure 21, β values approach the 100 mn/m boundary that distinguishes the L E from the L C phase prior to the observed local minimum. Up to this point the gradual increases in β would most likely come from adsorbed anions stabilizing the L E phase and

92 73 preventing more rapid β increases from lipid ordering. In approaching the L E -L C threshold the lipid ordering and packing becomes too great, leading to the anions being ejected. This caused a decrease in the β at 38 mn/m illustrated by the orange arrow in figure 21, most likely from a change in lipid packing once the anions were released, temporarily increasing the interfacial elasticity. Furthermore, the local minimum suggests that this process may not have been immediate, that anion release occurred over time instead of all at once. Had the process been from a single concerted event a kink would have been present instead of the valley observed in figure 21. This would indicate that anion exclusion may not have a distinct cut off but instead takes place over a small range of pressures. Once the anions were excluded from the monolayer, the rigidity rapidly increased until collapse, as the cause of earlier monolayer fluidity was removed. In regards to the final binary system, 3DPPC:2PSM monolayers had elastic properties similar to their pure lipid components based on the location of the local minima and the general shape of the curve (fig. 21). The 3DDPC:2PSM trace from figure 21 can be broken down into four sections with respect to the β value changes; the initial rise, followed by a decrease in slope, to the presence of a local minimum, and finally, rapid increase until collapse. Both pure DPPC and PSM films followed these trends as well. The initial β rise is in regards to the transition out of the gas phase into the L E state. Following that, the change in β tapers off as the film begins to transition into the L C phase. This transition has a marked increase in elasticity due to phase coexistence, resulting in a β value local minimum. Once all the molecules have entered the L C phase lipid ordering causes film rigidity and therefore a rapid increase to β. The L E -L C phase transition for the 3DPPC:2PSM system occurred at approximately the same surface pressure as individual PSM monolayers, which was roughly 15 mn/m compared to 6 mn/m for DPPC.

93 74 However, the maximum β value reached for 3DPPC:2PSM (168.1 ± 6.55 mn/m) was more similar to DPPC (158.6 ± 4.85 mn/m) than to PSM monolayers (204.0 ± 5.84 mn/m). From these two results, it appears that the interfacial elasticity of this binary system was more influenced by PSM at lower surface pressures and by DPPC at higher surface pressures. Table 6. The average compression modulus (β, mn/m) at different surface pressures (mn/m) and the average maximum compression modulus value (mn/m) for the binary lipid systems. The compression modulus was obtained from the compression isotherms using the equation in section Error is reported as the standard deviation (n 3). Lipid System β at 10 mn/m β at 20 mn/m β at 30 mn/m Maximum β (mn/m) (mn/m) (mn/m) (mn/m) 3DPPC : 2DPPE 69.6 ± ± ± ± DPPC : 1PGC 97.7 ± ± ± ± DPPC : 2PSM 47.5 ± ± ± ± DPPE : 3PGC ± ± ± ± DPPE : 1PSM 60.8 ± ± ± ± PGC : 2PSM ± ± ± ± 10.8 To summarize, based on the results from the six different binary systems, a few patterns have emerged. The films containing PGC had the highest compression modulus values, and therefore, were the least elastic and most rigid. As well, adding PGC to the other model lipids resulted in a condensing effect on the monolayer, shifting the isotherm to lower molecular areas.

94 75 PGC was the only lipid within the model systems that carried no local charges; therefore, electrostatic repulsion would in effect be zero. The van der Waals forces that order the hydrocarbon tails would not need to overcome any electrostatic repulsion, increasing the effectiveness of their interactions. Additionally, hydroxyl moieties on the sugar head group allow it to hydrogen bond, leading to further lipid ordering. When these two features are coupled, it is not surprising that PGC causes condensation and inelasticity to monolayers. The other trend suggested by the binary results was that DPPE may play a major role in maintaining the relative elasticity within the films. In all DPPE systems, changes to the compression modulus were small over larger surface pressure ranges when compared to the other lipids. This phenomenon would occur at different β values depending on the other lipid present. For instance, the plateau observed in the 3DPPC:2DPPE system occurred at a β of 160 mn/m, while in 1DPPE:1PSM monolayers the β fluctuated within 25 mn/m over surface pressures ranging from 10 mn/m to 40 mn/m. Compare this to 3DPPC:2PSM which, over the same surface pressures, had β changes from 48 to 153 mn/m. Therefore, while DPPE may not make each film in itself more elastic, it may buffer rapid ordering, evidenced by smaller increases in compression moduli over large surface pressure changes. 3.3 Tertiary and Quaternary Lipid Systems Compression isotherms of the more complex tertiary and quaternary mixtures exhibited the disappearance of more distinguishing isotherm features, such as shoulders or kinks (fig. 22). With the exception of the DPPC and PSM containing tertiary mixtures, the isotherms in figure 22 contained no obvious kinks or shoulders between take off and collapse. It was not surprising that individual lipid traits may be hard to distinguish in such mixtures. In the few cases that kinks

95 76 were observed, the lipids present had similar isotherms during pure lipid compressions. These two systems were 3DPPC:2DPPE:2PSM and 3DPPC:3PGC:2PSM. Of all the tertiary and quaternary mixtures, 3DPPC:2DPPE:2PSM was the least condensed based on the take off area. Once compressed to 92.3 ± 0.53 Å 2 /mol (table 7), the monolayer transitioned from the gas phase into the L E phase. The take off of this tertiary system was almost exactly equal to the average take off area of individual DPPC and PSM films, if the molar ratios are taken into account. More specifically, the take off of DPPC and PSM was 98.5 and 83.8 Å 2 /mol (table 3), respectively. The calculated take off average of a 3DPPC:2DPPE monolayer using these values would be 92.6 Å 2 /mol. With this in mind, it s possible that initial ordering of the 3DPPC:2DPPE:2PSM monolayer occurs between the choline based DPPC and PSM lipids first. This may be from the combination of DPPE having a smaller head group and hydrogen bonding potential where surface pressure increases from DPPE would most likely occur only at smaller molecular areas. Following take off, at approximately 2.4 mn/m the 3DPPC:2DPPE:2PSM monolayer began to transition from the L E phase into the L C state. This was evidenced by both a kink and flatter slope in the isotherm (fig. 22, green arrow). Due to the apparent shallowness of the slope following the kink, the transition into the L C phase was most likely not rapid, allowing for L E -L C phase coexistence. This transition resembled that of DPPC based on the molecular area and surface pressure in which it occurred. Individually, DPPC transitioned from the L E to L C at approximately 5 mn/m and 80 Å 2 /mol (fig. 15). In the tertiary mixture, this transition was shifted to larger molecular areas and lower surface pressures (fig. 22). As the 3DPPC:2DPPE:2PSM monolayer completed its transition into the L C phase, the surface pressure gradually increased until collapse at 62.0 ± 0.60 mn/m (table 7). Overall, surface pressure increases occurred over

96 77 larger molecular areas for this system, indicating it was more fluid and less condensed than the other tertiary and quaternary mixtures. The next most fluid complex system was 3DPPC:3PGC:2PSM (fig. 22). The isotherm took off at the second largest molecular area for the tertiary systems at 81.6 ± 1.53 Å 2 /mol. Following this transition from the gas phase, surface pressures increased until reaching approximately 1.3 mn/m and a molecular area of 75.6 Å 2 /mol. At this point, like 3DPPC:2DPPE:2PSM, the 3DPPC:3PGC:2PSM monolayer underwent L E -L C phase mixing (fig. 22, purple arrow). This phase coexistence was only evident in the two tertiary systems that contained both DPPC and PSM. For the complex films to undergo this transition there appears to be a necessity for the tertiary mixture to consist of both choline containing lipid species independent of the nature of the third component. The lack of the characteristic kink associated with the L E -L C phase coexistence in the remaining two tertiary systems supports this statement. Additionally, the condensing effect of PGC is evident in the 3DPPC:3PGC:2PSM monolayer. Compared to 3DPPC:2DPPE:2PSM, the location of the L E -L C plateau for 3DPPC:3PGC:2PSM has shifted to smaller areas by 10 Å 2 /mol, occurring also at a lower surface pressure (1.0 mn/m). As the film was compressed, gradual surface pressure increases occurred until collapse at 65.6 ± 0.54 mn/m (table 7). Of all the 15 different individual, binary, tertiary, and quaternary systems, this was the highest collapse pressure and therefore, the most stable monolayer.

97 78 Figure 22. Average compression isotherm (n 3) for the four different tertiary and quaternary (3DPPC:2DPPE:3PGC:2PSM) mixtures on a 1x PBS subphase. 3DPPC:2DPPE:3PGC (red), 3DPPC:2DPPE:2PSM (green), 3DPPC:3PGC:2PSM (purple), 2DPPE:3PGC:2PSM (orange), and quaternary mixture (blue). Arrows highlight plateau regions. Substituting PSM with DPPE to get 3DPPC:2DPPE:3PGC, lead to a less fluid and more condensed monolayer (fig. 22). For 3DPPC:2DPPE:3PGC, take off occurred at a molecular area over 15 Å 2 /mol smaller than 3DPPC:3PGC:2PSM, at 64.9 ± 1.63 Å 2 /mol (table 7). Like other DPPE and PGC monolayers, surface pressure increases were more rapid following take off, with

98 79 no evidence of phase mixing based on the isotherm. While pure DPPC monolayers did exhibit phase mixing, the results from the DPPC binary mixtures with either DPPE or PGC abolished this, evidenced by the disappearance of the kink and plateau from these isotherms (fig. 19A). In the tertiary mixture 3DPPC:2DPPE:3PGC, this result was, unsurprisingly, carried forward. The 3DPPC:2DPPE:3PGC monolayer reached collapse at a surface pressure of 61.5 ± 0.57 mn/m (table 7). Like the other tertiary systems, the collapse pressure was higher than individual lipid monolayers. Combination of different lipid species therefore appears to have a stabilizing effect on the films. 2DPPE:3PGC:2PSM was the most rigid and most condensed of the complex mixtures (fig. 22). This was expected based on the previous experiments. Of the binary mixtures, 2DPPE:3PGC experienced surface pressure changes over the smallest molecular areas, taking off at the smallest molecular area, 57.7 ± 1.28 Å 2 /mol, thus making it the most condensed of those systems. Additionally, PSM monolayers were more rigid than DPPC films due to hydrogen bonding in the lipid interface region. It stands to reason that of the tertiary mixtures, the one containing DPPE, PGC, and PSM would continue this trend and be the most rigid. The surface pressure then steadily increased until collapse at 57.5 ± 2.08 mn/m (table 7). Along with being the most rigid film of the tertiary mixtures, it was also the least stable, having the lowest collapse pressure. It is important to note, however, that a collapse of 57 mn/m is still fairly high, and that least stable is very much a relative term. When combining all the model lipids into the quaternary mixture, the result was the dark blue compression isotherm from figure 22. This monolayer transitioned out of the gas phase at a molecular area of 76.3 ± 2.96 Å 2 /mol (table 7). Taking into account the take off areas of the individual lipids and their molar ratios, the ideal take off area for the quaternary system would be

99 Å 2 /mol. The similarity between these values suggests that lipid mixing at the take off molecular area was close to ideal. Following take off, surface pressure increases were gradual and smooth without apparent kinks, along with no sudden surface pressure increases. From the take off area to approximately 58 Å 2 /mol the isotherm slope was at its most gentle. It s likely that during this portion of the isotherm, regions of the monolayer existed in both the L E and L C states, due to the small increases in surface pressure. However, the isotherm represents the overall film characteristics, and may not allow identifying distinct transitions from the L E to L C phase. Additionally, while half of the lipid species present had characteristic kinks in their compression isotherm, the other half do not. As evidenced in the tertiary systems, these transition kinks were lost when the lipid components responsible for this phenomenon did not represent the majority of the lipids. From the isotherm, however, it can be seen that at initial surface pressures the quaternary film was more fluid than the DPPE and PGC containing tertiary systems. Furthermore, as surface pressures approached collapse, the quaternary system was the least condensed, demonstrated by the film occupying the largest molecular area. The quaternary monolayer collapsed at 61.6 ± 0.73 mn/m, which was comparable to the other complex systems tested (table 7). Generally speaking, the quaternary system had properties intermediate to its lipid components.

100 81 Table 7. Results from the compression isotherms of the tertiary and quaternary lipid systems. Ratios represent the molar fractions of each lipid. Isotherms were completed on a 1X PBS buffered subphase at room temperature. Error is represented by the standard deviation; n 3. Lipid System Take Off Area (Å 2 /mol) Collapse Pressure (mn/m) 3DPPC : 2DPPE : 3PGC 64.9 ± ± DPPC : 2DPPE : 3PSM 92.3 ± ± DPPC : 3PGC : 2PSM 81.6 ± ± DPPE : 3PGC : 2PSM 57.7 ± ± 2.1 Quaternary 76.3 ± ± Tertiary and Quaternary Lipid Compression Modulus At first inspection, the most striking difference when comparing the compression modulus of the binary mixtures to the tertiary and quaternary systems is the shape of the overall curves. For the more complex mixtures, there appears to be symmetry associated with β vs. surface pressure graphs (fig. 23). With the exception of 3DPPC:2DPPE:2PSM, as the β increased and the films became more rigid, a maximum was reached at surface pressures more than 15 mn/m below collapse. Furthermore, the decrease in β associated with collapse was similar to the initial rise. This gave the curves a somewhat symmetrical mountain like shape. This phenomenon was most clearly seen in the 3DPPC:2DPPE:3PGC and 3DPPC:3PGC:2PSM systems. In both of these mixtures, the β values steadily rise, reach a maximum, and then steadily decrease. However, 3DPPC:2DPPE:3PGC had more gradual β changes evident by the much rounder peak (fig. 23, red arrow).

101 82 The reason for this behaviour is twofold. First, as different lipid species are added to the mixture there is a packing or averaging effect on the phase behaviour. The isotherm, and therefore the compression modulus graph, is the result of the surface activity of all the lipids present. From the individual isotherms it is clear that the physical characteristics of each lipid are different. For instance, when the lipids were combined, the different interfacial properties led to more heterogeneity in the monolayer with regions of different packing. This abolished distinguishable isotherm features such as kinks because of interfacial averaging of the different lipids. The second cause for the apparent symmetry is that the complex tertiary and quaternary mixtures were collapsing more gradually. As the monolayer collapses, there is either a fast downward drop in surface pressure or a plateau region as films are compressed past their collapse point. The resulting change in the isotherm slope during collapse causes a drop in the compression modulus. Additionally, immediately prior to collapse, the rigidity reaches a maximum due to chain ordering, seen as a peak in the β vs surface pressure figure. For the tertiary and quaternary mixtures, small regions of the film may be collapsing earlier or forming multilayers as the collapse pressure is approached. This is evident in the isotherms by a more rounded and gradual slope change during collapse. If collapse becomes more gradual, the decrease in β following the maximum will also be more gradual, leading to the symmetry in the β vs surface pressure figure, unique to the complex systems. More importantly, the β peaks for the complex mixtures occur between mn/m, which happens to be at pressures slightly above the natural open eye surface pressure [17]. It is possible then that the polar lipid layer is helping to prevent sudden collapse of the natural tear film during a blink or other disruptive events like eye movement or environmental effects. The mechanism for this may be similar to another lipid monolayer, lung surfactant, which is able to form protein mediated multilayers at

102 83 high surface pressures to prevent collapse (reviewed in [111]). Furthermore, with the recent discoveries of protein insertion into the tear film lipid layer, there is a need to explore these interactions in more detail. Figure 23. Representative compression modulus curves for the different tertiary and quaternary lipid systems. 3DPPC:2DPPE:3PGC (red), 3DPPC:2DPPE:2PSM (green), 3DPPC:3PGC:2PSM (purple), 2DPPE:3PGC:2PSM (orange), and quaternary (blue). The black line represents the compression modulus value (100 mn/m) in which monolayers transition from the L E to the L C phase. Arrows highlight specific points of interest such as local minima.

103 84 Of all the tertiary systems, 2DPPE:3PGC:2PSM was the most inelastic from initial surface pressure increases until collapse (fig. 23). 2DPPE:3PGC was the most inelastic binary systems, followed by 3PGC:2PSM. This trend clearly continued when these lipids were combined into their tertiary mixture. The slope from the β surface pressure graph for 2DPPE:3PGC:2PSM would have been continuous until the peak if not for a shoulder at a surface pressure of approximately 13 mn/m (fig. 23, orange arrow). This interfacial elasticity change occurred at the same surface pressure as the L E -L C phase transition of pure PSM films. Thus, the respective β change in figure 23 was likely associated with PSM ordering during this phase transition. From this point forward the rigidity increased until a peak was reached at a surface pressure of approximately 27 mn/m. After reaching a maximum, β values decreased at approximately the same rate that they increased previous to the peak, resulting in the mountain shape described in the previous paragraph. Based on the surface pressure-area isotherm from figure 22, collapse occurred at 57.5 ± 2.08 mn/m (table 7), almost 30 mn/m greater than where the β peak was observed. This indicates that collapse was possibly occurring over larger surface pressures as opposed to at one specific point, possibly due to partial collapse of a lipid component. It appears that broadening or averaging of different physical properties occurs not only with respect to the phase behaviour discussed earlier, but also in regards to film stability. Collapse nucleation sites may be occurring at these lower surface pressures, changing the interfacial elasticity and causing a decrease in β, until total collapse occurs exhibited by the horizontal slope in the isotherm. Both DPPE and PGC films collapsed at surface pressures below 50 mn/m, while PSM did so at 60 mn/m (table 3). PGC and DPPE molecules within the film may therefore be undergoing collapse as surface pressures approach these values. It appears then that the collapse behaviour of monolayers becomes more complex with increasing lipid diversity.

104 85 This phenomenon was also evident in the tertiary system 3DPPC:2DPPE:3PGC (fig. 23). Rapid β value increases at low surface pressures indicate that the film was fairly inelastic; the L E to L C compression modulus threshold of 100 mn/m was exceeded prior to surface pressures reaching 8 mn/m. Additionally, this curve was almost a perfectly inversed U shape, with no distinguishing features like kinks or shoulders besides the peak β value. The rate of β increase slowed down at a surface pressure of approximately 16 mn/m. Here, the films elasticity decreased more gradually until a maximum β of ± 9.29 mn/m (table 8). Following this maximum, the β values decreased at a similar rate to their initial increase. This behaviour again mimicked that of 2DPPE:3PGC:2PSM in terms of the films rigidity and complex interfacial behaviour during collapse for the same reasons as previously illustrated. Separating DPPE and PGC in the tertiary mixture resulted in more elastic films, as was the case for the 3DPPC:3PGC:2PSM system (fig. 23). Unlike the previous two films discussed, a local β minimum at a surface pressure of approximately 2.4 mn/m was observed (fig. 23, black arrow). This corresponds to the plateau region at 76 Å 2 /mol in the isotherm (fig. 22, purple arrow). The increase in film elasticity was, as mentioned previously, the result of clear phase coexistence within the monolayer. The surface pressure associated with this phase mixing was significantly depressed when compared to the two individual lipids, DPPC and PSM, which exhibited similar behaviour. For these lipids, phase mixing was present at 6 and 13 mn/m, respectively. Within the 3DPPC:3PGC:2PSM mixture, it is possible that both PSM and DPPC were prevented from forming the L E phase because of PGC induced rigidity, ordering the film and causing early promotion of the L C state at lower surface pressures. This makes sense when comparing this to the binary results where 3DPPC:2PSM exhibited phase coexistence at surface pressures only 2 mn/m smaller than that of PSM alone. Furthermore, binary mixtures of PGC

105 86 with either DPPC or PSM abolished the appearance of the L E -L C phase coexistence at all surface pressures. Following this phase mixing region, 3DPPC:3PGC:2PSM β values increased steadily until the maximum was reached at ± 6.49 mn/m (table 8), followed by a rapid decrease. Excluding the small dip at 2 mn/m, the overall shape of this curve was fairly symmetrical, most likely again from the complex collapse behaviour outlined earlier. The final and by far most elastic tertiary mixture was 3DPPC:2DPPE:2PSM (fig. 23). The compression modulus did not pass the 100 mn/m L E -L C barrier until surface pressures were in excess of 25 mn/m. This films fluidity was most likely imparted by the interactions between DPPE and PSM molecules. β values from the 1DPPE:1PSM binary mixture remained under the 100 mn/m threshold until surface pressures were above 40 mn/m. Pure DPPE or PSM crossed this boundary at lower surface pressures, at 30 and 18 mn/m respectively. Therefore, based on these previous results, the combined effect of DPPE and PSM on film elasticity was carried forward to the 3DPPC:2DPPE:2PSM monolayer. Similar to the 3DPPC:3PGC:2PSM system, there was a dip in the β values at the exact same surface pressure, 2.4 mn/m, in the 3DPPC:2DPPE:2PSM mixture (fig. 23, black arrow). However, unlike 3DPPC:3PGC:2PSM, an additional shoulder was observed in 3DPPC:2DPPE:2PSM at roughly 17 mn/m (fig. 23, green arrow), only 1 mn/m above the transition in 3DPPC:2PSM monolayers. The presence of more unique features in the 3DPPC:2DPPE:2PSM graph may be an indication that these three lipids are mixing less ideally than in mixtures made with PGC. As mentioned previously, ideal mixing seems to average the interfacial properties of pure and binary lipid systems, thus removing any kinks or shoulders. The result of this is lateral heterogeneities from the different components not mixing together, leading to regions with different packing and phase behaviour. With this in mind, the first dip in the 3DPPC:2DPPE:2PSM β vs surface pressure figure may be from DPPE

106 87 lipids transitioning out of the gas phase. This is based on the pure lipid isotherms where surface pressure increases occurred at much smaller areas in DPPE films than either DPPC or PSM due to head group size differences. Prior to this first minimum, the initial pressure increases would, therefore, be most likely from DPPC and PSM entering the L E phase. At 17 mn/m, the DPPC and PSM lipids may then be transitioning out of the L E state to the more ordered L C phase, causing the shoulder and increasing the change in β values as seen in figure 23 (green arrow). β values continued to increase until reaching a maximum at ± 20.4 mn/m. This was 40 mn/m lower than the next most elastic tertiary system, 3DPPC:2DPPE:3PGC. Combining all four lipid species into the quaternary mixture led to a film of intermediate elasticity when compared to the tertiary systems (fig. 23). The initial rate of β increase for this system, based on their relative slopes, was almost halfway between the more rigid 3DPPC:2DPPE:2PGC and the more fluid 3DPPC:3PGC:2PSM. A shoulder was observed at a surface pressure of approximately 12 mn/m indicating a transition from the L E state to the L C phase (fig. 23, dark blue arrow). After which, β increases were rapid until the maximum was observed at ± 12.3 (table 8). Like all the other films tested, following this peak, the β steadily decreased. The interfacial elasticity for each of the tertiary and quaternary systems over a range of surface pressures has been summarized in table 8.

107 88 Table 8. The average compression modulus (β, mn/m) at different surface pressures (mn/m) and the average maximum compression modulus value (mn/m) for the tertiary and quaternary lipid systems. The compression modulus was obtained from the compression isotherms using the equation in section Error was recorded as the standard deviation (n 3). Lipid System β at 10 mn/m β at 20 mn/m β at 30 mn/m Maximum β (mn/m) (mn/m) (mn/m) (mn/m) 3DPPC : 2DPPE : 3PGC ± ± ± ± DPPC : 2DPPE : 3PSM ± ± ± ± DPPC : 3PGC : 2PSM ± ± ± ± DPPE : 3PGC : 2PSM ± ± ± ± 10.9 Quaternary ± ± ± ± Compression Isotherm Summary In summary, PGC and DPPE had a condensing effect on the monolayers examined. This resulted in surface pressure increases occurring over smaller molecular areas. Additionally, DPPC and PSM films were slightly more stable, collapsing at higher surface pressures. Although, all lipids tested had fairly high collapse pressures in general. In regards to the natural tear film, the high collapse pressures indicate that the component lipids are able to form highly stable films. Furthermore, this stability was not negatively impacted when these lipids were combined into the ratios found in vivo. In terms of the interfacial elasticity, DPPE films were the most elastic and fluid while PGC were the least. This effect was most likely due to DPPE having a higher affinity for anions

108 89 in the subphase leading to anion adsorption that caused stabilization of the more fluid L E phase. On the other hand, DPPC and PSM were of intermediate elasticity to the other two lipids. The most striking observation was the presence of gradual collapse evident in the tertiary and quaternary systems. As mentioned in the introduction, tear film break up time has serious consequences on the healthy state of the eye. Rapid break up often leads to disease states such as DES. The polar lipid layer of tear film may then be forming multilayers at higher surface pressures or collapsing more gradual, thus preventing sudden tear film break up during a blink. Indeed, tear film models incorporating DPPC and non-polar molecules like cholesterol esters, and tri-acyl-glycerols, and meibomian extracts, have been shown to form multilayers upon compression when in the presence of high concentrations of ectoine [112, 113]. It then appears that both the polar lipid only films and mixtures containing polar and non-polar lipids have some functionality regarding tear film collapse inhibition at high surface pressures.

109 90 Chapter Four: Film Response to Multiple Compressions 4.1 Surface Pressure-Area Isocycles The compression and expansion of Langmuir monolayers is a good mimic of blinking, albeit at a much reduced speed. The barrier from a Langmuir trough compresses a film over minutes while people can blink their eyes multiple times each second. Each lipid system was compressed and expanded five times to determine how the lateral packing changes over multiple blinks. Shifts in the molecular area or changes in isotherm shape indicate that packing differences occur between compression and expansion of the monolayer. The difference between compression and expansion is referred to as hysteresis (fig. 24A). The blue compression isotherm of figure 24A does not overlap with the red expansion isotherm, thus indicating hysteresis. If there was no hysteresis, these isotherms would overlap completely. Generally speaking, hysteresis occurs when the current thermodynamic state of the system depends on its past state. For our purposes, the presence of hysteresis indicates that films are susceptible to deformation during compression and expansion. The less hysteresis the more resistant the film is to deformation and vice versa. Furthermore, looking at how the hysteresis changes between the first and last isocycles will provide insight into film reorganization and stability over multiple blinks. For instance, a smaller hysteresis within the 5 th cycle relative to the 1 st cycle would imply that lipid packing differences between expansion and compression were decreasing. This would suggest that the major packing changes or film deformation occurred during the initial compression-expansion cycle and that lipid reorganization was approaching equilibrium.

110 91 Figure 24. Examples illustrating which part of the isocycle belongs to the compression or expansion trace (A) and what is meant by the 1 st and 5 th cycles (B). The arrows in panel B illustrate comparisons within a cycle (the longer arrow) and between cycles (the shorter arrow). Finally, looking at the differences between the entire 1 st and 5 th cycles was determined to explore how the lipid packing differed in the final cycle compared to the 1 st cycle (fig. 24B). For instance, a small difference between the 1 st and 5 th cycles would indicate a films ability to resist deformation over multiple cycles or blinks. This analysis also relates to the reversibility of isothermal compression and expansion. Reversible deformation occurs if the hysteresis present within a single isocycle was large but had little differences between cycles, indicating that major packing changes from compression or expansion were most likely reversible. Figure 24B illustrates this possibility as the purple isocycle overlaps more closely with the green isocycle than it does with its own compression and expansion isotherm, evidenced by the small and large arrows respectively. The film was resisting permanent deformation because the lipid packing

111 92 remained relatively unchanged over multiple compressions and expansions based on the similar isocycles. Therefore, these studies provide evidence into which lipids were more important in maintaining film stability over multiple blinks To quantify these results, the root mean squared deviation (RMSD) was also reported for each lipid system, calculated as described in section The RMSD reports on how different two data sets are. In our case, the data sets examined were the RMSD between the compression and expansion of the 1 st cycle, the compression and expansion of the 5 th cycle, and between the entire 1 st and 5 th cycles. For the last data set, the RMSD from the entire 1 st and 5 th cycles was completed by comparing the surface pressure changes as the film undergoes compression and expansion from the first cycle with the surface pressures changes from the 5 th cycle, at specific molecular areas. In other words, this analysis compares one entire cycle with another one, and thus reports on how much the film organization has been impacted over continuous compression and expansions. In terms of film organization, the higher the RMSD the larger the difference between the data sets and therefore, the higher the cycle hysteresis. Conversely, the lower the RMSD, the fewer the changes to lipid packing and therefore, the more resistant the film was to deformation Individual Lipid Systems From the single lipid systems, the expansion curves from both the 1 st and 5 th cycles generally followed those of their respective compression curves (fig. 25). That is, while the expansion traces mostly occurred over smaller molecular areas relative to the compression traces, which indicates hysteresis, there were similar inflection points between the two. The expansion isotherms lagged behind the compression isotherms in the sense that over the same

112 93 change in surface pressure, film expansion occurred over smaller molecular areas than compression. However, this could also be explained in that over the same molecular area difference, surface pressure changes were less rapid during compression. Either way, this hysteresis, as described previously, was most likely the result of intermolecular forces causing increased cohesion between lipid molecules. This was not an artifact of the barrier speed because the films were expanded at the same rate that they were compressed. The monolayer attractive forces were stronger during expansion than compression possibly due to ordering. Compressing the film to smaller molecular areas increased the intermolecular interactions and ordering between the lipids such that film re-spreading occurred slower than the speed of the trough barrier as it opened up to larger areas. If the attractive forces remained the same over compression, the expansion isotherm would follow the compression isotherm identically, as the barrier speed was the same for both compression and expansion. For both the 1 st and 5 th cycles of DPPC (fig. 25A) the hysteresis was smallest when the film was most fluid, at surface pressures below 10 mn/m. This was especially so for the 5 th cycle. During both the L E state and the L E -L C phase coexistence pressures, the hysteresis between the 5 th compression and expansion was abolished. While in the L C state, the hysteresis was more pronounced for both the 1 st and 5 th DPPC cycles. Furthermore, there was a clear shift of the entire 5 th cycle to smaller molecular areas compared to the 1 st cycle. These observations were confirmed by the RMSD values for DPPC monolayers (table 9). The RMSD from the first cycle was 2.87 ± 0.45, while the 5 th cycle RMSD was 2.69 ± These values were not statistically significant indicating that the hysteresis remained relatively unchanged over multiple cycles and suggests that packing differences between compression and expansion remained constant regardless of the cycle. Additionally, the RMSD between the 1 st and 5 th cycles (3.33 ±

113 ) was larger than either cycle on their own. This higher inter-cycle RMSD was due to the shift to smaller areas as the film underwent multiple compression and expansions and not to changes in the overall isocycle shape. Therefore, while the packing differences between compression and expansion did not appear to change due to the low intra-cycle RMSDs (the RMSD between the compression and expansion from the same cycle), cycling led to film condensation evident by the 5 th cycle shifting to smaller molecular areas. This was most likely due to tighter packing following each cycle, accounting for a smaller effective area for each lipid. Furthermore, the shift to smaller molecular areas was probably not due to lipids being lost to the subphase, which would be unlikely do the hydrophobicity of the hydrocarbon tails. However, the possibility of lipid loss was calculated based on both the average molecule area (A m ) and the trough area (A t ) at the tear film physiological surface pressure of 30 mn/m between the 1 st cycle and the 5 th cycle: [ ( ) ( )] [ ( ) ( )] Within each square bracket is the total lipid present for that cycle at 30 mn/m. More specifically, A t,1st is the trough area based on the barrier position when the surface pressure reads 30 mn/m in Å 2, while A m,1st is the average molecular area at 30 mn/m in molecules/å 2, both during the 1 st cycle. Multiplying the trough area by the reciprocal of the average molecular area will give you the total amount of lipid molecules present. The second square bracket is the same except that the values are from the 5 th cycle. In all 15 systems, the difference between these two was determined to be zero, further suggesting that shifts in the molecular area between the isocycles were from packing differences and not lipids being ejected from the monolayer.

114 95 The only exception to the shape similarities between the expansion and compression isotherms was the 1 st cycle from DPPE (fig. 25B). There was an obvious hysteresis at higher pressures during the 1 st cycle (fig. 25B, black arrow). On the other hand, the 5 th expansion followed closely to the 5 th compression. There were, however, no apparent places of overlap within the cycles, indicating a higher hysteresis between compression and expansion of DPPE monolayers. Indeed, DPPE had the highest RMSD within the 1 st and 5 th cycles of the individual lipids systems, 5.31 ± 0.66 and 4.65 ± 0.60 respectively (table 9). The hysteresis and high intracycle RMSD indicate large packing changes between compression and expansion. On the other hand, there was no complete 5 th cycle shift to smaller areas, as was the case with DPPC. In fact, excluding the large inflection in the 1 st expansion, many regions of the 1 st cycle overlapped with the 5 th cycle. This was further evidenced by the small RMSD value between the 1 st and 5 th cycles at 1.81 ± 0.90 (table 9). The small difference between the 1 st and 5 th cycle either means that reorganization was slight over multiple compressions and expansions or that any apparent film deformation during compression was reversible. Finally, while the 5 th compression takes off at smaller areas than the 1 st cycle, the cause was most likely not due to lipid loss. This was not the case because at higher surface pressures, the 5 th and 1 st compressions begin to overlap. Furthermore, the majority of the 1 st and 5 th expansion isotherms overlap, which wouldn t be so if lipids had been ejected between or during cycling.

115 96 Figure 25. Selected surface pressure-area isocycles of the single lipid systems. (A) DPPC (B) DPPE (C) PGC (D) PSM. The first cycle is shown in blue and the fifth cycle in red. Each film was compressed until 15 mn/m before collapse and expanded until zero surface pressure was reached. The lipid system is shown above each graph. n 3 Of all the single lipid systems, PGC had the least pronounced hysteresis for both the 1 st and 5 th cycles (fig. 25C). This was confirmed by the RMSD calculations which were 2.69 ± 0.46 and 2.24 ± 0.31 respectively (table 9). The hysteresis was largest at lower surface pressures and smallest at higher surface pressures. Furthermore, the expansion isotherms closely followed the compression trace. When comparing both cycles, their differences were more pronounced during compression than expansion. Overlap of the 1 st and 5 th compression however, did occur at high

116 97 surface pressure ranges. On the other hand, the expansion isotherms overlapped nicely with one another at pressures in excess of 10 mn/m. The small RMSD value between the 1 st and 5 th cycles (1.76 ± 0.37, table 9) reflected this overlap. Finally, PGC reorganization over multiple compressions and expansions was minor because the calculated RMSD values were all relatively low. The pure PSM isocycles had properties intermediate to the other individual lipid systems (fig. 25D). Within the 1 st cycle, the hysteresis between compression and expansion remained constant with no apparent points of isotherm overlap following initial surface pressure increases. The differences between the compression and expansion isotherms from the 5 th cycle were minor relative to those from the 1 st cycle. In fact, at surface pressures below 5 mn/m the compression and expansion isotherms of the 5 th cycle overlapped. The RMSD from the 5 th cycle was lower than the 1 st cycle, confirming more packing similarities within the 5 th cycle relative to the 1 st. Finally, the entire 5 th cycle was shifted to smaller molecular areas when compared to the 1 st cycle. As was the case with DPPC, the packing between lipids became tighter over multiple compressions and expansions evidenced in the shift to lower molecular areas between the 1 st and 5 th cycles.

117 98 Table 9. The RMSD values from the pure lipid systems. The 1 st Cycle RMSD column describes the 1 st compression and expansion. The 5 th Cycle RMSD column describes the 5 th compression and expansion. The Inter-Cycle RMSD column is the overall difference between the 1 st and 5 th cycles. n 3 Lipid System 1 st Cycle RMSD 5 th Cycle RMSD Inter-Cycle RMSD DPPC 2.87 ± ± ± 0.59 DPPE 5.31 ± ± ± 0.90 PGC 2.69 ± ± ± 0.37 PSM 3.36 ± ± ± 0.58 The calculation of RMSD for both intra- and inter-cycles allows for a direct comparison between the different systems and provides an assessment of possible trends. For instance, the intra-cycle RMSD decreased for all individual lipid systems between the 1 st and the 5 th cycle (table 9). The RMSD decrease is an indication that reorganization was occurring over subsequent cycles. This suggests that the reorganization was moving towards equilibrium, with less packing changes occurring after each subsequent compression and expansion. That is, the packing became more consistent between compression and expansion moving from the 1 st to the 5 th cycle. From table 9, it was clear that the RMSD within cycles was the highest for DPPE and the lowest for PGC. In regards to DPPE, the large intra-cycle RMSD would arise from substantial cohesion between molecules, possibly due to the relatively smaller size of the PE head group, the hydrogen bonding potential, and buffer effects outlined in chapter 3, section 3.1. The hysteresis observed between the compression and expansion isotherms from figure 25B additionally

118 99 confirms the cohesion between DPPE molecules. Interestingly, both DPPE and PGC however, had the smallest inter-cycle RMSD, which arises from a high similarity between the 1 st and 5 th cycles. This would indicate that these two films were able to resist deformation over continuous compression and expansion cycles. Another explanation would be that any lateral reorganizing within cycles was reversed by the beginning of the next cycle. The hysteresis between compression and expansion indicates intra-cycle packing differences while the overlay between cycles however, suggests similar inter-cycle packing. Therefore, the deformation during compression must be relatively reversible to give rise to these behaviours. PGC may be resisting deformation altogether because the RMSD was consistently low for both the two intra-cycle values and the single inter-cycle RMSD value, comparatively speaking. DPPE on the other hand had larger intra-cycle RMSD values but a low inter-cycle RMSD indicating that packing was similar between cycles but not within them suggesting that reversible deformation was occurring. DPPC and PSM, on the other hand, exhibited RMSDs in between DPPE and PGC values when exclusively comparing intra-cycle values. However, DPPC and PSM had larger inter-cycle RMSD values than both DPPE and PGC. This observation may indicate film deformation as larger packing changes appear to be occurring over subsequent cycles, leading to greater intercycle differences for DPPC and PSM. Additionally, DPPC and PSM both experienced a shift to smaller molecular areas during the isocycles, suggesting tighter packing between lipid molecules. Tighter packing may have implications on the physiology of tear film, as protein insertion may be dependent on a more expanded lipid film. Comparatively speaking, it therefore appears that PGC and DPPE may provide greater stability to the tear film over multiple blinks.

119 Binary Lipid Systems The binary lipids experiments were conducted to determine if the trends observed for pure lipids were affected by the presence of other lipids. Figure 26 contains the isocycle results from the six different binary systems. Progressively smaller areas were observed for 3DPPE:2DPPE and 3DPPC:2PSM as the isocycles continued from the 1 st to the 5 th cycle (fig. 26A,C). For 3DPPC:2DPPE, the compression and expansion curves had similar shapes to one another with a slight hysteresis occurring at approximately 10 mn/m. However, there was an inflection point in the 5 th expansion isotherm at approximately 30 mn/m (fig. 26A, black arrow), resulting in a relatively larger hysteresis within this cycle at higher surface pressures. The RMSD of the 1 st cycle was slightly higher than that of 5 th cycle, 4.03 ± 0.83 vs ± 0.77 (table 10), however this was not significant. The hysteresis from figure 26A suggests increased cohesion following compression that remained relatively constant between cycles because the intra-cycle RMSD values did not change significantly. During the 5 th cycle, the compression and expansion isotherms had smaller molecular area differences than within the 1 st cycle. This would suggest a lateral reorganization that resulted in tighter packing during film cycling. The inter-cycle RMSD for 3DPPC:2DPPE was 4.14 ± 0.73 (table 10), over two times larger than the inter-cycle RMSD value of DPPE. From these results, DPPC had a negative impact on DPPE film stability over multiple compression-expansion cycles. 3DPPC:2PSM was the other binary system that exhibited a shift to smaller molecular areas (fig. 26C). The magnitude of this shift was represented by an inter-cycle RMSD value of 4.77 ± 1.06 (table 10). Based on this value, the 3DPPC:2PSM shift was larger than previously observed for 3DPPC:2DPPE films. This was not surprising because DPPC and PSM, as pure lipid films, experienced the greatest reorganization over multiple cycles (table 9). The apparent

120 101 increase in film cohesion of the 3DPPC:2PSM binary film was striking. The RMSD within the 1 st and 5 th cycles was 5.02 ± 0.21 and 3.75 ± 0.07 (table 10), respectively. This difference was significant to p<0.01. Both of these values were larger than the individual DPPC and PSM intracycle RMSDs. In figure 26C, both the 1 st and 5 th expansion closely follows the shape of their respective compression traces. The difference within the cycles arose from tighter packing, evidenced by the film s decreased molecular area at similar surface pressures between expansion and compression. Therefore, cohesion between lipid molecules increased in these mixtures. However, the 1 st cycle RMSD was larger than the 5 th cycle indicating that deformations were greater during the 1 st compression. Finally, like 3DPPC:2DPPE, the large RMSD between the 1 st and 5 th cycles of 3DPPC:2PSM represents lipid reorganization over multiple cycles. Common to both mixtures was DPPC. This lipid, at least in binary mixtures, appears to disrupt a films ability to resist deformation, evident by the largest inter-cycle RMSD values in these two systems. Subsequently, 1DPPE:1PSM and 3PGC:2PSM showed an increased resistance to deformation. The expansion isotherms from both the 1 st and 5 th cycles of 1DPPE:1PSM closely followed their compression traces (fig. 26E). Compression and expansion from both cycles shared similar slopes with no additional inflection points. The intra-cycle RMSD was 3.98 ± 0.67 for the first and 3.47 ± 0.63 for the 5 th cycle (table 10), which were not significantly different. These RMSD values indicate that the hysteresis remained relatively the same, suggesting similar packing. However, some film deformation occurred progressing from the 1 st to the 5 th cycle, evident by film condensation towards smaller molecular areas at all pressures. The inter-cycle RMSD of 1DPPE:1PSM was 2.30 ± 0.64, over 1.8 units smaller than the value for 3DPPC:2DPPE. Therefore, while PSM reduced the ability of DPPE films to resist deformation, the impact was less so compared to that of DPPC.

121 102 Figure 26. Selected surface pressure-area isocycles of the binary lipid systems. (A) 3DPPC:2DPPE (B) 1DPPC:1PGC (C) 3DPPC:2PSM (D) 2DPPE:3PGC (E) 1DPPE:1PSM (F) 3PGC:2PSM.The first cycle is shown in blue; the fifth cycle is in red. Each film was compressed until 15 mn/m before collapse and expanded until zero surface pressure was reached. The lipid system is shown above each graph. n 3

122 103 The RMSD value between the 1 st and 5 th cycle of 3PGC:2PSM was almost identical to that of 1DPPE:1PSM, at 2.30 ± 0.93 (table 10). Like 1DPPE:1PSM, the RMSD between the 1 st and 5 th cycle of 3PGC:2PSM was primarily the result of differences between compression and not the expansion isotherms. During the 5 th compression the film was more condensed, taking up less area at lower surface pressures when compared to the 1 st compression. Although, there were packing similarities at pressures above 35 mn/m evidenced by overlap between compression and expansion (fig 2F, black arrows). The differences in the expansion part of the cycles were minor in comparison to the compression curves. Overlap between the 5 th and 1 st expansions occurred multiple times. The few instances when the expansion isotherms did not coincide, the differences were less than a 2 Å 2 /mol shift. For 3PGC:2PSM films, however, large differences occurred within cycles and not between them. 3PGC:2PSM had the highest first cycle RMSD of the binary systems at 5.19 ± 0.88 (table 10). Residual packing changes from compression remained through most of the expansion isotherm, resulting in the relatively high RMSD value. The higher similarity between the cycles than within them would suggest that the lateral changes from compression were relatively reversible. The larger intra-cycle RMSD and hysteresis indicate packing differences between compression and expansion. However, had these packing changes persisted or had their extent become greater, there would have been a larger difference between the cycles and a larger inter-cycle RMSD. Of the six binary systems, 1DPPC:1PGC exhibited greater resistance to deformation (fig. 26B). The expansion and compression isotherm of the 1 st cycle were very similar. There was a slight hysteresis at intermediate pressures, occurring between 10 and 40 mn/m (fig. 26B, black arrow). Similarities in the lateral organization between the 1 st compression and expansion translated to the relatively low 1 st cycle RMSD value of 3.71 ± 0.46 (table 10). To an extent,

123 104 these similarities were carried forward to the 5 th cycle of 1DPPC:1PGC which had an intra-cycle RMSD of 3.04 ± 0.62 (table 10). The intra-cycle RMSD values were not significantly different suggesting that the cohesion between lipids was relatively unaffected from cycling. Furthermore, both the 1 st cycle and 5 th cycle had very similar traces to one another, suggesting little lateral reorganization. The inter-cycle RMSD value of 1.77 ± 0.36 (table 10) reflected this similarity. The low RMSD value further indicates that 1DPPC:1PGC film integrity was not impacted by multiple compression and expansions as much as the other binary systems. On the other hand, 2DPPE:3PGC films exhibited even more overlap between the 1 st and 5 th cycle traces (fig. 26D). At pressures greater than 25 mn/m the compression and expansion isotherms from both cycles were approximately equal. However, hysteresis was observed at pressures below this value (fig. 26D, black arrow). The RMSD for the 1 st and 5 th cycles of 2DPPE:3PGC was 3.34 ± 0.12 and 2.82 ± 0.06 respectively, which were not significantly different. These values were the lowest relative to the other binary systems indicating less cohesion and reorganization within the 2DPPE:3PGC films. Furthermore, the inter-cycle RMSD was 1.48 ± 0.35 (table 10), which was also the lowest of the binary systems. 2DPPE:3PGC films were, therefore, the most resilient to deformation of all the binary mixtures.

124 105 Table 10. The RMSD values from the binary lipid systems. The 1 st Cycle RMSD column describes the 1 st compression and expansion. The 5 th Cycle RMSD column describes the 5 th compression and expansion. The Inter-Cycle RMSD column is the overall difference between the 1 st and 5 th cycles. n 3 Lipid System 1 st Cycle RMSD 5 th Cycle RMSD Inter-Cycle RMSD 3DPPC : 2DPPE 4.03 ± ± ± DPPC : 1PGC 3.71 ± ± ± DPPC : 2PSM 5.02 ± ± ± DPPE : 3PGC 3.34 ± ± ± DPPE : 1PSM 3.98 ± ± ± PGC : 2PSM 5.19 ± ± ± 0.93 Some trends observed in the single lipid mixtures were carried forward to the binary systems. For instance, there was an apparent decrease of the intra-cycle RMSD values between the 1 st and 5 th cycles. Additionally, films containing DPPC most likely resulted in tighter lipid packing following multiple compression and expansions. This was based on an entire 5 th cycle shift to smaller molecular areas when compared to the 1 st cycle. DPPC binary systems, therefore, appear to have underwent relatively larger lateral reorganization over multiple 'blinks'. On the other hand, PGC binary films appeared to resist deformation the best, continuing the trend observed with pure PGC films.

125 Tertiary and Quaternary Lipid Systems Generally, there was limited hysteresis in the more complex tertiary and quaternary mixtures (fig. 27). 3DPPC:2DPPE:2PSM was, however, an exception to this trend. There was a large hysteresis within both the 1 st and 5 th cycle over the entire range of molecular areas (fig. 27B) with RMSD values of 6.29 ± 0.21 and 5.72 ± 0.53 respectively (table 11). Although, unlike some of the individual and binary systems that had high RMSD values, the 5 th cycle of 3DPPC:2DPPE:2PSM was not entirely condensed to areas smaller than the 1 st cycle (fig. 27B, arrow). At large areas, the lipids in the 1 st cycle were more expanded than in the 5 th cycle. However, at surface pressures greater than approximately 15 mn/m, while the lipids from the 1 st cycle still took up a larger area than the 5 th, there was less of a packing difference evident by the partial overlap between the isocycle traces. This would suggest that the lateral organization was most impacted by cycling when the film was more fluid, which would be at lower surface pressures. The inter-cycle RMSD of 3DPPC:2DPPE:2PSM was 4.34 ± 0.43 (table 11). This RMSD was the highest of all the more complex systems. The relatively high inter-cycle RMSD and clear difference between thr isocycle traces suggests that 3DPPC:2DPPE:2PSM experienced the greatest deformation over multiple compression and expansions. The deformation resulted in tighter packing between lipids because the 5 th cycle had condensed in relation to the 1 st cycle.

126 107 Figure 27. Selected surface pressure-area isocycles of the tertiary and quaternary lipid systems. (A) 3DPPC:2DPPE:3PGC (B) 3DPPC:2DPPE:2PSM (C) 3DPPC:3PGC:2PSM (D) 2DPPE:3PGC:2PSM (E) 3DPPC:2DPPE:3PGC:2PSM.The first cycle is shown in blue and the fifth cycle in red. Each film was compressed until 15 mn/m before collapse and expanded until zero surface pressure was reached. The lipid system is labelled above each graph. n 3

127 108 Generally speaking, the remaining four mixtures exhibited similar lateral reorganization properties. For instance, there was slight deformation after the 1 st cycle compared to the 5 th cycle. Lipids during the 1 st compression were more expanded compared to the 5 th compression at larger molecular areas. When comparing the expansion isotherms, the packing differences were even less, exhibited by a very slight molecular area shift between the 1 st and 5 th cycles. Furthermore, the compression and expansion curves within the cycles generally shared the same shape. An exception to this trend was observed for 3DPPC:2DPPE:3PGC (fig. 27A). For both the 1 st and 5 th cycles, there was a hysteresis at approximately 25 mn/m due to an inflection point within the expansion isotherms. The RMSD of the 1 st cycle of 3DPPC:2DPPE:3PGC was the second highest at 5.26 ± 0.17 (table 11). Although there was a relatively large difference within the cycles, the inter-cycle RMSD was lower at 2.98 ± 0.26 (table 11). This was also the case for the remaining tertiary and quaternary systems that contained PGC. The compression and expansion isotherms between the cycles were more alike than the compression and expansion curves within the cycles (fig. 27A,C,D,E). This would mean that the 1 st compression isotherm was more similar to the 5 th compression isotherm than it was to the 1 st expansion isotherm. Additionally, the 1 st expansion was more similar to the 5 th expansion than it was to the 1 st compression. This assessment was confirmed by calculating the RMSD values. The inter-cycle RMSD was always lower than values from within the cycles. In the case of 2DPPE:3PGC:2PSM and the quaternary systems the difference was almost two RMSD units smaller (table 11). This indicates that the lipid packing during compression remained similar over multiple compressions. The same could be applied for the packing during expansion over multiple expansions. On the other hand, different curves for compression and expansion indicate film packing differences between compression and expansion, which was confirmed by higher

128 109 intra-cycle RMSDs. However, the overall film behaviour in the complex PGC mixtures did not change drastically from cycle to cycle, and therefore, these films were stable over multiple cycles and resisted deformation. This was most obvious in relation to the results from the 3DPPC:2DPPE:2PSM mixture. 3DPPC:2DPPE:2PSM had the relatively highest inter-cycle RMSD at 4.34 ± Adding PGC to this mixture, to get the quaternary system, resulted in an almost 3 unit drop in the RSMD to 1.65 ± 0.51 (table 11). In fact, the low inter-cycle RMSD of the quaternary mixture was second only to 2DPPE:3PGC (1.48 ± 0.35, table 10). However, there was no significant difference between these two values. On the other hand, the quaternary intercycle RMSD was significantly lower than the bottom 40% of the systems tested (p<0.05), suggesting that the biomimetic composition was effective in maintaining film integrity over multiple blinks. Table 11. The RMSD values from the tertiary and quaternary lipid systems. The 1 st Cycle RMSD column describes the 1 st compression and expansion. The 5 th Cycle RMSD column describes the 5 th compression and expansion. The Inter-Cycle RMSD column is the overall difference between the 1 st and 5 th cycles. n 3 Lipid System 1 st Cycle RMSD 5 th Cycle RMSD Inter-Cycle RMSD 3DPPC : 2DPPE : 3PGC 5.26 ± ± ± DPPC : 2DPPE : 3PSM 6.29 ± ± ± DPPC : 3PGC : 2PSM 3.59 ± ± ± DPPE : 3PGC : 2PSM 5.05 ± ± ± 0.50 Quaternary 3.69 ± ± ± 0.51

129 110 To determine the impact of lipid species on film deformation, table 12 lists the average RMSD for each of the four lipids studied in regards to the 1 st cycle, the 5 th cycle, and between cycles. The values were determined by averaging the RMSD for each system containing that specific lipid. For instance, all systems containing DPPC such as 3DPPC:2DPPE, 3DPPC:2DPPE:2PSM, etc. had the RMSD averaged for the 1 st cycle, the 5 th cycle and between the 1 st and 5 th cycles. From these results, DPPE had the largest intra-cycle RMSDs (1 st cycle was 4.75 ± 1.0 and 5 th cycle was 4.10 ± 0.92) while PGC had the lowest (1 st cycle was 4.12 ± 1.04 and 5 th cycle was 3.34 ± 0.92). The relatively large RMSD values indicate that DPPE films were the most cohesive after compression, and PGC the least cohesive. In other words, the lipid ordering from compression resulted in slower respreading during the expansion of DPPE films relative to other lipids examined. There was, however, no significant difference between an intracycle RMSD of one lipid species with another. This would indicate that while there indeed was cohesion evident in the systems examined based on the hysteresis, the actual differences between them were minor. Furthermore, while the observed hysteresis for each system was not the same based on the isocycle traces, the differences were averaged out when the systems were grouped according to the 4 different lipid classes used. That is to say, while individual differences between systems may be large, for instance the 1 st cycle RMSD difference between PGC (2.69 ± 0.46) and 3DPPC:2DPPE:2PSM (6.29 ± 0.21), there was no clear trend regarding which of the four lipid species continually had low hysteresis based on the insignificant RMSD differences. In terms of the inter-cycle RMSD, however, there was significant difference between systems containing DPPC and those containing PGC, p<0.05 (table 12). The inter-cycle RMSD relates to how different the isocycles were from each other, which was the result of lateral reorganization during multiple compression and expansions. The higher average inter-cycle

130 111 RMSD of DPPC containing systems, 3.33 ± 1.16 (table 12), would suggest that these films undergo greater packing changes from cycling. Looking at the isocycle traces, these differences generally arose from tighter packing in the 5 th cycle compared to this 1 st, evidenced by a shift to smaller molecular areas. Furthermore, the smaller inter-cycle RMSD with PGC containing films indicate greater similarities between the 1 st and 5 th isocycle traces suggesting less lateral reorganization between cycles. That is to say that film deformation was significantly more prominent with DPPC films than with PGC. These results suggest that PGC may have a greater impact in maintaining tear film integrity during blinking in comparison to DPPC. PSM and DPPE containing systems, on the other hand, had inter-cycle RMSD values of 3.09 ± 1.11 and 2.79 ± 1.10 (table 12) respectively. However, the difference between these values was not significant, suggesting a negligible impact between PSM and DPPE films on resisting deformation. Table 12. The average RMSD of each lipid studied taken from all systems that had that lipid present, excluding the quaternary system. The bolded values were significantly different from each other (p<0.05). n=7 Lipid System 1 st Cycle RMSD 5 th Cycle RMSD Inter-Cycle RMSD DPPC 4.40 ± ± ± 1.16 DPPE 4.75 ± ± ± 1.10 PGC 4.12 ± ± ± 0.52 PSM 4.64 ± ± ± 1.11

131 Isocycle BAM Images As described in the methods sections 2.2 and 2.7, BAM was used to visualize the lateral organization of the 15 different systems as they underwent five compression and expansion isocycles. Micron scale lipid domain changes between the cycles would be detected through BAM, providing further insight into the impact of blinking on the organization of tear film models based on polar lipids. BAM images were collected during the 1 st compression and expansion, as well as the 5 th compression and expansion. Additionally, images were taken at either similar molecular areas or similar surface pressures for more accurate comparisons. Before the results are addressed, it is important to describe what monolayer domains are. In terms of detection by BAM, these are the regions on the image that reflect light more intensely than the surrounding bulk lipid, due to more ordered hydrocarbon tails protruding farther into the air. Domains at the air-water interface arise when thermodynamic phase separation occurs due to competing short range interactions [114]. Line tensions created from van der Waal forces promote phase separation while energetically unfavourable oriented dipole-dipole forces disrupt domain formation [114]. The line tension, or hydrocarbon chain length mismatch, at domain boundaries represents an unfavourable energetic cost, leading to minimization of the boundary lengths [114, 115]. Thus, line tension favours domains with circular morphology. However, in the presence of lipids that have unfavourably oriented dipole-dipole moments, minimizing electrostatic repulsion results in elongated and branched shapes [114] Individual Lipid Systems Upon compression, DPPC monolayers formed domains at surface pressures of approximately 7 mn/m (fig. 28A). These structures had an asymmetrical multi-lobed

132 113 morphology that formed larger aggregates as compression continued. The domains themselves belong to the more ordered liquid-condensed (L C ) phase while the surrounding bulk lipid was in the more fluid liquid-expanded (L E ) phase. Previous results of DPPC domains had less branching in comparison to the results obtained here [116]. However, it has also been found that divergent domain shapes are obtained with different compression speeds and trough types [79]. Phase heterogeneities continued as the film was compressed through the natural tear film pressures (fig. 28E,I). During the 1 st expansion, the lobed domains at lower surface pressures remained, but they had become less numerous and more condensed (fig. 28B). By the 5 th compression, the lobed domains at 6mN/m had shrunk but appear to have a similar frequency to that in the 1 st expansion (fig. 28C). The observed hysteresis from the isocycle results indicate that DPPC experienced cohesion following compression. Furthermore, the smaller and more compact domains evident in the BAM images of DPPC isocycles reflect this tighter packing. By the 5 th expansion the domains at this surface pressure had become even more condensed compared to the 1 st expansion, and had additionally formed more continuous branched chains (fig. 28D). The smaller domains may be a result of tighter packing within the domains evident by the shift to smaller molecular areas in the 5 th isocycle trace of figure 25A. Finally, at surface pressures closer to the biologically relevant value of 30 mn/m, the DPPC films appeared more homogenous with cycling. More specifically, comparing the BAM images from the last row of figure 28, individual domains became less distinct between the 1 st and 5 th cycles, suggesting better packing between the lipids.

133 114 Figure 28. BAM images from the DPPC isocycles. The figure is split into columns containing images from the different isocycle regions, labelled at the top of the figure. Each image is 218 x 271 microns; the scale bar is 50 microns.

134 115 Unlike DPPC, DPPE monolayers did not exhibit any domain formation throughout the isocycle imaging (fig. 29). At initial take off surface pressures, a continuous film had formed with minor defects highlighted in the orange box (fig. 29A). Of the pure lipid systems, isocycles of DPPE had the largest intra-cycle hysteresis. The increased cohesion between lipids was also clearly evident in the BAM images. At zero surface pressure and molecular areas over 50 Å 2 /mol greater than the initial surface pressure take off, regions enriched in lipids were evident (fig. 29D). These images correlate well with the isocycle results that suggested lipid ordering from the earlier compressions were carried forward to later cycles. Furthermore, at the tear film physiological surface pressure, the BAM images illustrated similar lateral packing regardless if it was the 1 st compression or the 5 th (fig. 29I,K). While there were packing differences between compression and expansion of the same isocycle, evidenced by both the intra-cycle RMSD and the lipid aggregation at zero surface pressure, the DPPE packing differences between cycles at surface pressures near 30 mn/m were relatively minor. It is possible that had there been greater deformation from cycling, either the inter-cycle RMSD would be relatively higher or the BAM images would have reflected this.

135 116 Figure 29. BAM images from the DPPE isocycles. The figure is split into columns containing images from the different isocycle regions, labelled at the top of the figure. Each image is 218 x 271 microns; the scale bar is 50 microns. Inset is an enlarged image from the orange box.

136 117 BAM images of PGC monolayers illustrated phase homogeneity and lack of domain formation throughout both compression and expansion cycles (fig. 30). Like DPPE, at large areas and low surface pressures images showed lipid ordering. For instance, during the 1 st compression at 0.1 mn/m the bright reflection represents the lipid film and the dark region on the left would be buffer (fig. 30E). Furthermore, the lateral organization was more of a mesh like network at pressures below 20 mn/m (fig. 30B, orangesquare). Typical gas phase structures look like the large soap bubbles observed in the 1 st compression at Å 2 /mol (fig. 30A). The contrast in film organization between the 1 st compression (fig. 30A) and 1 st expansion (fig. 30B) indicates cohesion, as the film remained ordered even at very large molecular areas following compression. The much smaller hysteresis in the 5 th cycle of PGC compared to the 1 st cycle (fig. 25C) suggests similar packing between the 5 th compression and expansion. Similar lateral organization between the 5 th compression and expansion was further confirmed through BAM (fig. 30C,D). The typical gas phase structures from the 1 st compression were absent in subsequent cycles, replaced instead by the more ordered lipid packing organization. Finally, PGC had the lowest inter-cycle RMSD indicating similar packing throughout the five isocycles (table 9). Images I through L in figure 30 further suggest this, as very little lateral reorganization was observed. Finally, at pressures greater than 20 mn/m, near the natural tear film surface pressure, PGC films were homogenous with no obvious defects.

137 118 Figure 30. BAM images from the PGC isocycles. The figure is split into columns containing images from the different isocycle regions, labelled at the top of the figure. Each image is 218 x 271 microns; the scale bar is 50 microns. Inset is an enlarged image from the orange box.

138 119 For the most part, BAM revealed no large microscopic changes to the lateral organization of PSM films over multiple cycles (fig. 31). At near zero surface pressures, the film appeared relatively homogenous with small circular defects highlighted by the orange box (fig. 31A). At surface pressures found near the inflection point in the PSM isotherm (~15 mn/m), small circular domains began to form. These structures were approximately 1 µm in diameter and formed regardless of compression or expansion at the isotherm inflection point pressures (fig. 31E). At the natural tear film surface pressures, further lipid ordering occurred until a homogenous film resulted (fig. 31I,K). As mentioned earlier, compression and expansion shared similar topographical features. The one exception to this occurred during the initial expansion of the 5 th cycle. At 50.5 Å 2 /mol larger aggregates remained following compression, found within the green box (fig. 31L). The 5 th PSM isocycle had shifted to smaller molecular areas suggesting tighter lipid packing from cycling. The lipid aggregates observed in the 5 th expansion may be from the lateral reorganization during cycling resulting from tighter lipid packing. Both the higher inter-cycle RMSD and the appearance of different domains in later cycles suggest that PSM films were relatively less resistant to deformation following multiple compression and expansions.

139 120 Figure 31. BAM images from the PSM isocycles. The figure is split into columns containing images from the different isocycle regions, labelled at the top of the figure. Each image is 218 x 271 microns; the scale bar is 50 microns. Insets represent enlarged images from the boxes.

140 Binary Lipid Systems Two distinct lipid populations were observed within 3DPPC:2DPPE films at surface pressures less than 1 mn/m (fig. 32A). The lipids demixed into separate phases as more ordered lipids will be primarily found in the domains, while the more fluid lipid population will exist in the surrounding bulk fraction. At this initial surface pressure, domain formation was primarily driven by line tension according to the circular domain morphology. From an image alone, it is not clear if the domains contain exclusively DPPC, DPPE, or a mixture. Based on the compression modulus from the previous chapter, it is more likely that the domains mostly contain the more rigid DPPE molecules at this pressure. However, at pressures of approximately 8 mn/m the domain morphology changed as distinct lobes began forming (fig. 32D). Individual DPPC monolayers exhibited phase separation at surface pressures slightly below those observed in figure 32, unlike DPPE monolayers which did not form domains. However, the DPPC domain shapes were more circular (fig. 28) with no small protrusions such as those in 3DPPC:2DPPE. Fluorescence microscopy of dimyristoyl-pe (14:0,14:0-PE) monolayers exhibited L C morphologies similar to the domains in figure 32E [117]. If the domains from the 1 st compression of 3DPPC:2DPPE were most likely composed of DPPE lipids, then it would suggest that DPPC influenced the packing between DPPE molecules to cause the domain formations that were otherwise not observed in the pure DPPE system. Due to the non-circular morphology of the domains in figure 32E, it was most likely that the lipid dipoles were reoriented following compression. This caused electrostatic repulsion between DPPE molecules within the L C phase of the image and therefore small domain protrusions to form. Domain protrusions were also evident between 6 and 9 mn/m in the 5 th cycle (fig. 32G,H), however, the domains themselves were quite different from those in the 1 st cycle. The individual domains

141 122 from the 1 st cycle had been replaced instead by more continuous network lipid structures in the 5 th. The observed domain fusion was most likely from large lateral reorganizations over continued compression and expansion leading to changes in packing between the DPPE and DPPC lipids. Within both cycles, the domains started to coalesce as surface pressures increased past 20 mn/m (fig. 32I,K). Interestingly, near the natural tear film surface pressures, the 1 st compression appeared to form a more optimally packed monolayer compared to the 5 th compression, evidenced by the relatively continuous film in figure 32I. From the 3DPPC:2DPPE isocycle traces, the 5 th cycle had shifted to smaller molecular areas by a larger extent than some of the other binary systems (fig. 26A). The packing differences observed between the 1 st and 5 th compression images may therefore be from lipids within the L C domains packing tighter together, preventing a more homogeneous film from forming during the 5 th compression. The type of packing will most likely have physiological implications such as tear protein insertion [51], which may suggest that tighter packing from blinking may inhibit protein insertion.

142 123 Figure 32. BAM images from the 3DPPC:2DPPE isocycles. The figure is split into columns containing images from the different isocycle regions, labelled at the top of the figure. Each image is 218 x 271 microns; the scale bar is 50 microns.

143 124 Domains were also observed near the initial take off pressures of the 1DPPC:1PGC system (fig. 33A). At these pressures, the two distinct lipid populations represent demixing between DPPC and PGC molecules. Like the images from 3DPPC:2DPPE, it is most likely that these domains were primarily comprised of the more rigid PGC lipids. During the 1 st cycle, branched structures formed via lobe elongation of the initial domains (fig. 33E,F). Branched domains such as the ones observed here are characteristic of ceramide monolayers [118]. This suggests that DPPC existed mostly in the fluid phase while the domain morphology was primarily the result of PGC lipids, leading to the snowflake like domains. Similar to 3DPPC:2DPPE, binary mixtures of DPPC appear to be promoting initial demixing and dipole reorientation leading to non-circular domain formation. For both of these systems, the domains were most likely not comprised of DPPC but either DPPE or PGC because the morphologies had similarities to domains of other PE or ceramide monolayers, respectively. However, over multiple cycles the domain morphology became smaller and rounder, along with an increase in frequency (fig. 33G,H). The more circular domain shape within the 5 th cycle suggests lateral reorganization resulting in less electrostatic repulsion. The lateral reorganization may be from the different lipid species mixing better as the result of continuous compression and expansions of the film. Similar to the previous film, the domains had condensed in the 5 th cycle relative to the 1 st cycle at the physiologically relevant pressures (fig. 33I,L).

144 125 Figure 33. BAM images from the 1DPPC:1PGC isocycles. The figure is split into columns containing images from the different isocycle regions, labelled at the top of the figure. Each image is 218 x 271 microns; the scale bar is 50 microns.

145 126 Unlike the previous two binary systems, 3DPPC:2PSM exhibited limited phase separation at initial surface pressures during both the 1 st and 5 th compressions (fig. 34A,C), suggesting more ideal mixing between these lipids. This was not surprising given that DPPC and PSM had similar rigidities at these pressures, based on the compression modulus results from chapter three, section There were slight film defects evident by the small holes at initial take off pressures highlighted by the orange box (fig. 34A). Once these holes had disappeared, small domains aligned in a string formation were observed at approximately 15.0 mn/m (fig. 34E). These domains remained throughout expansion, arranging into linear formations that were most evident at approximately 11 mn/m (fig. 34B,D). As cycling continued, the spheroid domains developed into more jagged, star shapes (fig. 34G) with the string like orientation disappearing until the 5 th expansion (fig. 34D). The linear domain arrangements suggest an extra level of organization above domain morphology that was most likely from long range interactions between the domains. The long range interactions would be from weak dipole-dipole repulsions as each domain effectively acts as one large dipole due to the lipid organization within it [115]. It is possible that the domain dipoles were aligning to form this string like network to reduce dipole-dipole repulsion. Furthermore, after multiple compression and expansions there appears to be some lateral reorganizations causing the string orientation to disappear (fig. 34G,H). The isocycle traces from figure 26C showed further evidence of packing changes between the 1 st and 5 th cycles suggesting that cycling of 3DPPC:2PSM did cause some film deformation. Finally, increasing the surface pressure past 20 mn/m near to the natural tear film pressures, led to domain coalescence (fig. 34K). Compared to the previous binary systems at this surface pressure, 3DPPC:2PSM appeared to form a more evenly spread L C film due to the lack of apparent distinct domain boundaries during the compression isotherms.

146 127 Figure 34. BAM images from the 3DPPC:2PSM isocycles. The figure is split into columns containing images from the different isocycle regions, labelled at the top of the figure. Each image is 218 x 271 microns; the scale bar is 50 microns. Inset is an enlarged image from the orange box.

147 128 2DPPE:3PGC formed relatively more homogenous monolayers at all pressures and areas regardless of how many cycles had been completed (fig. 35). The apparent single lipid population suggests more ideal mixing between DPPE and PGC molecules. However, similar to both pure DPPE and PGC films, the defects or spots found in figure 35A,C correspond to lipid free areas due to a non-fully formed film. These spots appeared larger in diameter during the 5 th cycle most likely due to increased cohesion between lipid molecules. (fig. 35C). The film defects disappeared when compressing the film to pressures greater than 15 mn/m (fig. 35I,K). Furthermore, while the previous two binary mixtures containing either DPPE or PGC exhibited domains, the effect was abolished in 2DPPE:3PGC. This was similar to pure PGC and DPPE films. In the instances where PE and ceramide domains did form, DPPE or PGC were mixed with DPPC. This would further suggest, as previously noted, that DPPC promotes lateral packing changes that make it possible for DPPE and PGC to organize into domains. The driving force behind this may be the result of DPPC's relative fluidity compared to PGC or DPPE, causing these lipids to aggregate and remain together during compression. Due to their comparable rigidities, binary mixtures of DPPE and PGC did not undergo phase separation and therefore, did not form any domains.

148 129 Figure 35. BAM images from the 2DPPE:3PGC isocycles. The figure is split into columns containing images from the different isocycle regions, labelled at the top of the figure. Each image is 218 x 271 microns; the scale bar is 50 microns. Insets represent enlarged images from the boxes.

149 130 Similar to mixing DPPE with DPPC, the 1DPPE:1PSM binary system exhibited lipid heterogeneities at initial (<1 mn/m) surface pressures (fig. 36A,C). Based on the compression modulus results from chapter three, section PSM was far less rigid than DPPE at surface pressures below 15 mn/m. As previously mentioned, domain formation occurs due to phase separation between a rigid population and a fluid population, whereby more rigid molecules are located within the domains. Therefore, the domains must be primarily of DPPE origin. Indeed, the domains in figure 36E resemble the DPPE domains from 3DPPC:2DPPE in figure 32E. As surface pressures exceeded 23 mn/m, the star like domains began to coalesce (fig. 36E). During the 1 st expansion, the film separated into circular domains (fig. 36F). The circular domains remained during subsequent cycling with no apparent change in size or frequency, evident in the 5 th compression and expansion columns of figure 36. The circular domain morphology suggests permanent reorganization following the 1 st cycle such that either dipole interactions were minimized or line tension increased relative to the initial lipid packing. Furthermore, film defects appeared during the initial pressure increase of the 5 th compression, observable within the orange box and highlighted by the red arrow (fig. 36C). These holes may be an indication of film breakdown and instability from cycling. The isocycle results of 1DPPE:1PSM indicate that the film was more condensed during the 5 th compression relative to the 1 st (fig. 26E). Therefore, subsequent compression and expansion may have led to increased cohesion between molecules resulting in the observed defects and possible film instability within the 5 th cycle.

150 131 Figure 36. BAM images from the 1DPPE:1PSM isocycles. The figure is split into columns containing images from the different isocycle regions. Frame C's insert is a magnification of the orange box. Each image is 218 x 271 microns; the scale bar is 50 microns. Inset is an enlarged image from the orange box.

151 132 Similar to the 1DPPE:1PSM mixture, 3PGC:2PSM films initially existed in two separate lipid populations (fig. 37A). The domains, however, with 3PGC:2PSM were more spherical, smaller, and had a higher frequency. Additionally, domain coalescence was initiated fairly rapidly, at pressures of approximately 7 mn/m (fig. 37E). Expanding the film to zero surface pressure within the first cycle led to phase separation but not clear domain separation (fig. 37B). More specifically, the boundaries between individual domains were not obvious, instead forming a continuous ordered phase suggesting increased lipid cohesion. This ordered lipid network continued into the 5 th compression (fig. 37G). However, the most striking difference between the 1 st and 5 th cycles was the presence of two lipid phases among large dark areas observed at zero surface pressure and molecular areas greater 65 Å 2 /mol (fig. 37C, green box). These dark spots correspond to the subphase, or areas that lack lipids, indicated by the absence of a reflection. The appearance of the subphase within the 5 th cycle indicates that 3PGC:2PSM films did not expand completely following expansion of the available trough area relative to the 1 st cycle. The film from the 1 st cycle lacked any dark spots implying that it covered the subphase entirely at these molecular areas. The cohesion between molecules, therefore, increased in the 5 th cycle compared to the 1 st cycle. The appearance of these defects within the 5 th cycle again, suggests film deformation and instability. From these results it appears that 5 cycles or 'blinks' appear to have the largest impact on the stability of PSM binary films. However, the 5 th cycle defects did not appear in the 3DPPC:2PSM binary mixture implying that the instability was more pronounced when the accompanying lipid was more rigid, such as with DPPE or PGC.

152 133 Figure 37. BAM images from the 3PGC:2PSM isocycles. The figure is split into columns containing images from the different isocycle regions, labelled at the top of the figure. Each image is 218 x 271 microns; the scale bar is 50 microns. Insets represent enlarged images from the boxes.

153 Tertiary and Quaternary Lipid Systems Similar to most of the binary mixtures, phase separation was evident within the tertiary and quaternary systems at or before initial surface pressure increases. For the 3DPPC:2DPPE:3PGC system, large asymmetrical domains were initially observed at pressures between 0 and 1 mn/m and areas of approximately 85 Å 2 /mol (fig. 38A). Domain aggregation occurred at approximately 5.0 mn/m, forming long continuous stretches of ordered lipids (fig. 38E). Furthermore, at this surface pressure, small protrusions formed on the continuous lipid domains indicated by the red squares in figure 38. This phenomenon was previously described in regards to the 3DPPC:2DPPE system, suggesting that the domain changes at pressures near 5 mn/m were the result of DPPE ordering. Initial expansion of the 3DPPC:2DPPE:3PGC film led to domain separation and a more uniform circular morphology (fig. 38F). After subsequent compression and expansion cycles, domain separation was abolished, existing more akin to an ordered network even when at zero surface pressures (fig. 38C,D). However, lobe formation at 3 mn/m continued, as noted during the 1 st cycle (fig. 38G). Like the PSM binary systems, circular defects appeared again in the 5 th cycle suggesting film instability over subsequent compression and expansion (fig. 38C, orange box). Although the most striking observation of the 3DPPC:2DPPE:3PGC BAM images were the bright dots that appeared as the film neared coalescence at 27 mn/m (fig. 38I, green boxes). These structures are generally related to the onset of monolayer collapse. However, from the isotherm results, 3DPPC:2DPPE:3PGC collapsed at 61.5 ± 0.6 mn/m (section 3.1), more than 30 mn/m greater than the onset of the bright spots observed with BAM. The bright spots could, therefore, be the result of multilayer formation. This relates well with the idea of buffered collapse first mentioned in chapter 3, section 3.2, based on the compression modulus results of the tertiary and quaternary systems.

154 135 Figure 39C includes the compression modulus versus surface pressure graph of the 3DPPC:2DPPE:3PGC system to provide context to the reader. The reversible formation of multilayers may act as "shock absorbers" allowing for continued film compression to smaller areas that may have otherwise resulted in film collapse. Using the relative intensity of reflected light, 3D images can be constructed from the BAM images (fig. 38I). The brighter the intensity the more the film protrudes out of the subphase. Figure 39A illustrates that the bright spots observed within the 1 st compression of 3DPPC:2DPPE:3PGC were relatively taller than surrounding bulk lipid (fig. 39B). The relative difference between the panels was based on the 'Image scan graylevel' which is a measure of the observed light intensity. The lipid within the bright spots extended into the air over two times greater than the film regions lacking these domains based on the 'Image scan graylevel' between figure 39A and 39B. This behaviour was similar to that observed with another tear film model system containing a mixture of DPPC, cholesterol esters, and tri-acy-glycerols in which protrusions were observed in the presence of high concentrations of ectoine [112]. However, the results presented in this thesis suggest that multilayer formation may be possible even in the absence of the non-polar tear film lipids, evident by the bright domains observed from the 3DPPC:2DPPE:3PGC system. Finally, the substantially brighter area highlighted by the black arrow in figure 38I corresponds to a dust particle.

155 136 Figure 38. BAM images from the 3DPPC:2DPPE:3PGC isocycles. The figure is split into columns containing images from the different isocycle regions, labelled at the top of the figure. Each image is 218 x 271 microns; the scale bar is 50 microns. Insets represent enlarged images from the boxes.

156 137 Figure 39. 3D images of a bright spot domain (A) and of the surrounding L C phase (B) from 3DPPC:2DPPE:3PGC films at 27.1 mn/m during the 1 st compression. (C) Representative compression modulus curve from the 3DPPC:2DPPE:3PGC system. The blue line represents the surface pressure (27 mn/m) where the potential multilayer formation occurs. Asymmetrically branched domains that were larger and of heterogeneous size formed at surface pressures below 5 mn/m during the 1 st compression isotherm of the 3DPPC:2DPPE:2PSM film (fig. 40A). Continued compression led to a domain size increase until partial coalescence as pressures increased past 30 mn/m (fig. 40I). The observed domain shapes

157 138 had more branching and were progressively narrower, similar to the morphology of either PE or SM domains [117, 119]. Unlike the previously examined 3DPPC:2DPPE:3PGC system that had a network like organization by the 5 th cycle, no domain fusion occurred over subsequent compressions for 3DPPC:2DPPE:2PSM. The lack of PGC, the only completely uncharged lipid studied, may be the reason why 3DPPC:2DPPE:2PSM maintains domain separation throughout all cycles (fig. 40E-H). Within the 3DPPC:2DPPE:3PGC system, PGC molecules may be moving in between the zwitterionic PE and PC lipids, reducing the repulsive electrostatic forces and allowing the domains to come together into long network like patches. In the case of 3DPP:2DPPE:2PSM, some lateral reorganization occurred over the cycles, evidenced by a decrease in the length of the domain branches and an increase in the domain radius (fig. 40G,H). However, the domain repulsion was not reduced sufficiently that would allow for domain fusion as was seen in 3DPPC:2DPPE:3PGC films at the same surface pressures. Furthermore, the bright spots that accompanied the 1 st compression of the 3DPPC:2DPPE:3PGC system were not present in the 3DPPC:2DPPE:2PSM film (fig. 40I). However, during the 5 th compression, approximately four or five bright spots were observed, but did not have the same intensity or frequency as those from the previous system (fig. 40K, green box). The 3D analysis of these brighter spots suggests very minor protrusion into the air relative to the surrounding lipid (fig. 41). Furthermore, the hysteresis was largest for 3DPPC:2DPPE:2PSM isocycles (fig. 27B) suggesting that the presence of these domains may be more related to the onset of collapse.

158 139 Figure 40. BAM images from the 3DPPC:2DPPE:2PSM isocycles. The figure is split into columns containing images from the different isocycle regions, labelled at the top of the figure. Each image is 218 x 271 microns; the scale bar is 50 microns. Inset represents enlarged image from the green box.

159 140 Figure 41. 3D analysis of the bright circular domain from the 5 th compression of the 3DPPC:2DPPE:2PSM film at 32.2 mn/m. 3DPPC:3PGC:2PSM also formed branched domains at pressures below 5 mn/m (fig. 42A), although they were smaller in size compared to the structures observed with the previous 3DPPC:2DPPE:2PSM films. This further suggests the condensing effect of PGC in mixtures first evident in the isocycles from section 4.1. Overall, 3DPPC:3PGC:2PSM had slight lateral reorganizations between the 1 st and 5 th cycles. For instance, the frequency, size, and shape of domains remained fairly constant, with slightly increased branching and narrowing during the 5 th compression and expansion. The domain similarities between compression and expansion were consistent with the RMSD results from table 11, which showed that 3DPPC:3PGC:2SM had the relatively lowest intra-cycle RMSD values. Furthermore, this domain morphology was consistent with SM domains observed in the previous systems and the literature [119]. Unlike the previous PGC containing tertiary system, 3DPPC:2DPPE:3PGC, domains from 3DPPC:3PGC:2PSM remained un-fused over multiple cycling. The domains did, however, begin to coalesce when

160 141 surface pressures increased past 30 mn/m (fig. 42I,K) but separated once pressures dropped below 10 mn/m (fig. 42J,L). This contrasts with the previous hypothesis that PGC caused domains to fuse into long continuous patches following film cycling. This discrepancy may arise for two reasons. The first reason is that although PGC was present in both films, the dipoles from the 3DPPC:3PGC:2PSM domains themselves may have been stronger than in 3DPPC:2DPPE:3PGC, causing domain separation due to long-range dipole-dipole repulsion. The second reason may be due to the differences with PSM lipids versus DPPE, as the composition difference between the two tertiary systems was a swap between DPPE and PSM. At surface pressures below 20 mn/m, DPPC had similar rigidity to PSM, while DPPE was more like PGC, based on the compression modulus data for section In regards to 3DPPC:3PGC:2PSM, DPPC-PSM mixing may therefore, be excluding PGC due to DPPC and PSM having more similar rigidities at these pressures. If PGC was excluded, then the mechanism proposed earlier by which PGC inserts and reduces dipole-dipole repulsions resulting in domain aggregation, was restricted. Furthermore, the bright spots associated with multilayer formation did not occur during compression of the 3DPPC:3PGC:2PSM films.

161 142 Figure 42. BAM images from the 3DPPC:3PGC:2PSM isocycles. The figure is split into columns containing images from the different isocycle regions, labelled at the top of the figure. Each image is 218 x 271 microns; the scale bar is 50 microns.

162 143 Replacing DPPC with DPPE to give rise to the 2DPPE:3PGC:2PSM system resulted in a heterogeneous film at initial surface pressure increases (fig. 43A). Two distinct lipid populations were observed until the film was compressed past the natural tear film surface pressures in both the 1 st and 5 th compression (fig. 43I,K). From the compression modulus results (section 3.3.1) the brighter regions on the BAM image would be mostly comprised of DPPE and PGC molecules as they were more rigid than PSM at these surface pressures. As well, SM domain morphology generally consists of snowflake like shapes [119] relatively distinct from the domains observed in figure 43. Furthermore, the lateral organization of 2DPPE:3PGC:2PSM films remained relatively unchanged over multiple cycles, with a few exceptions. First, there was a condensation of the rigid lipid phase at larger molecular areas suggested by the increased area of the more fluid phase seen as the grey area highlighted by the orange arrow (fig. 43C,D). This may be the result of increased cohesion between the rigid lipid molecules due to lipid reordering following compression and expansion cycles. Secondly, the increased cohesion between lipid molecules was most likely the cause of the small holes observed within the 5 th expansion (fig. 43D). These defects indicate that the film did not entirely cover the subphase to the same extent it did during the first cycles, suggesting decreased film stability. Thirdly, the bright spots indicating multilayer formation were observed only during the 5 th compression, which were more numerous than those observed in 3DPPC:2DPPE:2PSM films (fig. 43K). The 3D analysis of these bright domains suggests small protrusions similar to those from 3DPPC:2DPPE:2PM (fig. 44). The 3D images from both 2DPPE:3PGC:2PSM and 3DPPC:2DPPE:2PSM exhibited domains much shorter than those obtained from the 3DPPC:2DPPE:3PGC film. Furthermore, because of the small defects seen in figure 43D, the multilayer formation evident during the 5 th compression of 2DPPE:3PGC:2PSM was most likely from film instability. These small holes appeared during

163 144 the expansion following the observation of multilayer formation. This further suggests a possible five 'blink' limit to film stability, as the defects appeared in the other tertiary images following the 5 th compression.

164 145 Figure 43. BAM images from the 2DPPE:3PGC:2PSM isocycles. The figure is split into columns containing images from the different isocycle regions, labelled at the top of the figure. Each image is 218 x 271 microns; the scale bar is 50 microns. Inset represents enlarged image from the green box

165 146 Figure 44. 3D analysis of the circular bright domain from the 5 th compression of the 2DPPE:3PGC:2PSM film at 27.5 mn/m. Initial compression of the quaternary system resulted in phase separation at surface pressures below 1 mn/m (fig. 45). During the first compression, however, two domain morphologies appeared. The first a smaller more circular shape, highlighted by the black arrow, and a second larger and lobed structure, indicated by the red arrow (fig. 45A). This was not unreasonable considering the diversity of lipids present. Further compression resulted in more homogenous domain morphology suggesting greater lipid mixing (fig. 45E). Furthermore, this trend continued during the 1 st expansion (fig. 45F) suggesting that mixing between lipid species remained. The domains from the 1 st cycle resembled more closely to the morphology from the PE and GC binary systems but were larger in size with less branched and shorter lobes. After multiple cycles, there was slight domain aggregation observed during the 5 th compression at zero surface pressure (fig. 45C). The extent of aggregation, however, was limited in that no long continuous patches were evident. Lipid packing changes also led to larger domain radii with

166 147 increased domain branching (fig. 45G, orange arrow). SM films generally exhibit longer and more branched domains [119] implying that lateral reorganization may have increased by the presence of PSM within the domains. Finally, the bright spots observed at pressures greater than 25 mn/m were also seen within the quaternary system during both cycles (fig. 45I,K, green boxes). Like 2DPPE:3PGC:2PSM films, the bright spots were more numerous in the 5 th cycle than the 1 st. The relative sizes of the small circular shapes were similar between the 1 st and 5 th compression of the quaternary system (fig. 46). These bright domains were evident within the 1 st compression in only the quaternary and 3DPPC:2DPPE:3PGC films suggesting that their formation may be dependent on the presence of DPPE, as they were also completely absent in 3DPPC:3PGC:2PSM films. Furthermore, the defects observed in some of the previous tertiary systems during the 5 th expansion were absent in the quaternary system (fig. 45D). This would imply that the quaternary system may be relatively resistant to the limited life of these domains over continued compression and expansion.

167 148 Figure 45. BAM images from the 3DPPC:2DPPE:3PGC:2PSM isocycles. The figure is split into columns containing images from the different isocycle regions, labelled at the top of the figure. Each image is 218 x 271 microns; the scale bar is 50 microns. Insets represent enlarged images from the boxes.

168 149 Figure 46. 3D analysis of the circular bright domain from the 3DPPC:2DPPE:3PGC:2PSM (quaternary) film during the 1 st compression at 28.2 mn/m (A) and the 5 th compression at 31.9 mn/m (B). 4.3 Compression Isocycle Summary Comparing the isocycle RMSD results to the isocycle BAM images suggests that the largest lateral changes between the 1 st and 5 th cycles occurred with films containing either DPPC or PSM. DPPE and PGC containing films were more homogenous, with fewer morphological differences between the cycles. For instance, BAM images from the binary system of 2DPPE:3PGC showed almost no difference between the 1 st and 5 th cycles (fig. 35). This film also had the lowest inter-cycle RMSD of all 15 systems suggesting the similar lipid packing between the 1 st and 5 th cycles. Additionally, DPPC monolayers had the highest statistically significant inter-cycle RMSD values, while PGC had the lowest. One of the highest inter-cycle RMSD values was from 3DPPC:2DPPE (table 10). The BAM images of this system (fig. 32) exhibited large lateral reorganizations that resulted in domains completely different between the cycles.

169 150 This would suggest that PGC may have the largest positive impact on tear films stability over multiple blinks, while DPPC may destabilize it. More generally speaking, film instability was also apparent within the 5 th cycle of some of the binary and tertiary systems, evident by small holes exposing the subphase. This would imply a limited lifetime for the biomimetic films in which they break down after approximately 5 'blinks', consistent with the natural tear film disruption [62]. The BAM images also paralleled the isocycle observations in terms of the intra-cycle results in which comparisons were made between compression and expansion of the same cycle. For 13 of the 15 systems, the intra-cycle RMSD values were not significantly different between the 1 st and 5 th cycle. This would suggest that packing changes between compression and expansion were similar in both the 1 st and 5 th cycles. The BAM images generally confirmed this as domain morphology was more similar within cycles and that domain shape changes were most apparent when comparing different cycles. Using 3DPPC:2DPPE as an example, the domain shapes from this system were generally round with similar frequencies in either the 1 st compression or expansion (fig. 32). In contrast, the domain fusion within the 5 th cycle led to long patches of L C ordered lipids. While this organization was dissimilar from the 1 st cycle, there was no apparent difference between the 5 th compression and expansion images. This not only suggests that both the isocycle RMSD and BAM analyses complement one another but also that it's not a single blink impacting film integrity but most likely a series of blinks. Finally, multilayer formation was observed in all of the tertiary and quaternary systems, excluding 3DPPC:2DPPE:3PGC, evident by small bright domains at higher surface pressures (~30 mn/m) but still far below actual collapse (~60 mn/m). The potential collapse buffering discussed in chapter three in regards to the tertiary and quaternary mixtures may function

170 151 through this reversible multilayer formation revealed from the BAM images. Furthermore, the compression modulus results from section of 3DPPC:2DPPE:2PGC suggested that this system had the most gradual collapse of the tertiary and quaternary systems. This tertiary system also exhibited the greatest degree of multilayer formation during the 1 st compression evidenced by the largest number of the bright circular domains that based on the 3D analysis also protruded into the air the most. Combining the compression modulus and the BAM results further implies multilayer formation as a possible mechanism by which the natural tear film inhibits collapse.

171 152 Chapter Five: Lysozyme Injection Studies Recent discoveries have suggested that tear proteins play a critical role regarding tear film organization and function (reviewed in [25]). Monolayer injection studies offer a straightforward method for determining the extent of protein interaction with tear film lipids whereby the protein of interest is examined in terms of its insertion to the films upon injection into the subphase [120]. Initial studies have focused on tear lipocalin and lysozyme insertion to meibomian extract monolayers [50, 51, 53]. These important works confirm both the surface activity and film insertion of the selected tear proteins but do not provide a more detailed analysis to identify preferential or even specific interactions of lipids and proteins. Furthermore, initial studies with tear lipocalin and lysozyme in the presence of specific lipid types [52, 82], do not match the scope of the experiments investigated here. Here we expand on the prior lysozyme experiments by investigating its interactions with the remaining known polar lipids from tear film. Lysozyme was chosen over lipocalin because it was more accessible which was necessary for the amount of protein needed to complete all of the injection experiments. The surface activity of lysozyme was confirmed through protein injection into an aqueous solution within a Langmuir trough in the absence of lipids (fig. 47). The average surface pressure increase was 9.0 ± 0.4 mn/m. Our results indicated that lysozyme was indeed surface active but values were lower than reported previously in the literature (20 32 mn/m [24, 38]). It has to be emphasized that both of these studies used far higher concentrations of lysozyme (3.2 mg/ml vs 0.05 mg/ml) and far greater time scales (hours versus minutes). It is likely that higher levels of surface activity would have been observed at longer time points as the surface pressure continued to trend upwards at the trial end (fig. 47). However, as it was discussed previously in chapter one

172 153 that the tear film is constantly recycled and reformed, our studies focused on time lines that were more comparable with the in vivo conditions. Figure 47. Surface activity of 50 µg/ml of lysozyme determined by an increase in surface pressure following protein injection into a 1x PBS buffer subphase in the absence of a lipid film and measured by a surface pressure sensor. 5.1 Individual Lipid Systems Protein interactions were studied at three different surface pressures of pre-spread lipid films in order to survey a broad range of tear film states. The fully formed tear film on the open eye has a surface pressure range between mn/m [17]. Therefore, lysozyme injections at 10 mn/m would correspond to the initial stages of film formation, 20 mn/m to more

173 154 intermediate stages, and finally, 30 mn/m was used to represent the upper pressure range of a fully formed tear film. The insertion of lysozyme to lipid monolayers was first examined in the presence of a single lipid species (fig. 48). Significant lysozyme insertion occurred at a surface pressure of 10 mn/m for all of the single lipid films (p>0.05) in a decreasing magnitude of PGC > DPPE > PSM > DPPC. Lysozyme insertion to PGC monolayers was significantly higher (p<0.05) than the remaining individual lipid systems at 10 mn/m, equal to 6.3 ± 0.4 mn/m (fig. 48C). Furthermore, PGC additionally saw a significant increase (p<0.05) of 2.1 ± 0.6 mn/m at 20 mn/m, while all other single lipids showed minor insignificant increases (fig. 48). DPPC monolayers, on the other hand, had the lowest surface pressure increase at 10 mn/m, equal to 2.6 ± 0.5 mn/m (fig. 48A). At 30 mn/m, DPPC was the only single lipid to have a significant decrease (p<0.05) in the surface pressure following lysozyme injection.

174 155 Figure 48. Single lipid system lysozyme injections. The x-axis represents the different initial surface pressures prior to lysozyme injection. For each initial surface pressure, the final lysozyme concentration was 0.05 mg/ml. The y-axis is the change in the initial surface pressure following lysozyme injection. (A) DPPC (B) DPPE (C) PGC (D) PSM. Significance was determined in relation to the control, * p<0.05, ** p<0.01. The error bars represent the standard deviation. n 3. Previous literature has shown that structural rearrangements exposing the inner hydrophobic regions of lysozyme results in its irreversible adsorption to hydrophobic surfaces [121]. The primary driving force for lysozyme insertion into lipid monolayers would then be the result of hydrophobic interactions between the lipid hydrocarbon tails and the hydrophobic fragments within lysozyme. It would stand that lipid species that allow the greatest access to their hydrophobic tails, or the fewest barriers, would also have the greatest insertion. For

175 156 instance, the isoelectric point (pi) of lysozyme is 11, giving it a net positive charge at the subphase ph of 7.4. While all the lipids in this study are neutral, they all carry a local positive and negative charge within their head groups, with the exception of PGC. The positive charge is found at the terminal end of the head group, on the amine, while the negative charge is found on the phosphate connecting the head group to the glycerol/sphingosine backbone. Prior to lysozyme denaturation and insertion to the film, adsorption needs to occur. One possible energy barrier to initial adsorption could be overcoming the electrostatic repulsion between the positive lipid head group charge the surface positive charge on lysozyme. PGC, the lipid species carrying no formal charges, had the greatest significant pressure increase following injection compared to the other lipids studied. With no localized charges, PGC may be providing the greatest access for the hydrophobic interactions required for lysozyme insertion. The next possible barrier to lysozyme insertion is lipid head group size and orientation. In regards to DPPE, DPPC, and PSM films, the head group orients almost parallel to interface plane due to the positively charged amine interacting closely with the negatively charged phosphate linker of adjacent lipid molecules [106, 122]. DPPE films had the second highest interaction with lysozyme. Even though the head group orientations are the same between DPPE, PSM, and DPPC, the choline moiety from PSM and DPPC would provide greater steric hindrance than ethanolamine for incoming protein side chains during lysozyme insertion. It is possible then that the head group size accounts for the greater lysozyme insertion for DPPE over PSM and DPPC by providing less of a barrier for insertion. Unlike the three phospholipids, NMR studies on PGC have shown that the head group points normal to the surface plane, allowing for maximum hydration of the four glucose hydroxyls by the bulk subphase [105]. By aligning vertically, the effective size of the PGC head group is reduced in terms of increased space between adjacent

176 157 glucose head groups. It is possible then, that the head group orientation may also be promoting lysozyme insertion into PGC films, in addition to the lack of local charges present on PGC. The last major difference between the lipid species examined here come from the potential to hydrogen bond to lysozyme. Initial adsorption to the lipid film may be promoted through hydrogen bonds. For instance, both PGC and DPPE have head group hydrogen bonding groups from the sugar hydroxyls and the primary amine, respectively; these lipids also exhibited the highest lysozyme adsorption. PSM has the ability to hydrogen bond as well, however, this is conferred from the amide and hydroxyl groups in the sphingosine backbone, not the head group. Furthermore, PGC insertion was significantly higher than PSM (p<0.05). Therefore, hydrogen bonding in the lipid backbone region may not be as important as hydrogen bonding in the lipid head group, due to a decreased access to lysozyme hydrogen bonds. Finally, while both PGC and DPPE exhibited the greatest lysozyme adsorption, PGC values at 10 mn/m were significantly higher than DPPE (p<0.05). If hydrogen bonding to lysozyme played a role in protein insertion, such as through initial interaction with the film, than it stands that PGC would have greater insertion than DPPE. As mentioned in the previous paragraph, the glucose moiety in PGC points vertically into the subphase while the primary amine of DPPE is almost horizontal, bonding to the phosphate of the nearest lipid molecule. PGC hydrogen bond exchange from water to lysozyme would have negligible energy differences because of the bond similarities. For DPPE, however, there is both an electrostatic interaction and a hydrogen bond between the positive amine and negative phosphate suggesting an enthalpic penalty when breaking these interactions to hydrogen bond to lysozyme. Additionally, this interaction would bring the DPPE lipids tighter together, providing further steric hindrance to lysozyme insertion.

177 158 Furthermore, PGC was the only single lipid film to have significant lysozyme adsorption at 20 mn/m. Lipid packing at this pressure would be greater than at 10 mn/m, limiting lysozyme s access to the hydrocarbon tails required for insertion. Lipid head group size and orientation may have more of an impact at 20 mn/m than at 10 mn/m due to the tight lipid packing. This suggests that significant space between PGC lipids still exist at this surface pressure, allowing for lysozyme insertion. While on the other hand, larger head groups or those oriented parallel to the surface plane would therefore, inhibit insertion by crowding an already tightly packed film. Indeed, DPPC, DPPE, and PSM films had no significant lysozyme insertion at 20 mn/m. At 30 mn/m, lipid packing was too tight in any of the four films for lysozyme insertion, evidenced by a negative pressure change (fig. 48). The decrease in pressure occurred very gradually (fig. 49). Evaporation was determined to not be a factor affecting the very slight pressure drop as the position of the Wilhelmy plate within subphase did not change over the time period for all trials. The 30 minute time period would most likely have been too short to allow for much evaporation to occur. Furthermore, had the films been instable, the surface pressure drop would have been more sudden, indicative of film collapse. Combining all the results together would suggest that while hydrophobic attraction drives protein insertion, the process may be encouraged by either a smaller head group size or favourable head group orientation. Additionally, barriers to insertion are predominately steric restrictions from tight lipid packing, with penalties possibly from charge-charge repulsion as well. Finally, tear film rupture may be required for protein insertion because significant interactions between lysozyme and lipid films occurred at 10 mn/m but were lacking at the natural tear film surface pressure of 30 mn/m.

178 159 Figure 49. Surface pressure change following lysozyme injection at 30 mn/m in a PSM monolayer. The figure is used to emphasize the gradual decrease in surface pressure over 30 minutes. 5.2 Binary Lipid Systems Like the individual lipid systems, all of the binary mixtures exhibited lysozyme adsorption when the surface pressure was adjusted to 10 mn/m (fig. 50). This observation was the greatest in 3DPPC:2DPPE films (fig. 50A) where the surface pressure increased significantly by 4.1 ± 0.6 mn/m (p<0.005). This surface pressure increase was significantly higher than all other binary systems (p<0.05), with the exception of 2DPPE:3PGC films. At both 20 and 30 mn/m, lysozyme injection did not change the surface pressure of 3DPPC:2DPPE significantly from the control. 2DPPE:3PGC films had the second largest surface pressure increase at 10 mn/m of 2.6 ± 1.4 mn/m (fig. 50D) following lysozyme injection, which was significant from

179 160 the control (p<0.05). There was also an increase of 1.4 ± 1.7 mn/m at 20 mn/m, which was not found to be significant. Of all the binary mixtures, 3DPPC:2PSM had the smallest observed pressure increase after lysozyme injection at 10 mn/m, equal to 1.2 ± 0.6 mn/m (p<0.05) (fig. 50C). This was significantly smaller (p<0.01) than the observed pressure change from 3DPPC:2DPPE films, but represented an insignificant difference to the other binary systems. Individually, DPPC and PSM films had the smallest interaction with lysozyme at 10 mn/m, so it is not surprising that a binary mixture of these lipids would have the lowest adsorption. However, with the exception of 3DPPC:2DPPE, there was no significant difference between any of the binary films when lysozyme was injected at 10 mn/m. This would suggest that the individual characteristics of the lipids studied, in regards to lysozyme adsorption, were averaged out at these particular ratios.

180 161 Figure 50. Binary lipid system lysozyme injections. The x-axis represents the different initial surface pressures prior to lysozyme injection. For each initial surface pressure, the final lysozyme concentration was 0.05 mg/ml. The y-axis is the change in the initial surface pressure following lysozyme injection. (A) 3DPPC:2DPPE (B) 1DPPC:1PGC (C) 3DPPC:2PSM (D) 2DPPE:3PGC (E) 1DPPE:1PSM (F) 3PGC:2PSM. Significance was determined in relation to the control, * p<0.05, ** p<0.01, *** p< The error bars represent the standard deviation.

181 162 On the other hand, some trends can still be proposed based on the observed data. For instance, every binary mixture exhibited lysozyme insertion at surface pressures equal to 10 mn/m. At pressures of 20 mn/m and 30 mn/m, this interaction was abolished. This would suggest that tighter lipid packing prevents lysozyme insertion. In regards to the binary results, the lysozyme exclusion pressure would most likely exist between 10 and 20 mn/m. This further indicates that lysozyme most likely inserts into broken or not fully formed films in vivo. Additionally, it is possible that blinking may eject inserted lysozyme from tear film due to higher pressures that would be in excess of the natural open eye pressure of 30 mn/m. It was interesting that binary mixtures containing PGC generally had the weakest lysozyme interaction while individual PGC films experienced the greatest. It was suggested in section 5.1 that the vertical orientation of the glucose head group may be promoting lysozyme insertion. In the PGC binary systems, it is possible that the glucose orientation was impacted by other lipid species that resulted in a conformation not present in pure PGC films. The rigid glucose ring would most likely inhibit lysozyme insertion should it become oriented more parallel to the film surface, which is what may be occurring in the PGC binary films based on the decreased lysozyme insertion. On the other hand, lysozyme insertion into DPPE films was less impacted than PGC when in binary mixtures. As discussed previously, the ethanolamine head group in pure DPPE films is almost horizontal to the surface plane from interactions with the neighbouring lipid`s phosphate group. The almost horizontal orientation of the lipid head group however would have a greater impact on lysozyme insertion depending on the size of the head group. Therefore, if the glucose is rearranged such that its orientation moves more perpendicular to the monolayer when in binary mixtures, the head group size would most likely inhibit lysozyme insertion. This may be the reason why there was a larger change in lysozyme insertion

182 163 between the pure and binary lipid results for PGC films and not DPPE. That is to say, if the head groups were oriented similarly, DPPE films would have greater insertion because of less steric hindrance preventing the lipid and protein hydrophobic groups from interacting. 5.3 Tertiary and Quaternary Lipid Systems Continuing the trend, the tertiary and quaternary lipid mixtures all exhibited significant pressure increases (p<0.05) following lysozyme injection at 10 mn/m (fig. 51). The quaternary mixture had the largest increase, while 3DPPC:3PGC:2PSM had the smallest, increasing by 4.5 ± 1.0 mn/m and 2.0 ± 0.1 mn/m, respectively (fig. 51C,E). 3DPPC:2DPPE:PSM films had the second lowest adsorption with the pressure increasing by 2.4 ± 0.4 mn/m (fig. 51B). The quaternary system increase was significantly larger (p<0.05) than both of these systems. Lysozyme adsorption at pressures equal to 20 mn/m was also observed with 3DPPC:2DPPE:2PSM films showing a 1.7 ± 0.7 mn/m increase, significant to p<0.05 (fig. 51B). Generally speaking, the tertiary systems appeared to have greater lysozyme insertion compared to the binary systems based on greater surface pressure increases at 10 mn/m with some observed at 20 mn/m as well, specifically from 3DPPC:2DPPE:2PSM. Referring to the BAM images from section 4.1.3, the tertiary and quaternary systems exhibited complex packing behaviour based on the presence of multiple lipid populations (2DPPE:3PGC:2PSM) and the small defects found within domains (3DPPC:2DPPE:2PSM, and quaternary). The different observed populations within the BAM images may be from lipids clustering together in different combinations, which would affect lysozyme insertion. For instance, at 10 mn/m 2DPPE:3PGC:2PSM had the greatest pressure change of all the tertiary systems, increasing by 3.2 ± 1.0 mn/m. If the DPPE and PSM lipids were packing together based on the propensity of

183 164 their head groups to bond with a neighbouring lipid s phosphate, it is possible that PGC molecules may tend to pack together, separate from the other lipid species. The regions rich in PGC may emulate the behaviour of the pure PGC film, resulting in increased lysozyme insertion based on the glucose head group orientation discussed previously. Furthermore, the packing defects observed surrounding the domains of some of the tertiary and quaternary systems may enhance lysozyme insertion. Not packing ideally would create space between lipids, providing less of a barrier for insertion. Previous literature has shown that peptide insertion occurs at membrane defect sites [123]. Therefore, it may be a combination of both film packing defects and single lipid enriched regions that resulted in increased lysozyme insertion at 10 mn/m with the quaternary system compared to the binary and tertiary systems. On the other hand, lysozyme injections at both 20 and 30 mn/m had observable pressure decreases. There was no significant difference between the pressure changes at 20 mn/m and 30 mn/m. The decrease in pressures was gradual, similar to what was observed in figure 49, suggesting that films were not collapsing over the trial period. The decrease in pressure may be from packing rearrangements occurring over the trial period. Previous experiments have shown that without blinking, the natural tear film is stable for up to 50 seconds in healthy individuals [64]. Thus, slight instability of our tear film polar layer biomimetic at the natural surface pressure was not surprising as each trial lasted over 30 times longer than the upper range of the natural tear film break up time.

184 165 Figure 51. Tertiary and quaternary lipid system lysozyme injections. The x-axis represents the different initial surface pressures prior to lysozyme injection. For each initial surface pressure, the final lysozyme concentration was 0.05 mg/ml. The y-axis is the change in the initial surface pressure following lysozyme injection. (A) 3DPPC:2DPPE:3PGC (B) 3DPPC:2DPPE:2PSM (C) 3DPPC:3PGC:2PSM (D) 2DPPE:3PGC:2PSM (E) 3DPPC:2DPPE:3PGC:2PSM. Significance was determined in relation to the control, * p<0.05, ** p<0.01, *** p<0.005.the error bars represent the standard deviation.

185 166 The most obvious trend for all 15 systems tested was that every single film exhibited lysozyme insertion at 10 mn/m. This would suggest that lysozyme penetration into tear film occurs during the initial stages of film formation or that film break up is required for insertion. In descending order based on the magnitude of surface pressure increases at 10 mn/m, the top three films can be ordered as following: PGC > DPPE > Quaternary. PGC increases were significantly larger than DPPE values (p<0.05). However, PGC values were not significantly greater than quaternary increases, due to the larger standard deviation associated with the quaternary data set. On the other hand, the systems with the smallest pressure increase at 10 mn/m, in descending order, were 3PGC:2SM > 3DPPC:3PGC:2PSM > 3DPPC:2PSM. There was, however, no significant difference between these values. These results suggest that lysozyme insertion favours lipids that provide the greatest accessibility to their hydrocarbon tails. In terms of the top three systems, this was facilitated by either head group orientation (PGC), small head group size (DPPE), or lipid demixing/packing defects (quaternary). On the other hand, PSM and DPPC systems that had the smallest pressure increases at 10 mn/m, suggesting that these lipids prevent or inhibit insertion. Additionally, in binary and some tertiary mixtures, lysozyme propensity to insert into PGC films was decreased. This was evidenced in PGC-PSM mixtures, such as 3DPPC:3PGC:2PSM and 3PGC:2PSM, generally having lower pressure increases compared to the other films studied. This is most likely from other lipids packing more tightly with PGC or a change in the PGC s head group orientation towards to monolayer plane. Both situations would cause steric hindrance between the film and lysozyme. Furthermore, lysozyme s preference to insert less into PSM films compared to the others was most likely due to positioning of the hydrogen bonding groups within PSM. All the hydrogen bonding potential is found in the

186 167 interface region of PSM at the amide and hydroxyl groups. These hydrogen bonds increase lipid order, induce tight packing, and concomitantly prevent lysozyme insertion to the same as extent as seen for the other lipids. Additionally, the choline head group in both DPPC and PSM is oriented more parallel to the film surface, in a similar fashion to DPPE. However, choline is a larger head group and was most likely why PSM and DPPC had decreased lysozyme insertion. These results suggest that tighter lipid packing and large head group size prevents lysozyme insertion (fig. 52). Figure 52. Cartoon showcasing the different head group size and orientations of the lipids used in the polar layer biomimetic. The hydrocarbon tails were drawn at the same distance from each species. A vertical orientation (PGC) or a small head group (DPPE) allows for greater hydrophobic interactions between lysozyme and the lipid hydrocarbon tails.

187 Monolayer Rigidity and Lysozyme Adsorption Comparisons Insertion of some proteins into monolayers have been previously shown to depend on lipid ordering, with more insertion occurring with increasing monolayer compressibility or fluidity [124, 125]. The lateral compressibility of the 15 lipid systems studied was determined in chapter three but presented as the reciprocal, or the compression modulus (β). This parameter is more sensitive to subtle packing changes [72], whereby the lower β, the higher the compressibility. In order to assess the correlation between monolayer rigidity and the extent of lysozyme insertion, the change in surface pressure following lysozyme injection was plotted against the β determined at that initial pressure for each specific lipid film (fig. 53). More specifically, as discussed in the section 5.1, three initial surface pressures were investigated for possible lysozyme interaction, 10, 20, and 30 mn/m. The linear regression of these results suggest very little or no correlation between monolayer rigidity and lysozyme insertion. There was a very slight upward slope in figure 53A. The R 2, or the coefficient of determination, was, however, very low at , suggesting that there is very limited correlation between lysozyme insertion and monolayer rigidity. Furthermore, the R 2 value approaches zero when investigating the relationship between adsorption and β at 20 mn/m (fig. 53B) and 30 mn/m (fig. 53C). These results would suggest that there is no trend between the fluidity of the monolayer and lysozyme insertion, at least in terms of the surface pressures and lipid species examined here.

188 169 Figure 53. Change in initial surface pressure following lysozyme injection ( Π) versus compression modulus (β). The compression modulus was taken from the values at the surface pressure prior to injection. These pressures were; 10 mn/m (A), 20 mn/m (B), and 30 mn/m (C). Each point represents one of the 15 different lipid systems.

189 170 Chapter Six: Differential Scanning Calorimetry Results 6.1 Individual Lipid Systems The phase transition of meibomian extracts is broad, having a melting range between C [40]. The average corneal surface temperature is 34.3 C [89] suggesting the potential for coexisting phases within the tear film. Additionally, the individual tear film polar lipids melt at far higher temperatures, ranging from 41 C to 89 C [88, 90]. Differential scanning calorimetry was conducted on the tear film polar lipids to determine how their melting temperatures were impacted by the biomimetic lipid ratios of tear film. Thermograms of the individual lipid systems are in figure 54. The melting temperature (T m ) of the pure lipids, corresponding to the transition from the gel to liquid crystalline phase, agreed well with their literature values [88, 90, 126]. Furthermore, the pre-transitions of the DPPC and PGC multilamellar vesicles (MLVs) from the lamellar gel phase to ripple gel phase were observed (fig. 54, red and purple arrows respectively), confirming the purity of the lipid samples. These pre-transitions occur at 35 ºC for DPPC and 65 ºC for PGC [90, 127]. Figure 54 also illustrates the wide range of melting temperatures for the model polar lipids. DPPC and PSM had the same T m at 41 ºC (table 13). DPPE exhibited a T m of 61.6 ºC, with PGC the highest at 85.9 ºC (table 13). Moreover, the choline containing lipids had very sharp transitions indicated by their small temperature at halfheight values (T 1/2 ), which was 0.6 ºC for DPPC and 0.3 ºC for PSM (table 13). For DPPE and PGC the T 1/2 was slightly larger at 2.3 ºC and 1.3 ºC respectively (table 13), which agrees with the literature values for PGC but was marginally off from the DPPE value of 1.3 ºC [90, 128]. PGC had the highest enthalpy of the observed endothermic transition at 11.2 kcal/mol (table 13). The glucose head group of PGC can make extensive hydrogen bonds which most likely accounts

190 171 for both the high T m and the large enthalpy. Prior to the melting temperature, it is believed that the glucose head group may be somewhat immobilized by the hydrogen bonding network formed with neighbouring lipids [90]. Following the transition into the liquid crystalline state, the loss of internal ordering may allow for greater hydration of the head group as more hydrogen bonds are made with the bulk water [90]. The other hydrogen bonding lipids present, DPPE and PSM, had a relatively lower ΔH of transition, which was 5.55 kcal/mol for both lipid species (table 13). This lower change in enthalpy may be the result of larger hydrocarbon chain constraints in DPPE and PSM lipids in the fluid liquid crystalline state compared to the hydrocarbon tails in PGC due to interactions of DPPE and PSM with the phosphate of neighbouring lipid molecules [127]. Salt bridges from the primary amine of DPPE and hydrogen bonding from the interface of PSM may maintain lipid ordering following the transition, resulting in a smaller energy difference between the two phases and therefore a relatively lower ΔH compared to PGC. DPPC on the other hand, had a larger ΔH compared to DPPE and PSM, at 8.43 kcal/mol (table 13). As DPPC cannot hydrogen bond, the energy difference between the ordered gel state and the fluid liquid crystalline state would be larger as the constraints to the acyl chains would be less than DPPE and PSM following the transition [127].

191 172 Figure 54. DSC thermograms of the pure lipid systems. DPPC (red), DPPE (green), PGC (purple), and PSM (blue). The final concentration was 1 mg/ml of lipid. The arrows, red for DPPC and purple for PGC, represent observed lamellar gel to ripple gel phase pre-transitions. The red shaded box represents the melting range of the natural tear film.

192 173 Table 13. DSC data for the enthalpy, T m, and T 1/2 of the pure lipid MLVs. System Enthalpy (kcal/mol) T m (ºC) T 1/2 (ºC) DPPC DPPE PGC PSM Binary Lipid Systems While the pure lipid DSC experiments indicate that we were working with pure lipid samples, these results confirm existing data but cannot be extrapolated to tear film. More complex mixtures of these lipid species however represent novel and interesting avenues to be investigated. Figure 55 contains the scans of the different model lipids in their binary mixtures. Moving from the systems with lowest T m 's to the highest, the 3DPPC:2PSM binary system melted at 38.0 ºC (fig. 55, purple line). This mixture additionally had the sharpest transition, indicated by a T 1/2 value of 0.7 ºC suggesting high degree of cooperative melting usually only observed in pure synthetic lipid MLVs (table 14). This was not surprising as DPPC and PSM had the same T m in pure MLVs, most likely from similarities in lipid structure. Furthermore, the T m of the 3DPPC:2PSM binary mixture was slightly lower than that of the individual components indicating that possible packing irregularities and interactions between the lipids may have decreased their relative rigidities when mixed together. The comparatively lower ΔH of the binary system further supports this as there would be less of an energy difference between the gel and liquid crystalline phase of 3DPPC:2PSM accounting for the smaller change in enthalpy during this transition. Furthermore, this T m fell completely within the natural tear film melting

193 174 range, which may indicate that DPPC and PSM mixtures help fluidize the otherwise very rigid polar lipids. Lastly, the pre-transition observed in pure DPPC MLVs was abolished within the 3DPPC:2PSM system indicating that there was no large aggregation of DPPC lipids that could independently enter the ripple phase, further suggesting a high degree of mixing between DPPC and PSM. 3DPPC:2DPPE MLVs had the next lowest transition temperature at 51.6 ºC (fig. 55, red line). This was almost exactly in the middle of the T m s between pure DPPC and DPPE MLVs. However, unlike the 3DPPC:2PSM system, there was very little cooperative melting with 3DPPC:2DPPE evidenced by a large T 1/2 at 7.8 ºC (table 14) and marked asymmetry in figure 55. Mixtures of disaturated 14:0 PE and PC also exhibited transition asymmetry and peak broadening [129] suggesting a pronounced effect on the transition to the liquid crystalline phase from DPPC and DPPE interactions or lipid demixing. The range from the onset to completion of the main transition for 3DPPC:2DPPE was quite prolonged, occurring between 20 and 55 ºC with a distinct shoulder at 41 ºC (fig. 55, red arrow). The large peak broadening may indicate a mixture with greater packing irregularities possibly causing large surface defects on the MLVs. Additionally, while the pre-transition of DPPC was abolished in the 3DPPC:2DPPE mixture, the shoulder at 41 ºC corresponds to the main DPPC transition suggesting populations of DPPC undergoing an endothermic event prior to DPPE. Furthermore, initial melting of DPPC exclusive populations may be somewhat expected as BAM images of 3DPPC:2DPPE films indicated phase separation between DPPE domains and bulk DPPC molecules (fig. 32). However, most of the MLV melting occurred at temperatures higher than the T m of DPPC indicating that DPPE interactions stabilized the gel phase, preventing DPPC from fully melting at 41 ºC.

194 175 1DPPE:1PSM MLVs also exhibited a slight shoulder in their DSC thermogram but at a slightly higher temperature of 47 ºC (fig. 55, orange arrow). Like in the previously described scan, the shoulder may correspond to populations rich in PSM completing the transition into the liquid crystalline phase and the onset of the main DPPE transition, based on the temperatures these events took place. The mixing of DPPE with PSM additionally had a broadening effect evident by a relatively larger T 1/2 at 7.6 ºC, further indicating less cooperative melting and disruption of the lamellar gel to liquid crystalline phase transition. Finally, PSM depressed the T m of DPPE from 61.6 ºC to 54.8 ºC (table 14).

195 176 Figure 55. DSC thermogram of the six binary systems in their specified molar ratios. 3DPPC:2DPPE (red), 1DPPC:1PGC (green), 3DPPC:2PSM (purple), 2DPPE:3PGC (light blue), 1DPPE:1PSM (orange), and 3PGC:2PSM (dark blue).the final lipid concentration was 1 mg/ml. The red shaded box represents the melting range of the natural tear film. 1DPPC:1PGC melted very closely to 1DPPE:1PGC at 54.6 ºC (fig. 55, green line). The DPPC pre-transition was abolished when mixed with PGC MLVs indicating that there were no distinct domains enriched in DPPC lipids that would cause the transition to and from the ripple phase. Furthermore, the main transition peak was asymmetrical and again fairly broad, relative to the other binary mixtures, based on a T 1/2 of 7.6 ºC (table 14). This suggests very limited cooperative melting of the 1DPPC:1PGC MLVs. Additionally, the asymmetry of the peak could

196 177 arise from regions containing predominately more fluid lipids that would begin melting at lower temperatures compared to regions containing more rigid species, which would melt at higher temperatures. Furthermore, the relatively lower enthalpy of this mixture indicates that DPPC disrupted the PGC packing. 3PGC:2PSM MLVs had the highest T m of the binary systems examined, melting at 58.7 ºC (fig. 55, dark blue line). Unlike the previous binary systems containing either DPPE or PGC, there was more relative cooperative melting present in the 3PGC:2PSM mixture evidenced by a threefold reduction in the T 1/2 to 2.5 ºC (table 14). The 3PGC:2PSM system had the second highest cooperativity of the binary mixtures, surpassed only by 3DPPC:2PSM MLVs. This makes sense as lipids with comparable structures would most likely behave similarly, specifically in regards to the main transition peak being less broad than in mixtures with dissimilar components. PGC is more structurally related to PSM compared to either DPPC or DPPE, as both PGC and PSM were the only lipids studied to have a sphingosine backbone. This dissimilarity between PGC and the glycerol based lipids most likely resulted in the smallest cooperativity for any of the other PGC binary mixtures. The thermogram from 2DPPE:3PGC MLVs had very minor Cp changes during heating, less than 5.0 x 10-6 kcal/mol/ºc accounting for the totally flat scan in figure 55 (light blue line). The heating DSC scan also exhibited exothermic effects suggesting the presence of a thermodynamically irreversible event prior to the first scans [130], as heating transitions are generally endothermic. Furthermore, the 2DPPE:3PGC MLVs did not readily form once resuspended in buffer, instead large aggregates remained even after many freeze-thaw cycles (greater than 5) and extensive sonication (greater than 10 minutes). DPPE and PGC are very rigid lipids which may be preventing them from forming the MLVs necessary for DSC. This may

197 178 also have implications with the natural tear film as excess PEs and GCs may lead to aggregation, preventing rapid spreading of the polar lipid layer, possibly resulting in a DES pathology or DES like symptoms. Indeed, it was reported for rabbit tear film that the severity of the pathological state was correlated with significant increases in the ceramide content [131]. Table 14. DSC data for the enthalpy, T m, and T 1/2 of the binary lipid MLVs. The ratios listed are the molar ratios. System Enthalpy (kcal/mol) T m (ºC) T 1/2 (ºC) 3DPPC : 2DPPE DPPC : 1PGC DPPC : 2PSM DPPE : 3PGC N/A N/A N/A 1DPPE : 1PSM PGC : 2PSM With the exception of 3DPPC:2PSM, the T m 's of the binary systems remained outside of the natural tear film melting range. DPPC and PSM were the most fluid relative to the other lipids studied. However, DPPC appeared to disrupt the main transitions of DPPE and PGC more so than PSM because the DPPC binary mixtures exhibited a greater melting temperature decrease. This was most evident when comparing the T m of the most rigid lipid studied, PGC. Pure PGC MLVs melted at 85.9 ºC. In the presence of DPPC, the T m was lowered to 54.6 ºC

198 179 while in the presence of PSM it was 58.7 ºC. This was also the case for DPPE which had a T m of 51.6 ºC with DPPC and 54.8 ºC with PSM compared to the T m of pure DPPE MLVs at 61.6 ºC. Furthermore, PSM appeared to have the least impact on PGC packing based on the threefold decrease to the T 1/2 in 3PGC:2PSM MLVs compared to 3DPPC:PGC, further suggesting that DPPC helps to relatively fluidize the rigid tear film polar lipids. In the end, however, the transition peaks were still above the natural tear film melting range indicating that more fluid lipids, such as those containing unsaturated acyl tails like oleic acid, are most likely present but remain unreported. For instance, unsaturated PCs and PEs along with ethanolamine plasmalogens (phospholipids with a vinyl ether linkage) have been identified within the rabbit tear film [132] which would most likely depress the melting temperature further. It is more than likely that 24% of the unknown phospholipid fraction from human tear film [30] would contain some of these more fluid lipids. 6.3 Tertiary and Quaternary Lipid Systems The tertiary and quaternary DSC thermograms exhibited more complex melting behaviour evidenced by greater peak asymmetry, presence of multiple shoulders, and in many cases, multiple transition peaks. The tables used in sections 6.1 and 6.2 have been dropped from this part because the values listed within them do not accurately describe the more complex lipid mixture endotherms. For instance as the T 1/2 is calculated from the half height, it would not take into account the broadening or multiple peaks observed in many of the tertiary and quaternary mixtures, especially when these features occur below the half height of the tallest peak. The thermogram of the 3DPPC:2DPPE:2PSM system exhibited a very broad transition with above zero Cp values between 36 and 56 C (fig. 56). There were also two peaks present, the lower temperature one at 38.8 C and a much broader peak at 52.7 C. Additionally, there

199 180 was a prominent shoulder observed at approximately one degree higher than the DPPC/PSM transition temperature (represented by the green line in figure 56). These multiple thermogram features indicate that different populations of lipid transitioning out of the gel phase at different temperatures, suggesting lipid demixing within the MLVs. Furthermore, BAM images from this system exhibited extensive phase separation that continued as surface pressures became greater than 30 mn/m. The low temperature endothermic event from figure 56 most likely corresponds to DPPC and PSM melting based on their own pure lipid transitions occurring at 41 C, which was slightly above the one observed in the 3DPPC:2DPPE:2PSM mixture (fig. 56, red arrow). Interactions within this system additionally appear to fluidize the component lipids based on the lower temperature peak occurring almost 3 C below the main transition of either pure DPPC or PSM. In fact, this initial thermotropic event falls partially within the natural tear film melting range, evident by the red shaded box (fig. 56). The broadness of this transition and presence of a shoulder also indicate that melting was not cooperative, possibly due to packing irregularities and lipid demixing impacting the transition. For the higher temperature transition at 52 C, the peak was smaller but less cooperative evident by the increased broadness (fig. 56). Furthermore, this peak occurred at 52.7 C indicating that if the lower temperature event corresponded to DPPC and PSM melting, this higher temperature transition would most likely come from DPPE lipids. This peak occurred approximately 9 C lower than the T m of pure DPPE. Thus, it appears that the 3DPPC:2DPPE:2PSM mixture had a larger impact on the gel to liquid crystalline transition for all the lipids, as the melting temperatures appear to have shifted to lower temperatures. This was more so for DPPE which appeared to melt almost 10 C lower, while DPPC and PSM T m s were depressed by approximately 3 C.

200 181 Figure 56. DSC thermogram of the tertiary system 3DPPC:2DPPE:2PSM. The vertical line represents the T m of pure DPPC and PSM (green line). The red shaded box represents the melting range of the natural tear film. Exchanging PSM for the more rigid PGC resulted in MLVs that experienced more cooperative melting (fig. 57). The multiple transition peaks observed in the previous tertiary system were abolished in the 3DPPC:2DPPE:3PGC mixture. The main transition for this system occurred at 54.8 C, with shoulders on either side of the peak. The lower temperature shoulder occurred at approximately 43 C, two degrees higher than the main transition of pure DPPC MLVs. As the T m s from the other component lipids occur more than 20 C higher, this feature most likely corresponds to initial melting of DPPC lipids. DPPE and PGC, therefore, appear to

201 182 be stabilizing DPPC molecules within the gel phase, preventing them from melting until higher temperatures. Additionally a higher temperature shoulder, whose onset occurred at 57 C and remained until approximately 61 C, was near the T m of pure DPPE MLVs (fig. 57, red line). This feature would most likely correspond to DPPE and PGC rich regions transitioning to the liquid crystalline phase that hadn t done so during the main transition. While this mixture increased the melting temperature for DPPC it appeared to depress the T m of both DPPE and PGC, based on where the main transition temperature and the higher temperature shoulder occurred. Furthermore, these lipids also appeared to mix better than those within the 3DPPC:2PPE:2PSM system, evident by the single main transition peak and smaller shoulders observed with the 3DPPC:2DPPE:3PGC MLVs. This was also evident from the BAM images of 3DPPC:2DPPE:3PGC films from section While there was early phase separation, domain mixing did occur at surface pressures greater than 5 mn/m, eventually forming a relatively homogenous film at pressures larger than 20 mn/m (fig. 38). Additionally, the relatively decreased peak broadening in figure 57 also indicates relatively less disruption of the gel to liquid crystalline transition, suggesting well packed MLVs. However, the main transition of the 3DPPC:2DPPE:3PGC system occurred almost 15 C higher than the upper limit from the natural tear film melting range.

202 183 Figure 57. DSC thermogram of the tertiary system 3DPPC:2DPPE:3PGC. The vertical lines represent the T m of pure DPPC (green line) and pure DPPE (red line). The red shaded box represents the melting range of the natural tear film. The thermogram of 3DPPC:3PGC:2PSM MLVs had very interesting melting behaviour (fig. 58). A relatively sharp transition occurred at almost exactly the T m of pure DPPC or PSM MLVs suggesting regions rich in these lipids melting cooperatively. Following this thermotropic event, a much broader transition occurred from approximately 42 C to about 55 C. The broadness and overall shape of this peak would most likely contain multiple sub-peaks. From the figure, it then appears that many different lipid populations formed within the MLVS. Almost no disruption of the DPPC and PSM main transition would suggest that regions relatively enriched in these lipids existed in order to account for the fairly sharp lower temperature transition.

203 184 Although the small enthalpy of this transition would indicate that it was not a major endothermic event. Furthermore, the apparent multiple sub-speaks within the higher temperature transition suggest that this event was not exclusively due to PGC melting. Therefore, there appears to be demixing occurring that possibly results in different lipid populations; corresponding to relatively pure DPPC and PSM, and another to a complex mixture of DPPC, PGC and PSM. Furthermore, sphingomyelin domains were evident in the BAM images of 3DPPC:3PGC:2PSM films throughout the entire compression (fig. 42). These domains remained separated at pressures larger than 30 mn/m, further suggesting lipid demixing. Lastly, interactions from the presence of the more fluid DPPC and PSM molecules appear to disrupt the main PGC transition, evident by both the peak broadening and the T m being depressed by approximately 37 C from the pure PGC transition. However, very little melting was evident in the natural tear film surface range (fig. 58, red shaded box); further suggesting that excess PGC results in a fairly rigid system..

204 185 Figure 58. DSC thermogram of the tertiary system 3DPPC:3PGC:2PSM. The vertical line represents the T m of pure DPPC or PSM (green line). The red shaded box represents the melting range of the natural tear film. Unlike the DSC scans from the previous tertiary systems, no shoulders or multiple peaks were observed for the 2DPPE:3PGC:2PSM system (fig. 59). The gel to liquid crystalline transition peaked at 56.4 C. This endothermic event was fairly cooperative with a T 1/2 of 2.5 C, lower than most of the binary T 1/2 values (table 14) and comparable to that of the pure DPPE thermogram (table 13). The apparent lack of multiple peaks, shoulders, and a relatively narrow transition would indicate that the component lipids mixed very well together, melted cooperatively, and had minimal disruption of the gel to liquid crystalline transition. Indeed, BAM images of 2DPPE:3PGC:2PSM further confirm extensive mixing, as distinct domains were

205 186 not observed at any surface pressures (fig. 43). Additionally, the 2DPPE:3PGC:2PSM mixture appears to increase the main transition of PSM lipids while concurrently decreasing the transition temperature of PGC lipids. The main transition of this system occurred approximately 15 C above that of pure PSM MLVs and roughly 30 C below PGC. Thus, possibly rigidifying the more fluid lipids and fluidizing the more rigid ones. Lastly, of all the tertiary mixtures, 2DPPE:3PGC:2PSM MLVs transitioned the furthest from the natural tear film melting range (fig, 57, red shaded box). This was possibly due to two reasons. The first is that this tertiary mixture contained all of the lipids studied that were capable of hydrogen bonding, which had a rigidifying effect. The other possibility is that DPPC is more effective at disrupting the gel to liquid crystalline phase transition of the more rigid DPPE and PGC lipids relative to PSM. This also most likely due to the fact that PSM can hydrogen bond and DPPC cannot.

206 187 Figure 59. DSC thermogram of the tertiary system 2DPPE:3PGC:2PSM. The vertical lines represent the T m of pure PSM (green line) and pure DPPE (red line). The red shaded box represents the melting range of the natural tear film. Similar to the other tertiary mixtures that contained both DPPC and PSM, the quaternary system had an additional minor peak at 41 C (fig. 60). While this transition wasn t as sharp as the corresponding peak from the 3DPPC:3PGC:2PSM mixture, its presence within the quaternary system suggests that there were still significant populations of DPPC and PSM melting prior to the other lipids present. The larger endothermic transition peaked at 52.0 C, with another shoulder present at approximately 57 C. This feature was similar to the one observed in the 3DPPC:2DPPE:3PGC system but with a lower magnitude. The higher temperature shoulder most likely arose from portions of the more rigid DPPE and PGC lipids

207 188 melting at a slightly higher temperature than the main transition. The shoulders at either end of the main peak and the relatively broad transition would suggest some demixing between the lipids and a non-cooperative transition. This was also observed in the BAM quaternary system images in which distinct lipid domains were observed prior to the initial pressure lift off which continued until approximately 20 mn/m (fig. 45). However, for a system containing four distinct lipid species with over a 40 degree T m difference between the highest and lowest melting temperatures, the transition was relatively speaking not that broad. In terms of all the lipids present, it appears that the peak broadening was most likely caused by DPPC. For instance, comparing the quaternary system to the 2DPPE:3PGC:2PSM mixture, the tertiary system without DPPC, the entire melting range was relatively narrower, occurring over 10 C compared to the almost 30 C difference in the quaternary system. This would suggest that DPPC disrupts the main transition of the lipids studied. Finally, there was again a slight overlap of the quaternary endotherm with that of the natural tear film melting range. The onset of melting, characterized by the initial increase in Cp, occurred at approximately 37 C (fig. 60). Therefore, the polar lipid layer than appears to have a degree of fluidity near the upper range of the tear film melting range. This makes sense as this layer generally provides structural integrity to the tear film when the eye is open and during blinking. The maintenance of film integrity would most likely be hampered if the polar lipids existed in a more fluid state. Furthermore, within the more complex mixtures, the T m of PGC was greatly reduced, causing these lipids to melt almost 30 C lower than they would in pure PGC MLVs. This would suggest when in the natural tear film ratios, the other lipids present disrupt the main transition of PGC causing it to become more fluid and melting at lower temperatures. If PGC is removed from the quaternary system to get the 3DPPC:2DPPE:2PSM system, the resulting mixture was the most fluid of the 15 systems tested

208 189 and the one with largest natural tear film T m overlap. Therefore, while it appears that the presence of PGC rigidifies the mixtures the extent was limited, with apparent fluidity near the natural tear film T m. If the fluidity was decreased to such an extent that film spreading was reduced, the result may be an exposed aqueous phase that could cause more rapid evaporation. Figure 60. DSC thermogram of the quaternary system 3DPPC:2DPPE:3PGC:2PSM. The vertical lines represent the T m of pure DPPC or PSM (green line) and pure DPPE (red line). The red shaded box represents the melting range of the natural tear film. The addition of lysozyme to the quaternary MLVs (30:1 lipid to protein ratio) resulted in very minor changes to the melting behaviour (fig. 61A). Both the main transition peak at 52.0 C

209 190 and the minor DPPC/PSM melting peak at 41 C remained unchanged in the presence of lysozyme. On the other hand, the enthalpy of the transition appeared to increase slightly based on the larger peak height when the protein was present (fig. 61B). Increasing the relative abundance of lysozyme to a final ratio of 15:2 lipid to protein broadened the gel to liquid crystalline transition slightly, which also had an accompanying increase to the enthalpy (fig. 61B, green scan). Generally though, lysozyme appears to have little impact on the quaternary system main transition. Based on the lysozyme injection results from section 5.3, there was an observable interaction between the quaternary system monolayers and lysozyme when surface pressures were equal to 10 mn/m. However, at pressures greater than this, no interaction was observed. It is generally accepted that the effective internal pressures of lipid bilayers are equivalent to monolayer surface pressures between 30 and 35 mn/m (reviewed in [133]). The tightly packed MLVs would most likely be preventing the soluble lysozyme from interacting and therefore disrupting the quaternary system s main transition.

210 191 Figure 61. DSC thermogram of the quaternary system 3DPPC:2DPPE:3PGC:2PSM in the presence of lysozyme at a 30:1 lipid to protein ratio (A). The vertical lines represent the T m of pure DPPC or PSM (green line) and pure DPPE (red line). The red shaded box represents the melting range of the natural tear film. Panel B represents overlay comparison of the lipid pure thermogram onto lysozyme and quaternary mixtures in their specified lipid to protein ratios. Quaternary only (blue), 30:1 lipid to protein (red), and 15:2 lipid to protein (green).