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1 BIOPHYSICAL CHARACTERIZATION OF BIS(MONOACYLGLYCERO)PHOSPHATE (BMP) MODEL LIPID MEMBRANES USING ANALYTICAL TOOLS By JANETRICKS NANJALA CHEBUKATI A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2009
2 2009 Janetricks Nanjala Chebukati
3 To my parents: M y mom, Ritah Nekesa Muchungi and m y dad, Gerishom Muchungi Chebukati. For your endless love and for never giving up on me. And t o the memories of: My nephew, Rocky Wasike ( 1993 1996). Even though you have been long gone, you are still dearly missed and fond memories of you are always close in our hearts. M y brother, Justus Wangila Wekesa ( baba Bryo 1966 2005). Thank you f or believing in me always We miss you so much. May you rest in peace dear brother, until we meet again.
4 ACKNOWLEDGMENTS None of this work would have been accomplished without the numerous people that the Lord Almighty graciously blessed me with, to help me along this journey I am indeed very grateful to God for blessing me with many opportunities for second chances. First and foremost, I would like to express my sincere gratitude to my advisor and mentor D r. Gail E. Fanucci, who took me in at a time when I most needed someone to believe in me as a n aspiring scientist Gail gave me a chance to learn and develop a renewed appreciation for science especially the science of biological membranes I am very grat eful for her direction, support and encouragement during my time in her research group, and for always making the time to chat over my many questions. Gails resourcefulness and stimulating ideas made everything seem very possible, even in the most diffi cult of situations. Most of all, I thank Gail for teaching me to not only be a better scientist, but to also be a better person, and to never stop dreaming big! I would also like to thank my committee members Dr Dan Talham, Dr. Nicolo Omenetto, Dr. John Eyler and Dr. Byung Ho Kang especially for making time out of their busy schedules to serve on my committee. I am very grateful for all their input. I would also like to express my gratitude to Dr. Joanna Long for all her input and suggestions during our lipid discussions. I am thankful to the entire Fanucci research group, both present and past members, for all their support during my time in the group. I am especially grateful to Mandy Blackburn, who always went to great lengths to simplify a lot of stu ff for me, and whose sense of humor always made for the many much needed light moments to Dr. Yong Ran for the many lipid discussions we had, and to Tom Frederick for all the
5 interesting BMP discussions. I am also thankful to several members of the c he mistry department who in one way or another made my graduate years a lot easier. I am especially grateful to Dr. Ben Smith, Lori Clark and Vivian Thompson for all their support. I am very grateful to all my friends both here in the US and back home in Ken ya, for supporting me every step of the way. I would like to thank my best friend and sister, Dr. Catherine Nabifwo Situma (Cate), for all her support and encouragement. Thank you Cate, for always being there, for the lots and lots of laughter and for bei ng both a friend in need and indeed! I would also like to thank my friends, Dr. Cecelia Njeru and her husband, Mr. Peter Njeru, for all their support friendship and encouragement I am especially grateful to Cecelia for all her advice and encouragement during some of my lowest moments. I also thank my dear friends Dr. William and his wife Susan Mkanta for being our fa mily away from home I am greatly indebted to William and Susan for being such true friends in both good and bad times, for constantly encouraging me, and for freely opening their home to us whenever we needed to get away from Gainesville once in a while! I am also very thankful to Dr. R ichard and his wife Jane Makopondo for their friendship and encouragement I would like to remember my friends Isabella Tembra and Gladys Moige for always encouraging me throughout the years. I would also like to remember our small group friends, especially Aaron and Kea ne Wilkinson Patr ick and Tarasue Maness Steve and Jenni Williams Natalie and Justine, for their fellowship and continued prayers. Our fellowship together made me always look forward to Friday evenings with lots of excitement and anticipation, and I wish we had started doing this much sooner than just six months ago!
6 Without a doubt, my family has been my stronghold and inspiration every step of this journey. I am greatly indebted to my dearest friend and husband, fellow scientist Dr. George Odhiambo Okeyo. I am very grateful to George for being there always, for providing a shoulder to lean on, and for listening when at times that was all I needed, especially after a futile day of trying unsuccessfully to capture electron microscopy images of my lipid vesicles I also thank George for all our scientific discussions, and for helping me better unde rstand and appreciate the science of ion channels. Above all, I am grateful to George for being a selfless understanding husband and a great dad to our daughters, and for always dreaming big with me! I would also like to thank my daughters, Lavender and Subi, for giving me the reason to wake up each day and push myself further, and for bringing lots of joy and laughter to my life. I especially thank Lavender for helping so much with Subi, for patiently bearing with me, and for learning independence at suc h an early age! Subi has been such a bright spot in all our lives, and I thank her for the happiness she has brought us all! I am also very grateful to my siblings back in Kenya, who have endured through a lot of difficulty, and in some cases sacrificed a lot, so that I could be where I am today. I would like to remember my late brother Justus Wangila Wekesa for always encouraging me and believing in me. I also thank my sister Violet Lukela for being very supportive, for encouraging me throughout the year s, and for her great sense of humor! I am also grateful to my brother Walter Lubisia for quietly encouraging me on, believing in me and for praying for me I thank my younger brothers Brawel Welikhe and Caleb Muchungi, and my younger sisters Esther Naliaka Joyce Nasimiyu, Peris Namae and
7 Mildred Nakhumicha especially for being such a big help with Lavender when she was younger and for never stopping to believe in me! I thank all my nephews and nieces who always believed in me, especially Harun, Bryan Laura and Ian. I would like to also thank my mother i n law, Peres Jura, my father in law Zephaniah Jura, my sister in law Jacqueline and her family, my brother in laws Victor, James and their families, for their love, support and prayers. Finally, and most i mportantly, I cannot express enough gratitude to the two people without whom I would never have made it to graduate sc hool in the first place I am very grateful to my parents ; my dad, Gerishom Muchungi Chebukati and m y mo m Ritah Nekesa Muchungi for t hei r unconditional love, support, prayers, for encouraging me to reach for the sky, for believing in me and for never, ever giving up on me! My deepest gratitude to my parents and the appreciation of my PhD journey, can be better expounded by the following p aragraph, which I hope may someday be an inspiration to some girl, so that she may see life as being full of possibilities and to never lose hope! In the year 1995, my parents displayed the biggest lov e a parent can have for a child at least in my opinion A rmed with nothing but faith and hope, they requested the Jomo Kenyatta University of Agriculture and Technology (JKUAT) administration to defer my admission to the university until the following year after I failed to report for admission as scheduled. Admission to the few Kenyan public universities is highly competitive and one can easily lose their slot if they dont report as required. At the time, my parents had no idea where I was, or if and when they could ever see me again, but still hoped and b elieved for the best. My absence had been caused by unusual circumstances that, for confidential purposes, cannot be disclosed here. Little did anyone know that such a simple gesture of a parents true love, would later define who I am today, and bear so m uch fruit as witnessed by this work among other things. Needless to say, I turned up almost a year later in my parents rural home in Kenya, a frustrated and hopeless single mother at 19. You can imagine my joy and relief when I learnt that because of my parents, my chance at the university was still available and that through my education, there was still hope for me for a better future! As if that was not enough, my parents readily took care of my child while I went back to school, as if she was their own! The day I received my readmission letter to Jomo Kenyatta University was one of the happiest days of my life, and that was also the beginning of
8 my many blessings for second chances, and since then, I have never looked back. After all, life is full of po ssibilities! I am forever indebted to my parents, for the task they took upon themselves to guide me, to sacrifice everything so I can have an education, for supporting me through thick and thin, for taking care of my daughter Lavender so I could go back to school, for believing in me always, and above all else, for loving me endlessly. All in all, I thank the Lord God Almighty for making all things possible!
9 TABLE OF CONTENTS Page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES .......................................................................................................... 12 LIST OF FIGURES ........................................................................................................ 13 LIST OF ABBREVIATIONS ........................................................................................... 17 ABSTRACT ................................................................................................................... 20 CHAPTER 1 INTRODUCTION TO LIPIDS .................................................................................. 22 Biological Lipids ...................................................................................................... 22 Description and Classification .......................................................................... 22 Functional Roles of Lipids in the Cell ............................................................... 25 Glycerophospholipids .............................................................................................. 29 Typical Structure and Anatomy ........................................................................ 29 Unusual Phospholipids ................................................................................... 32 Ether phospholipids ................................................................................... 32 Cardiolipin (CL) .......................................................................................... 34 Bis(monoacylglycero)phosphate (BMP) ..................................................... 35 Phospholipid Polymorphism ............................................................................. 37 Self assembly, organization and phase behavior ....................................... 38 Lipid vesicles (liposomes) as membrane models ....................................... 43 Bis(monoacylglycero)phosphate (BMP) .................................................................. 46 Role of BMP in the Late Endosome ................................................................. 49 Overview of Biophysical Studies of BMP .......................................................... 51 Scope of Dissertation .............................................................................................. 53 2 TECHNIQUES UTILIZED IN LIPID ANALYSIS ...................................................... 57 Dynamic Light Scattering (DLS) .............................................................................. 57 Theory .............................................................................................................. 57 Fundamentals of DLS ................................................................................ 59 Factors that affect the translational diffusion coefficient ............................. 60 Basis of intensi ty fluctuations in DLS ......................................................... 62 How the digital autocorrelator works .......................................................... 66 The auto correlation function (ACF) ........................................................... 68 Instrumental design for DLS ....................................................................... 71 DLS Applications .............................................................................................. 73 Transmission Electron Microscopy (TE M) .............................................................. 76 Theory .............................................................................................................. 76 The need for a vacuum system in TEM ..................................................... 81
10 Sample pr eparation methods in TEM ......................................................... 83 Negative staining ....................................................................................... 84 TEM Applications ............................................................................................. 85 Fluorescence Resonance Energy Transfer (FRET) ................................................ 87 Theory .............................................................................................................. 87 Principles of FRET ..................................................................................... 88 Methods for measurement of FRET ........................................................... 93 Biological Applications of FRET ....................................................................... 94 Chromatography Separations ................................................................................. 97 Thin Layer Chromatography (TLC) ................................................................... 98 Column Chromatography ............................................................................... 100 3 INVESTI GATION OF BMP VESICLE SIZE AND MORPHOLOGY IN MODEL MEMBRANES ....................................................................................................... 104 Introduction ........................................................................................................... 104 Experimental section ............................................................................................. 108 Materials and Reagents Used ........................................................................ 108 Hydrated Dispersions and Extruded Vesicle Preparation ............................... 109 Sample Preparation for Vesicleleakage Assays ............................................ 110 Instrumentation Used ..................................................................................... 111 Dynamic light scattering (DLS) ................................................................. 111 Negative staining transmission electron microscopy ............................... 112 Fluorescence measurements ................................................................... 112 Results and Discussion ......................................................................................... 113 Evaluation of Mixing Different Sizes of Neat POPC Vesicle Populations ....... 113 Comparison of Vesic le Stability: BMP versus POPC Vesicles ..................... 120 BMP Vesicle Leakage Assays under Acidic and Neutral pH Conditions ........ 122 Characterizat ion of BMP and POPC Hydrated Dispersions and Unilamellar Vesicles. ...................................................................................................... 126 Effect of pH .............................................................................................. 126 Effects of Ionic strength ........................................................................... 131 Characterization of Hydrated Dispersions and Extruded Vesicles of POPC Mixed With BMP and POPG ....................................................................... 133 Conclusions .......................................................................................................... 136 4 ANALYSIS OF CHANGES IN BMP VESICLE SIZE AND MORPHOLOGY IN THE PRESENCE OF GANGLIOSIDE GM1 AT LATE ENDOSOMAL pH 5.5 ....... 138 Introduction ........................................................................................................... 138 Experimental Section ............................................................................................ 141 Materials Used ............................................................................................... 141 Preparation of Hydrated Lipid Dis persions and Extruded Unilamellar Vesicles ....................................................................................................... 141 Instrumentation ............................................................................................... 142 Dynamic light scattering (DLS) ................................................................. 142 Negative staining transmission electron microscopy (TEM) .................... 143
11 Results and Discussion ......................................................................................... 143 Ch aracterization of BMP Hydrated Lipid Dispersions as a Function of pH ..... 143 Characterization of BMP:GM1 Hydrated Dispersions and Extruded Vesicles at Specific Concentrations ........................................................................... 146 Effect of GM1 and BMP Mixing with POPC Membranes ................................ 153 Conclusions .......................................................................................................... 157 5 EFFECT OF CHOLESTEROL MIXING WITH GM1 AND BMP ON PHOSPHOLIPID MODEL MEMBRANES AT LATE ENDOSOMAL pH 5.5 .......... 159 Introduction ........................................................................................................... 159 Experimental D etails ............................................................................................. 163 Materials Used ............................................................................................... 163 Hydrated Lipid Dispersions and Extruded Vesicle Preparation ...................... 163 Instrumentation ............................................................................................... 164 Dynamic light scattering (DLS) ................................................................. 164 Negative staining transmission electron mic roscopy (TEM) .................... 165 Results and Discussion ......................................................................................... 165 Characterization of BMP Vesicle Size and Morphology in the Presence of Cholesterol .................................................................................................. 165 Investigation of Vesicle Size and Morphology in POPC:CHOL:BMP Mixtures ....................................................................................................... 168 Conclusions .......................................................................................................... 175 6 SUMMARY AND FUTURE PERSPECTIVES ....................................................... 176 BMP Forms Small Stable Lamellar Vesicle Structures and Induces Formation of Small Vesicles when mixed with POPC ............................................................. 176 Ganglioside GM1 Leads to Formation of Small Homogenous Vesicles When Mixed with BMP ................................................................................................. 176 BMP Counteracts the Cholesterol Effect when Mix ed with GM1 and POPC ........ 177 Future Perspectives .............................................................................................. 178 APPENDIX STEP BY STEP ANALYSIS OF DYNAMIC LIGHT SCATTERING DATA ................... 180 LIST OF REFERENCES ............................................................................................. 183 BIOGRAPHICAL SKETCH .......................................................................................... 204
12 LIST OF TABLES Table page 1 1 Common classes of glycerophospholipids. ......................................................... 31 2 1 Types of lasers commonly used in DLS instruments. ......................................... 72 3 1 Summary of POPC vesicle diameters extruded with polycarbonate membranes of varying pore sizes. .................................................................... 114 3 2 Summary of BMP, POPC and POPG average vesicle diameter as a function of extrusion membrane pore diameters. ........................................................... 120 3 3 Summary of average vesicle diameters of BMP and POPC dispersions and extruded vesicles at neutral pH 7.4. ................................................................. 126 3 4 Average vesicle diameters of BMP and POPC hydrated dispersions and 400nm extruded vesicles at acidic pH 4.2. ............................................................. 129 3 5 Summary of average vesicle diameters of BMP vesicles hydrated without NaCl in the buffer. ............................................................................................. 132 3 6 Summary of the average vesicle diameters of POPC:BMP (85:15) hydrated dispersions and 400 nm extruded vesicles at neutral pH. ................................ 134 3 7 Summary of average vesicle diameters of POPG and POPC: POPG (80:20) vesicles. ............................................................................................................ 135 4 1 Summary of the average diameter of BMP hydrated dispersions at specific pH conditions. ................................................................................................... 145 4 2 Summary of DLS average vesicle sizes of BMP:GM1 hydrated lipid dispersions and 400nmextruded unilamellar vesicles at specific concentrations. ................................................................................................. 150 5 1 Average vesicle of BMP:CHOL (7:3) hydrated dispersions and extruded Vesicles. ........................................................................................................... 167 5 2 Average vesicle di ameters of POPC:CHOL (8:2) and POPC:BMP:CHOL (65:15:20) hydrated dispersions and extruded vesicles. ................................... 170 5 3 Vesicle size distributions of POPC:GM1:CHOL (70: 10: 20) and POPC:GM1:BMP:CHOL hydra ted dispersions and 400 nm extruded Vesicles. ........................................................................................................... 173 A 1 OriginPro 8 spreadsheet of the imported DLS raw data of 100 nm extruded BMP vesicles. ................................................................................................... 181
13 LIST OF FIGURES Figure page 1 1 General classification of eukaryotic lipids. Modified from Ref. (3). ...................... 22 1 2 Chemical structures of A) glycerol backbone, B) triacylglycerol and C) sphingosine backbone. ....................................................................................... 23 1 3 Enzymatic hydrolysis of phosphatidylinositol 4, 5, bisphosphate to diacylglycerol and 1, 4, 5 triphosphate. .............................................................. 27 1 4 Chemical structures of A) dolichol, B) vitamin A, C) vitamin E and D) vitamin K. ........................................................................................................................ 27 1 5 General structural anatomy of gly cerophospholipids. ......................................... 30 1 6 Chemical structures of common classes of glycerophospholipids. A) Phosphatidic acid. B) Phosphatidylcholine. C) Phosphatidylethanolamine. D) Phosphatidylserine. E) Phosphatidylglycerol. F) Phosphatidylinositol. ............... 31 1 7 Chemical structures of representative ether phospholipids. A) Platelet activating factor ( PAF). B) Plasmalogen. ............................................................ 32 1 8 Enzyme catalyzed biosynthesis of cardiolipin from POPG and CDP DAG. ........ 34 1 9 Chemical structures of A) BMP (sn 1, sn 1 stereoconfiguration) and B) phosphatidylcholine (sn 3 glycerophosphate stereoconfiguration). .................... 36 1 10 Different shapes of lipid polymorphisms in water (or aqueous environment). ..... 39 1 11 Morphology of different sizes/types of lipid vesicles (liposomes). A) Multilamellar vesicles (MLVs). B) Large unilamellar vesicles (LUVs). C) Small unilamellar vesicles (SUVs). ............................................................................... 41 1 12 Structural isoforms of BMP. A) (R, R) B) (R, S) and C) ( S, S) isomers. ............. 48 2 1 Schematic representation of a speckle pattern observed in DLS. Adapted from Ref. (119). .................................................................................................. 63 2 2 A) Destructive interference and B) constructive interference observed in a speckle pattern. .................................................................................................. 64 2 3 Typical intensity fluctuations for A) large particles and B) small particles. .......... 65 2 4 Schematic showing the fluctuation in the intensity of scattered light as a function of time. Adapted from Ref. (119). .......................................................... 66
14 2 5 Correlation spectrum for a sample containing A) large particles with long decay time and B) small particles with rapid decay time. ................................... 68 2 6 N umber, volume and intensity distributions of a bimodal mixture of 5 and 50 nm lattices present in equal numbers. A) Number distributions. B) Volume distributions. C) Intensity distributions. Modified from Ref. (119). ....................... 71 2 7 Typical experimental set up in a dynamic light scattering instrument. ............... 72 2 8 Layout of major components in a basic transmission electron microscopy (TEM) instrument. ............................................................................................... 78 2 9 A Jablonski diagram illustrating the coupled transitions involved between the donor emission and acceptor absorbance in FRET. ........................................... 89 2 10 A) Overlap of the emission spectrum of the donor and acceptor absorption spectrum results in FRET. B) Lack of overlap of the spectra means no FRET observed. ............................................................................................................ 90 3 1 Chemical structures of A) BMP, B) POPC and C) POPG. ................................ 105 3 2 DLS histograms of size distributions of POPC vesicles extruded with polycarbonate membranes of varying pore sizes. A) Black line, 30 nm. A) Red line, 100 nm. B) Green line, 400 nm. B) Blue line, hydrated unextruded dispersions. ...................................................................................................... 113 3 3 DLS histograms of populations of manually mixed POPC vesicles extruded with 30 nm and 400 nm por e size membranes and mixed in various volume/volume ratios in 5 mM HEPES buffer. .................................................. 115 3 4 DLS histograms showing populations of manually mixed POPC vesicles extruded with 100 nm and 400 nm pore size membranes in various volume/volume ratios. ....................................................................................... 118 3 5 Dynamic light scattering size distributions of A) 30nm extruded BMP and B) POPC vesicles monitored over a five week period. .......................................... 121 3 6 Chemical structures of A) calcein, B) ANTS, C) DPX and D) SDS. .................. 123 3 7 Schematic illustration of SDS detergent solubilizing the li posome and causing leakage of the vesicle contents. Figure adapted from Tom Frederick. ............. 123 3 8 Vesicle leakage assay for BMP vesicles. A) Neutral pH 7.4. B) Acidic pH 4.2. 124 3 9 DLS vesicle size distributions of BMP and POPC vesicles, under neutral pH 7.4. A) Hydrated dispersions. B) 400 nm Extruded vesicles. ............................ 126
15 3 1 0 Negative staining TEM images of BMP and POPC under neutral pH conditions. A) BMP hydrated dispersions. B) POPC hydrated dispersions. C) BMP 400 nm extruded vesicles. D) POPC 400 nm extruded vesicles. ........... 127 3 11 DLS measurements of BMP and POPC A) hydrated dispersions and B) 400nm extruded large unilamellar vesicles at acidic pH 4.2. .................................. 129 3 12 Negative staining TEM images of BMP an d POPC hydrated dispersions and 400nm extruded vesicles at acidic pH. A) BMP hydrated dispersions. B) BMP extruded vesicles. C) POPC hydrated dispersions. D) POPC extruded vesicles. ............................................................................................................ 130 3 13 Negative staining TEM images of BMP A) hydrated dispersions and B) 400 nm extruded vesicles. Vesicles were hydrated in buffer lacking NaCl salt. ...... 132 3 14 DLS histograms of BMP A) hydrated disp ersions and B) 400 nm extruded vesicles that were hydrated in the absence of NaCl. ........................................ 132 3 15 DLS size distribution of POPC:BMP (85:15) A) hydrated dispersions and B) 400nm extruded vesicles at neutral pH. .......................................................... 134 3 16 Negative staining TEM images of POPC:BMP (85:15) A) hydrated dispersions and B) 400 nm extruded vesicles at neutral pH. ............................ 134 3 17 DLS measurements of POPG and POPC:POPG (80:20) hydrated dispersions and extruded vesicles at neutral pH. ............................................. 135 4 1 Chemical structure of A) Phosphatidylcholine and B) BMP. ............................. 138 4 2 Negative staining TEM images of BMP lipid dispersions as a function of pH. .. 144 4 3 Dynamic light scattering size distri butions of BMP hydrated dispersions as a function of pH. .................................................................................................. 145 4 4 Chemical structures of A) BMP and B) ganglioside GM1. ................................ 147 4 5 N egative staining TEM images of hydrated BMP:GM1 dispersions at specific molar ratios at pH 5.5. ...................................................................................... 148 4 6 TEM images of 70:30 mol % BMP: GM1 lipid mixture, showing A) small, aggregated homogenous vesicles, and B) magnified images of the same vesicles. ............................................................................................................ 148 4 7 DLS size distribution histograms of BMP:GM1 A) Hydrated dispersions and B) 400 nm Extruded unilamellar vesicles at specific co ncentrations. ............... 150 4 8 Dynamic light scattering average vesicle diameters of POPC A), black solid line, hydrated dispersions, A), red solid line, unilamellar vesicles extruded
16 with 400 nm polyc arbonate membranes and B) TEM image of POPC hydrated dispersions. ....................................................................................... 153 4 9 Dynamic light scattering average vesicle diameters of POPC:GM1 (80:20) A) hydrated dispersions, B) 400nm extruded ves icles and C) TEM images of POPC:GM1 hydrated dispersions at pH 5.5. .................................................... 154 4 10 Average vesicle diameters of POPC:BMP:GM1 (70:15:15) A) hydrated dispersions, B) 400nm extruded vesicles and C) TE M images of POPC: BMP:GM1 hydrated dispersions. ...................................................................... 155 5 1 Chemical structures of A) Cholesterol, B) BMP, C) GM1 and D) POPC. .......... 166 5 2 Dynamic light scattering histograms of BMP:CHOL (7:3) A) hydrated dispersions and B) 400 nm extruded unilamellar vesicles at pH 5.5. ............... 167 5 3 Negative staining TEM images of BMP: CHOL (7:3) hydrated dispersions. ..... 168 5 4 Dynamic light scattering histograms of POPC:CHOL (8:2) A) hydrated dispersions B) 400nm extruded vesicles, POC:BMP:CHOL (65:15:20) C) hydrated dispersions and D) 400 nm extruded vesicles. Note the different scales on the x axes. ........................................................................................ 169 5 5 TEM images of A) POPC:CHOL (8:2) and B) POPC:BMP:CHOL (65:15:20) hydrated dispersions. ....................................................................................... 171 5 6 Dynamic light scattering histograms of A) hydrated dispersions of POPC:GM1:CHOL (70: 10: 20), B) 400 nm extruded vesicles of POC: GM1: CHOL, C) hydrated dispersions of POPC:GM1:BMP:CHOL (50:15:15:20) and D) 400 nm extruded vesicles of POPC:GM1:BMP:CHOL. Note the different scales on the x axes. .......................................................................... 172 5 7 Negative staining TEM images of A) POPC:GM1:CHOL (70: 10: 20) and B) POPC:GM1:BMP:CHOL (50:15:15::20) hydrated dispersions. ......................... 174 A 1 DLS raw data histograms (1, 2, and 3) of a sample of 100 nm extruded BMP vesicles in 5 mM HEPES, 100 mM NaCl and 0.1 mM EDTA, pH 7.4. .............. 180 A 2 Average vesicle diameter for 100 nm extruded BMP vesicles. ......................... 182
17 LIST OF ABBREVIATION S BMP Bis(monoacylglycero)phosphate LBPA L ysobisphosphatidic acid POPC 1 palmitoyl 2 o l eoyl phosphatidylcholine POPG 1 Palmitoyl2 Oleoyl sn G lycero 3 Phosphorac 1 Glycerol D PDMP D threo1 phenyl 2 decanoylamino3 morpholino1 propanol DPPC D ipal mitoylphosphatidylcholine CHOL Cholesterol PtdCho Phosphatidylcholine PtdEtn P hosphat id ylethanolamine PtdSer Phosphatidylserine PtdIns Phosphatidylinositol PG Phosphatidylglycerol PA Phosphatidic acid SM Sphingomyelin PAF Platelet activating factor AD Alzheimers disease NPC Niemann Pick type C DS Down syndrome CDP DAG C ytidinediphosphatediacylglycerol PAMs P ulmo nary alveolar macrophages CMC C rit ical micelle concentration SDS Sodium dodecyl sulfate MLVs Multilamellar vesicles LUVs Lar ge unilamellar vesicles
18 SUVs Sm all unilamellar vesicles MVBs M ultivesicular bodies MVEs Mul tivesicular endosomes DNA Deoxyribonucleic acid RNA Ribonucleic acid PCR Polymerase chain reaction LE L ate en dosome VBs Vesicular bodies BHK B aby hamster kidney DSC D ifferential scanning calorimetry LDL Low density lipoprotein DLS Dynamic light scat tering TEM Transmission electron microscopy FRET F luorescence resonance energy transfer FLIM FRET F luorescent lifetime imaging FRET TLC Thin layer chromatography PCS P hoton c orrelation spectroscopy QELS Q uasi elastic l ight scattering SLS S tatic light scattering ACF A uto correlation function NNLS N onnegative least squares PMT Photomultiplier tube CCDs Charge coupled devices Cryo EM C ryo electron microscopy Rf R etention factor
19 ANTS 8 amino napt halene1, 3, 6 trisulfonic acid DPX p xylenebis pyridinium bromide H EPES 4 (2 hydroxyethyl,) 1 piperazineethanesulfonic acid EDTA E thylenediamine tetraacetic acid
20 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy BIOPHYSICAL CHARACTERIZATION OF BIS(MONOACYLGLYCERO)PHOSPHATE (BMP) MODEL LIPID MEMBRANES USING ANALYTICAL TOOLS By Janetricks Nanjala Chebukati December 2009 Chair: Gail E. Fanucci Majo r: Chemistry Bis(monoacylglycero)phosphate (BMP) is a negatively charged phospholipid found in elevated concentrations in the late endosome. BMP has an unusual structure and stereochemistry that are thought to be responsible for important roles in the endosome, including structural integrity, endosome maturation, and lipid/protein sorting and trafficking. The main objective of the work reported in this dissertation was to characterize the morphology and size distribution of BMP model membranes as a functio n of pH, ionic strength, concentration and lipid composition, using dynamic light scattering (DLS) and transmission electron microscopy (TEM). Dynamic light scattering is a simple non invasive particle sizing technique that measures the hydrodynamic diameter of particles or macromolecules suspended in solution based on their interaction with light, whereas TEM provides valuable information on the morphology and sizes of particles. Results presented in this dissertation demonstrate that BMP forms small sta ble lamellar vesicle structures, and that BMP induces the formation of small vesicles when mixed with typical phosphatidylcholine ( POPC ) membranes at specific concentrations.
21 M orphological and size distribution studies on the interaction between BMP and g anglioside GM1 reveal that GM1 mixes with BMP at specific concentrations to form small (~100 nm) spherical shaped vesicles with a narrow size distribution. This specific mixture of GM1 with BMP may be important for in vivo vesicular trafficking and lipid sorting in the endosome/lysosome pathways. Finally, when ganglioside GM1, cholesterol (CHOL) and BMP lipids are incorporated in typical POPC dispersions at specific concentrations, they form vesicles with different morphology and size distributions. Taken t ogether, results from these investigations give a further understanding to the role that BMP, GM1 and cholesterol may play in the late endosome, and allow for possible future studies in using BMP vesicles for drug delivery applications.
22 CHAPTER 1 INTRODUCTION TO LIPI DS Biological Lipids Description and Classification Biological lipids are a diverse group of chemical compounds, commonly defined by their insolubility in water due to their nonpolar chemical composition (1) a nd their solubility in nonpolar organic solvents such as acetone, ether, chloroform and benzene (2). Eukaryotic lipids are generally classified according to their biological functions as illustrated in Figure 11. Storage Lipids Triacylglycerols Glycerol backbone Fatty Acid Fatty Acid Fatty Acid Membrane Lipids Phospholipids Glycolipids Sphingolipids Glycerophospholipids Sphingolipids Galactolipids Glycerol backbone Fatty Acid Fatty Acid PO4 Alcohol Fatty Acid Sphingosine backbone PO4 Choline Fatty Acid Sphingosine backbone Saccharide Glycerol backbone Fatty Acid Fatty Acid Saccharide SO4 Sterols Four fused carbon rings Alkyl side chain Polar head group (hydroxyl) Figure 11. General classification of eukaryotic lipids Modified from Ref. (3).
23 S torage lipids, composed of fats and oils, are the principal stored forms of energy in most organisms (3). Chemically, fats and oils are triacylglycerols that are also known as simple lipids (1) Fats are characterized by being solid at room temperature (20 C), whereas oils are liquid at room temperature (1) The structure of triacylglycerols (Figure 1 2 B ) consists of a glycerol backbone that has three fatty acid molecules attached to its hydroxyl groups through ester linkag es (3). A)H H C OH H C OH H C OH H 1 2 3B) C )1 1 2 2 3 3 Figure 12. Chemical structures of A) glycerol backbone, B) t riacylglycerol and C) sphingosine backbone. Using the stereochemical numbering (sn) system, the fatty acid molecules may be designated as sn1, sn2 and sn3(Figure 1 2B) In most triacylglycerols the fatty acids in the sn1 and sn3 positions are different resulting in two possible enantiomers with similar fatty acid profiles, both with the same fatty acid located in the sn2position (4).
24 Generally, variations in the saturation and hydrocarbon ch ain lengths of the fatty acids result in different forms of triglycerols with different physical properties such as fluidity and melting point (1). The most fundamental structure of the biological membranes of the cell is the lipid bilayer (a double layer of lipids), which is formed from membrane lipids that act as a barrier to the passage of polar molecul es and ions in and out of the cell (3), hence membrane lipids are another major class of lipids. Membrane lipids are also referred to as structural lipids and are amphipathic in nature, with one end of the molecule being hydrophobic while the other end is hydrophilic (1, 3) When membrane lipids are dispersed in water, they spontaneously self aggregate into bilayer or micellar structures as a result of the hydrophobic effect, a phenomenon that arises due to the energetically unfavorable contact between the hydrocarbon and water molecules, hence the lipid acyl chains are directed inward towards each other and the polar lipid headgroups are exposed to w ater (5). Membrane lipids obtained from eukaryotic cells can belong to one of the following general groups: glycerophospholipids, in which the hydrophobic regions are composed of two fatty acid chains bonded to a glycerol backbone, galactolipids or sulfolipids, which also contain two fatty acids esterified to a glycerol backbone but lack the characteristic phosphate head group found in phospholipids The other groups of eukaryotic mem brane lipids are the sphingolipids in which a fatty acid chain is joined to a sphingosine backbone and an amine or a saccharide head group and sterols, compounds that are characterized by a rigid system of four fused hydrocarbon rings (2, 3, 6, 7)
25 Functional Roles of Lipids in the Cell Due to their lipid component, biological membranes are flexible self sealing boundaries that form the permeability barrier for cells and organelles and provide the means to compart mentalize functions in the cell (3). As a support for both integral and peripheral membrane processes, the physical properties of the lipid component directly affect these processes in a complex manner; hence each specialized membrane of a cell has a unique structure, lipid and protein composition and function (3, 6) Additionally, within each membrane there exist microdomains such as lipid rafts, lipid domains and organizations of membraneassociated complexes wit h their own unique lipid composition. In essence, defining lipid function is challenging due to the diversity of chemical and physical properties of lipids and the fact that each lipid type potentially is involved at various levels of cellular function (6). Generally, lipids serve three main purposes. First, because of their relatively reduced chemical state, lipids are used for energy storage, principally as triacylgl ycerol and steryl esters, in lipid droplets. The reduced state of the carbon atoms of the fatty acids allows for the enhanced oxidation of triacylglycerols, yielding more than twice as much energy as the oxidation of carbohydrates (3). Triacylglycerol and steryl esters function primarily as anhydrous reservoirs for the efficient storage of caloric reserves and as caches of fatty acid and sterol components that are needed for membrane biogenesis (8, 9) Secondly, the matrix of cellular membranes is formed by polar lipids, which consist of a hydrophobic and a hydrophilic portion. The tendency of the hydrophobic moieties to self associate and the ability of the hydrophilic moieties to interact with aqueous environments and with each other are the physical bases for the spontaneous formation
26 of membranes (9). It is thought that this unique chemical property of amphi pathic lipids may have enabled the first cells to segregate their internal constituents from the external environment and is the same principle that enables the cell to produce discrete organelles (8, 9) Cell memb rane compartmentalization enables segregation of specific chemical reactions for the purposes of increased biochemical efficiency and restricted dissemination of reaction products. Lipids also provide membranes with the potential for budding, tubulation, f ission and fusion, characteristics that are essential for cell division, biological reproduction and intracellular membrane trafficking (9, 10) Although storage lipids and membrane lipids are major cellular compon ents, with membrane lipids making up 510% of the dry mass of most cells, and storage lipids more than 80% of the mass of an adipocyte, a third group of lipids are in much smaller amounts but have active roles in the metabolic traffic as metabolites and m essengers. Some of these lipids may serve as potent signals, such as hormones carried in the blood from one tissue to another or as intracellular messengers generated in response to an extracellular signal (1, 3) For instance phosphatidylinositol and its phosphorylated derivatives act at several levels to regulate cell structure and metabolism (3). Phosphatidylinositol bi sphosphate is hydrolyzed by p hospholipase C to yield two intracellular messengers, diacylglycerol and 1, 4, 5 triphosphate (3), as illustrated in Figure 13
2 7 HO Phospholipase C H2O+Phosphatidylinositol 4, 5, bisphosphate Diacylglycerol 1, 4, 5 triphosphate Figure 13. Enzymatic h ydrolysis of phosphatidylinositol 4, 5, bisphosphate to diacylglycerol and 1, 4, 5 triphosphate. Other lipids may function as enzyme cofactors in electrontransfer reactions in mitochondria or in the transfer of sugar moieties in a variety of glycosylation reactions. For instance d olichols (which are isoprenoid alcohols Figure 14 A ) are known to activate and anchor sugars on cellular membranes for use in the synthesis of certain complex carbohydrates, glycolipids and glycoproteins (3) D C B A Figure 1 4. Chemical structures of A) dolichol B ) v itamin A, C) vitamin E and D) v itamin K.
28 Another group consists of lipids with a system of conjugated double bonds that may function as pigments that absorb visible light. For example vitamin A (Figure 1 4B) furnishes the visual pigment of the vertebrate eye and is a regulator of gene expression during epithelial cell growth, Vitamin E (Figure 1 4C) protects membrane lipids from oxidative damage and vitamin K (Figure 1 4D) is essential in the blood clotting process (1, 2, 6, 7) Finally, lipids can act as first and second messengers in signal transduction and molecular recognition processes. The degradation of amphipathic lipids allows for bipartite signalling phenomena, which can be transmitted within a membrane by hydrophobic portions of the molecule and also propagated through the cy tosol by soluble (polar) portions of the molecule. In addition, some lipids function to define membrane domains, which recruit proteins from the cytosol that subsequently organize secondary signalling or effector complexes (6, 9, 11) For instance, phosphoinositols (PIs) are important in cell si gnalling and vesicle formation, both of which are key events in neurotransmission and in the transit of vesicles from the endoplasmic reticulum to Golgi (12, 13) All biological membranes consist of a lipid bilayer to which proteins and carbohydrates may be associated or covalently linked. Evidently, the roles of membrane lipids in cells have evolved from a simple physical barrier to a c ritical component in cell signaling and other cellular processes (13) It has long been established that lipids provide the physical support of organelle membranes, acting as a barrier for water soluble molecules and as a solvent for the hydrophobic domains of membrane proteins. By contributing to the intrinsic properties of membranes, such as thickness, asymmetry
29 and curvature, lipids can potentially regulate protein movement and distribution among other functions (14) The most abundant group of structural lipids in eukaryotic membra nes are the glycerophospholipids, which include but are not limited to phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn), phosphatidylserine (PtdSer), phosphatidylinositol (PtdIns) and phosphatidylglycerol (PG) (9). The structural differences in the headg roups of these glycerophospholipi ds are further illustrated in Figure 16 and Table 1 1 Because g lycerophospholipids were the basis of the work covered in this dissertation, they are discussed in detail in the following subsection. Glycerophospholipids T ypical Structure and Anatomy Glycerophospholipids (also called phosphoglycerides) are the most common class of na turally occurring phospholipids; phospholipids are lipids with phosphatecontaining head groups (15) These compounds are derivatives of sn glycero 3 phophoric acid (2). The stereospecific numbering (sn) system is used in phospholipid nomenclature. The glycerol is drawn in a Fischer project hydroxyl on the left and the sn1 position is then located at the top of this projection, while the sn 3 position is located at the bottom (the prefix sn is used before the name) as illustrated in Figure 15
30 sn1 sn2 sn3 OX Polar functional group Polar head group Glycerol backbone sn1 chain sn2 chain Hydrophilic portion Hydrophobic portion Figure 1 5 General structu ral anatomy of glycerophospholipids. Figure 15 shows the general anatomy of glycerophospholipids. One of the glycerol hydroxyl groups (at sn 3 position) is linked to the polar phosphatecontaining head group, while the other two hydroxyl groups (at sn 1 and sn 2 positions) are linked to hydrophobic acyl chains R1 and R2 (8). The hydrophobic portion contains saturated or cis unsaturated fatty acyl chains of varying lengths (9). In all naturally occurring glycerophospholipids, the polar group is attached to the sn3 position of the glycerol moiety except in the glycerobased lipids of archeabacteria (2). The phosphate group is then esterified to one of several functional groups The functional group is designated as X in Figure 15 and Table 11 ; replacing the X with a specific polar functional group results in different glycerophospholipids which explains the diversity in physicochemical properties and roles of membrane lipids (7).
31 B C D E F A Figure 16. Chemical structures of common classes of glycerophospholipids. A) Phosphatidic acid. B) Phosphatidylcholine. C) Phosphatidylethanolamine. D) Phosphatidylserine. E) Phosphatidylglycerol. F) Phosphatidylinositol. Table 11. Common classes of glycerophospholipids. Name of X Chemical Formula of X Name of Phospholipid Hydroxyl OH Phosphatidic acid Choline CH2 CH2 -+N(CH3) 3 Phosphatidylcholine Ethanolamine CH2 CH2 + NH3 Phosphatidylethanolamine Serine CH2 CH (COO-) + NH3 Phosphatidylserine Glycerol CH2 CHOH CH2OH Phosphatidylglycerol I nositol C 6 H 6 (OH) 6 Phosphatidylinositol Figure 16 and Table 1 1 illustrate the common classes of glycero phospholipids. The name of the polar functional group attached to the phosphate head group lends a further distinction to the nomenclature of the phospholipids For instance, in the absence of any alcohol or functional group, the phospholipid is called phosphatidic acid (PA, Figure 16A) Phosphatidate, the ionized form of PA is not foun d in large quantities in cell me mbranes, as it is largely a biosynthetic intermediate. If the alcohol esterified to the phosphate is choline or ethanolamine, the phospholipid is called phosphatidylcholine (or lecithin) or phosphatidylethanolamine (or cephalin) respectively. Phosphatidyl choline (PC, Figure 16B) a nd phosphatidylethanolamine (PE, Figure 16C) are among the most common of cell membrane phospholipids contributing prominently to the phospholipid bilayers found in most cellular membranes. PE c ontains a free
32 amino group which can be stripped of a proton at high pH (910) to give an uncharged, primary amine (7, 8) Other alcohols that may be esterified to the phosphate include the L amino acid serine, and sugars such as glycerol and inositol, resulting in the phosp holipids phosphatidylserine (PS, Figure 16D), phosphatidylglycerol (PG, Figure 1 6E) and phosphatidylinositol (PI, Figure 16F) respectively. All of these phospholipids contain only one phosphate. Unusual Phospholipids A number of special glycerophospholipids vary from the rest of the diacylglycerophospholipids in terms of thei r chemical structure and functionality. Ether phospholipids One such unusual group of glycerophospholipids are the ether phospholipids, a special class of phospholipids that are characterized by the presence of an ether bond at the sn1 position of the gl ycerol backbone instead of an ester bond like in the diacylglycerophospholipids (16) Some ether phospholipids may be saturated as in the alkyl ether lipids exhibited by the platelet activating factor (PAF, Figure 17A) or may con tain a double bond between C 1 and C 2 (3), adjacent to the ether bond, forming a vinyl ether linkage as in lipids that are referred to as plasmalogens (Figure 17B) (17) A B Figure 17. Chemical structures of representative ether phospholipids. A) Platelet activating factor ( PAF ). B) Plasmalogen.
33 The hydrophobic acyl chain at the sn1 position in plasmalogens consists of either C16:0 (palmitic acid), C18:0 (stearic acid) or C18:1 (oleic acid) carbon chains, whereas the sn2 posit ion is occupied by polyunsaturated fatty acids and the head group is either an ethanolamine or a choline (16) Eth er phospholipids can be enriched in various tissues or even different cell types within one tissue. For instance, ver tebrate heart tissue is uniquely enriched in ether lipids where about half of the heart phospholipids are plasmalogens (3). The hig hest content of ethanolamine plasmalogens (PE plasmalogen) is found in brain myelin, whereas heart muscle has a higher content of choline plasmalogens (PC plasmalogen). Moderate amounts of plasmalogens are also found in kidney, skeletal muscle, spleen and blood cells (16) Although the functional roles of plasmalogens are not yet fully understood, past studies have implicated plasmalogens as antioxidants, a function attributed to the presence of a vinyl ether bond that makes plasmal ogens more susceptible to oxidative attack compared to their 1acyl analogues (18) Plasmalogens are also mediators of membrane dynamics, in which the vinyl ether bond may affect the hydrophobic hydrophilic interface region of phosphol ipid aggregates (19) Plasmalogens have been implicated in various disease forms including the Zel lweger syndrome, a lethal autosomal recessive disorder in which peroxisome biogenesis is impaired, leading to a generalized loss of peroxisomal functions (20) Alzheimers disease (AD) (21) Niemann Pick type C (NPC) lipid storage disorder (22, 23) and Down syndrome (DS) (24 ) Additionally, the platelet activating factor (PAF) is a potent molecular signal that is released from leukocytes called basophils and is found to stimulate platelet aggregation
34 and the release of serotonin, a vasoconstrictor, from platelets (3). PAF also has a variety of effects on the liver, smooth muscle, heart, uterine and lung tissues and as such plays an important role in infla mmation and allergic response (25, 26) Cardiolipin (CL) Cardiolipin is another unique dimeric glycerophospholipid in which two phosphatidyl moieties are linked by a glycerol backbone (27, 28) Cardiolipin was first isolated from beef heart in the early 1940s (29) hence its name. This lipid is found exclusively in bacterial and mitochondrial membranes, which function in the generation of an electrochemical potential for substrate transport and ATP synthesis (27) Whereas cardiolipin is most abundant in mammalian hearts, with current commercial preparations of cardiolipin being derived from heart tissue, it is also found in all mammalian tissues throughout the eukaryotic kingdom that have mitochondria (27) Cardiolipin Phosphatidylglycerol, POPG Cytidinediphosphatediacylglycerol, CDP DAG +cardiolipin synthase catalysis Figure 18. Enzyme catalyzed biosynthesis of cardiolipin from POPG and CDP DAG
35 The chemical structure of cardiolipin (Figure 1 8) differs from t hat of most membrane phospholipids in that it exhibits a double glycerophosphate backbone and four fatty acyl side chains (29) The biosynthesis of cardiolipin in eukaryotic cells involves phosphatidylglycerol (PG) and cytidinediphosphatediacylglycerol (CDP DAG) (Figure 1 8) catalyzed by cardiolipin synthase on the inner face of the inner mitochondrial membranes (30) When treated with phospholipase D, cardiolipin yields two phospholipid products; phosphatidic acid and ph osphatidylglycerol (31) and conversely, cardiolipin can be chemically synthesized from phosphatidic acid and phosphatidylglycerol (32). Cardiolipin from mammalian tissues exhibits high specificity in the fatty acyl chain composition, being predominantly comprised of 18carbon unsaturated acyl chains, the vast majority of which are linoleic acid (18:2) (33, 34) The ability of cardiolipin to mediate the optimal function of numerous mitochondrial proteins and processes is attributed to its unique ability to interact with proteins and its role in maintaining inner membrane fluidity and osmotic stability (27, 35) Cardiolipin is reportedly required for the proper structure and activity of several mitochondrial respiratory chain complexes involved in the oxidative generation of ATP (34, 36) and has been proposed to participate directly in proton conduction through cytochrome bc1 (37) and prevent osmotic instability and uncoupling at higher respiration rates (38) Other important cardiolipin roles include a regulatory role in cytochrome c release (39, 40) which triggers some of the events in apoptosis (41) essential roles in mitochondrial biogenesis (27) and the stabilization of respiratory enzyme supercom plexes (42, 43) Bis(monoacylglycero)phosphate (BMP) Bis(monoacylglycero)phosphate (BMP) is a characteristic lipid of the endocytic degradative pathway that is found in the late endosome luminal membranes in
36 concen trations of approximately 15 mole percent (44) BMP was first isolated from pulmonary alveolar macrophages (PAMs) of lung obtained from pig and rabbit by Body and Gray (45) The chemical structure of B MP (Figure 1 9A ) differs from that of other glycerophospholipids in that BMP contains two glycero components, each with a single acyl chain (2, 8, 46) Additionally, BMP has an sn 1 glycerophospho sn 1 glycerol (sn1:sn1') stereoconfiguration that differs from the typical sn 3 glycerophosphate stereoconfiguration found in other glycerophospholipids as illustrated by the red circles in Figure 19B (47 50) Figure 19. Chemical structures of A) BMP (sn 1, sn 1 stereoconfiguration) and B ) phosphatidylcholine (sn 3 glycerophosphate stereoconfiguration). Because studies of BMP form the bulk of the work presented in this dissertation, it w ill be discussed in more detail later on in the introduction.
37 Phospholi pid Polymorphism Phospholipids are amphipathic in nature, consisting of a hydrophobic portion (the hydrocarbon chains), and a hydrophilic portion (the polar head group). This property a llows phospholipids to establish a hydrophobic barrier to permeability when in close proximity to an aqueous medium. To maintain the permeability barrier, lipids associate in structures that sequester the hydrocarbon portions in hydrophobic regions away fr om the aqueous medium so that only the polar groups then encounter the polar phase. Through this arrangement, the phospholipid molecule is able to satisfy the hydrophobic effect, which is the dominant driving force behind membrane lipid assembly (2, 7) Membrane lipids are polymorphic, which implies that they can exist in a variety of different kinds of organized structures or phases especially when hydrated in water (2). The structural organization that a polar lipid assumes in water is determined by its concentration and the law of opposing forces; the hydrophobic forces driving self association of hydrophobic domains versus steric and ionic repulsive forces of the closely associated polar domains opposing self association (6). At low concentrations, amphipathic molecules exist as monomers i n solution, but as the concentration increases, the molecules stability in solution as a monomer decreases until the unfavorable repulsive forces of polar domains are outweighed by the favorable self association of the hydrophobic domains. Eventually, any further increase in concentration results in the formation of increasing amounts of self associated monomers in equilibrium with a constant amount of free lipid monomer. This state of self association and the remaining constant free lipid monomer is refer red to as the critical micelle concentration (CMC) (6, 51)
38 Due to the increased hydrophobic effect, a larger hydrophobic domain results in a lower critical micelle concentration, while the larger the polar domain due to either the size of the neutral domains or charge repulsion for like charged ionic domains, the higher the critical micelle concentration .This is attributed to the unfavorable steric hindrance or charge repulsion in bringing these domains into close proximity (6). Below the CMC, the lipid exists solely as monomers in solution, and above the CMC, the monomer concentration remains constant and is equal to the CMC (5). Self assembly, organization and phase behavior Pure phospholipids are capable of undergoing transformations from one shape or morphology to another, resulting in what is termed as lipid polymorphism, which is the ability of lipids to take on structures of different shapes (52) Lipid phases can be divided into two g eneral types: normal and inverted phases. Normal phases are those in which the polar moiety of the lipid faces outward from the lipid m whereas the nonpolar portion of the molecule makes up the structure core. In inverted phases, the polar groups face inw ard and the nonpolar portion occupies the exterior of the structure (53) Som e of the factors that determine the type of phase or morphology formed by lipids include lipid structure, hydration and temperature (53) Given precise conditions and the nature of the lipids, several types of lipid aggregates or shapes can form when amphipathic lipids are hydrated in water or aqueous media (3), including m icelles, inverted micelles, lipid bilayers (lamellar phase), bicelles, normal or inverse hexagonal phases and lipid vesicles or liposomes as illustrated in Figure 110
39 Micelle Inverted micelle Bicelle Lipid vesicle/liposome Lipid bilayer L amellar phase Hexagonal phase Normal Hexagonal phase Inverse Figure 110. Different shapes of lipid polymorphisms in water (or aqueous environment) Micelles, lipid bilayers and vesicles (liposomes) are the structures most commonly formed by most phospholipids in water; hence a lot of studies on the applications of these model systems have been carried out Micelles (Figure 1 10) are spherical structures that contain about a dozen to a few thousand amphipathic molecules (3) that are arranged in water, with their hydrophobic regions aggregated in the interior, where water is excluded, and their hydrophilic head groups at the surface in contact with water. Mi celle formation is generally favored when the cross sectional area of the head group is greater than that of the acyl side chain(s).This is mostly the case in molecules such as free fatty acids, lysophospholipids (phospholipids lacking one fatty acid),
40 phospholipids with short alkyl chains (eight or fewer carbons) (3, 6) and detergent s such as sodium dodecyl sulfate (SDS) The overall structure of a micelle reflects the optimal packing of amphipathic molecules at an energy minimum by balancing the attractive force of the hydrophobic effect and the repulsive force of close head group association (3). The CMC for most lysophospholipids and detergents is in the micromolar to millimolar range (6). Under physiologically relevant conditions, most membrane lipids exist as lipid bilayers (2), as illustrated in Figure 110, in which two lipid monolayers or leaflets form a two dimensional sheet. Phospholipids with long alkyl chains do not form micelles but organize into bilayer structures, which allow tight packing of adjacent side chains with the maximum exclusion of water from the hydrophobic domain. Bilayer formation occurs most readily when the cross sectional areas of the head groups and the acyl side chains are similar, which is the case in glycerophospholipids and sphingolipids (3). In living cells, phospholipids are not found free as monomers in solution but are organized into either membrane bilayers or protein complexes (6). The bilayer sheet is relatively unstable because the hydrophobic regions at its edge are transiently in contact with water, hence it spontaneously folds back on itself to form a hollow sphere referred to as a vesicle or liposome (3), as shown in Figure 110. Formation of vesicles enables bilayers to lose their hydrophobic edge regions, achieving maximal stability in their aqueous environment. The lipid vesicles enclose water, creating a separate aqueous compartment (3) and this property can allow vesicles to be studied for various encapsulation applications, including use as drug delivery vehicles
41 C) SUV d = 20 50 nm B) LUV d = 0.1 1 m A) MLV d = 0.1 10 m Figure 111. Morphology of different sizes/types of lipid vesicles (liposomes). A) Multilamellar vesicl es (MLVs). B) Large unilamellar vesicles (LUVs). C) Small unilamellar vesicles (SUVs). Liposomes are typically spherical in shape and may contain a single layer or multiple layers of amphiphilic polymolecular membranes Liposomes that contain only a single bilayer membrane are called either small unilamellar vesicles ( SUVs which are less than 5 0 nm in diameter, Figure 111C ) or large unilamellar vesicles ( LUVs 10 0 100 0 nm in diameter Figure 111B ). When liposomes contain more than a single bilayer memb rane they are referred to as multilamellar vesicles (MLVs Figure 111A ) if all
42 layers are concentric, or multivesicular bodies (MVBs) when a number of randomly sized vesicles are enclosed in the int erior of another larger vesicle. MLVs can be obtained through lipid hydration in an aqueous solution of water or buffer. Lipid vesicles that are larger than 300 nm will scatter light sufficiently enough so as to be seen by the naked eye and such samples will have a cloudy white appearance (54) Phospholipid membrane systems have complex phase behaviors (54) existing either in the lamellar liquidcrystalline or in the lamellar gel phase (2) Fully hydrated bilayers composed of single phospholipid s pecies undergo a well defined thermotropic phase transition in which the lipid chains change from the ordered or gel state to the f luid or liquid gel phase is designa found in the gel phase of certain phospholipids (5). Because most membrane lipids undergo this kind of phase transition, it is one of the most intensively studied lipid phase transitions. During this phase transition, a relatively ordered gel state bilay er, in which the hydrocarbon chains exist predominantly in their rigid, extended, all trans conformation, is converted to a relatively disordered liquidcrystalline bilayer, in which the hydrocarbon chains contain a number of gauche conformers and the lipi d molecules exhibit greatly increased rates of intraand intermolecular motion (2) Thermodynamically, the gel to liquidcrystalline phase transition occurs when the entropic reduction in free energy arising from chain isomerism counterbalances the decrease in bilayer cohesive energy arising from the lateral expansion and from the energy cost of creating gauche conformers in the hydrocarbon chains (2, 8) Gel to liquid crystalline phase transitions can be induced by changes in temperature and
43 hydration or changes in pressure and in the ionic strength or pH of the aqueous phase (2, 7) Another important parameter for liposomal systems is the gel to liquidcrystalline transition temperature Tm, at which the bilayer los es much of its ordered packing structure due to a melting of the hydrocarbon chains. Ideally, longer hydrocarbon chain lengths translate into higher phase transition temperatures (54) Notably, most natural phospholipids contain hydrocarbon chains that are asymmetric, where the fatty ac id chains may differ in length. The degree of fatty acid unsaturation in the hydrocarbon chain also influences the phase transition, and the Tm increases with increasing saturation (2) Strong head group interactions may increase the phase transition temperature as well. Consequently, the phase transition behavior of liposomes with more than one lipid component can be quite complex. For instance, cholesterol is o ften added to liposomes to improve their in vivo and in vitro stability since it provides the membrane with rigidity by changing interactions between both the polar head groups and the hydrocarbon chain and a decrease in both Tm and the transition enthalpy is observed when cholesterol is added (2, 7, 54) Lipid v esicles (liposomes) as membrane models Liposomes, also known as lipid vesicles, are spherical, closed structures made of curved lipid bilayers that were firs t investigated in the early 1960s (55) Liposomes are predominantly composed of amphiphilic molecules such as phospholipids. Due to their amphipathic character, phospholipids have a strong tendency in the presence of a queous solutions (mostly water) to aggregate spontaneously to ordered, lamellar, bilayer structures (56)
44 Because liposomes are highly versatile struc tures, they have been utilized for research involving both pharmaceutical and analytical applications. For instance, phospholipid mixtures with different polar headgroups can be functionalized for conjugation or to reduce liposome aggregation and phospholi pids with hydrophobic regions of varying chain length and degrees of saturation can be used to modify the properties of the resulting liposomes (57) For example, cholesterol is often included with membrane phospholipids to reduce the membrane permeability towards encapsulated ma terials (57) The struct ure of lipid vesicles mimics that of the cell membranes, hence vesicles can be utilized as a more easily characterized vessel for studying interactions between membrane lipids and biomolecules such as DNA and proteins; permeability of ions and drugs (57, 58) ; and for elucidating the mechanism of action of pesticides and antibiotics on target organisms (59) According to literature, liposomes have been used as models in several studies for estimating the partit ioning of drugs into cells by surface plasmon resonance (60) and chromatography (61) Biological molecules may be associated with liposomes in several established ways; encapsulation within the aqueous inner cavity of the liposome (62) partitioning within the lipid tails of the bilayer (63) or covalent and electrostatic interactions with the polar headgroups of the lipids. Surface modification of liposomes can be achieved through the proper choice of lipids that allow conjugation to a variety of biorecognition molecules, such as phosphatidylethanol amine (PE) (64) Other functional groups that can be used for surface modification include a carboxy group (64 67) a maleimide
45 group (68) a protected disulfide group (57) and a hydroxyl group, using cholesterol or p olye thylene glycol derivatives (69) Heterobifunct ional cross linking agents have been heavily utilized in methods for the conjugation of molecular recognition elements such as receptors, enzymes, antibodies and nucleic acids (70) Generally, the reaction chemistry chosen depends on the functional groups available on the recognit ion site and the lipid bilayer, the desired orientation of the recognition element and the effects of the functionalized lipid and reaction conditions on liposome stability (66, 71) These modifications allow liposo mes to be targeted towards specific cell types and target organs or organelles, leading to a reduction in the apparent toxicity associated with nonlocalized therapies (72) Some of the compounds that are frequently used to allow the targeting of liposomes to specific cell types include peptides (73, 74) lectins (75, 76) antibodies (77) and folate (78) Because liposomes have an inner hydrophilic cavity, a wide variety of hydrophilic molecules can be encapsulated within this cavity, including pharmaceutical compounds, enzymes, DNA molecules, vaccines, fluorescent dyes, electrochemical and chemiluminescent markers (57, 58) Previous studies have shown that the bilayer structure can potentially prolong the longevity of the encapsulated molecules by shielding them from destructive entities within the body. Chaize et al reported that the activity of the pesticide target enzyme acetylcholinesterase can be retained when encapsulated in liposomes despite the presence of proteolytic enzymes in the surrounding media (79) In other studies, liposomeencapsulated RNA was reported to be protected from RNase present in the external solution (80) and the oxidation of heme gr oups present in hemoglobin was minimized when encapsulated within liposomes (81)
46 The application of liposomes in therapeutics stems from the fact that the encapsulation of drugs within liposomes results in their delayed release which is beneficial for reducing toxic effects and maximizing the therapeut ic index of drugs (82 84) The FDA has approved several pharmaceutical compounds that use liposomes as a drug delivery system, including doxorubicin, daunorubicin, amphotericin B, morphine, and cytarabine which are used for the treatment of refractory ovarian and breast cancers, Karposis sarcoma, fungal infections, management of post surgical pain, and neoplastic and lymphomatous meningitis, respectively (85) Some drugs can also be associated with the lipid bilayer through electrostatic interactions (86) Additionally, the sequestration of various molecules within liposomal cavities has been utilized for a variety of unique applications. For instance, DNA has been encapsulated into liposomes for use as an internal control for real time PCR (8 7) and in similar studies, reagents have been entrapped in liposomes to allow for internal DNA transcription (80) and replication (88) Hemoglobin based blood substitutes have also been encapsulated within liposomes to enhance their stability and clinical utility (89) Bis(monoacylglycero)phosphate (BMP) Bis(monoacylglycero)phosphate (BMP), also erroneously termed as lysobisphosphatidic acid (LBPA) is a unique, negatively charged phospholipid that was first detected in pulmonary alveolar macrophages (PAMs) of lung from pig and rabbit by Body and Gray (45) It has since been postulated to be synthesized from phosphatidylglycerol (PG) or lyso phosphatidylglycerol in vivo (90, 91) BMP has also been found in other tissues and cell types such as the brain, l iver, and kidney of a number of different species (92) It usually represents less than 1 mol% of the total phospholipid mass (45, 46) although increased BMP contents have been found in
47 several lipidosis and i n response to some pharmaceutical agents (93) In the late endosome (LE), however, the concentration of BMP is near 15 mol % of the total lipid content of the organelle and can comprise as much as 70 mol % of the lipid composition of the intraendosomal vesicles (94 96) Endosomes are prelysosomal organelles that serve as intracellular sites for the sorting, distribution, and processing of receptors, ligands, fluid phase components, and membrane proteins internalized by endocytosis. E ndosomes are responsible for many of the critical events that regulate the trafficking, sorting, and processing o f internalized macromolecules. T heir acidic internal pH is also needed for the dissociation of incoming ligand receptor complexes, allowing the receptors to return to the cell surface and the ligands to be transported to lysosomes for degradation (5) Endosomes can be identified by the presence of vesicular bodies (VBs) inside the lumen of the limiting membrane or by lipid composition (44, 97) Whereas early endosomes have a limiting membrane with a lipid composition very similar to that of the plasma membrane, late endosomes are characterized by an absence of a significant amount of cholesterol and a relatively high concentration of BMP (97) During endosome maturation, the internal pH undergoes a series of changes from that of the neutral cytosol to that of the acidic lysosome. It is within late endosome intraluminal membranes that BMP is first found in relatively higher concentrations and is believed to be partly responsible for the formation of multivesicular liposomes that resemble multivesicular endosomes (98) BMP has an unusual structure that differs from that of other phospholipids from two aspects (Figure 1 12) First, unlike other phospholipids, it has two glycero components, each with a single acyl chain and secondly, BMP isolated from biological
48 sources differs from the typical sn 3 glycerophosphate struc tures exhibited by most other glycerophospholipids (2, 8, 46) in that it possesses an unusual sn 1 glycerophosphosn 1 glycerol (sn1:sn1) stereoconfiguration (47 50, 99, 100) However, the sn 3: sn 1 configuration has been reported for BMP isolated from baby hamster kidney (BHK) cells, although it is stipulated that this configuration is an intermediate in the synthesis of the sn1:sn1 BMP (48) (R) (S) (R) (S) (S) (R)A B C Figure 112. Structural isoforms of BMP A) (R, R), B) (R, S) and C) (S, S) isomers. Body and Gray (45) first identified BMP as a structural isomer of POPG, and since then, v arious structural isomers of BMP have bee n obtained by varying the location of the two acyl chains or by altering the sn stereochemistry (101) Three structural
49 isoforms of B MP are shown in Figure 112; t he (S,R) isomer, sn (3 oleoyl 2 hydroxy) glycerol 1 phosphosn 3' (1' oleoyl 2' hydroxy) glycerol (ammonium salt), (1,3 dioleoyl sn3:sn1or BMP18:1) (Figure 112A) ; the (R,R ) is omer, sn (3 oleoyl 2 hydroxy) glycerol 1 phosphosn 3' (1' oleoyl 2' hydroxy) glycerol (ammonium salt) (1,1 dioleoyl sn3:sn3) (Figure 1 12B) and the (S,S) isomer, sn (3 oleoyl 2 hydroxy) glycerol 1 phosphosn 3' (1' oleoyl 2' hydroxy) glycerol (ammoniu m salt) (3,3 dioleoyl sn1:sn1) (Figure 1 12C). The synthetic S, R isomer (sn3:sn1) was used in studies that were performed in this dissertation because of its commercial availability and is designated throughout this dissertation as BMP. Role of BMP i n the Late E ndosome Late endosomes are prelysosomal endocytic organelles that are defined by the time it takes for endocytosed macromolecules to be delivered to them (102) Late endosomes are usually loaded 430min after endocytic upt ake in mammalian cells, although the delivery time may vary between cell types and between cells in culture and in living tissue (103) Electron microscopy studies have revealed that late endosomes are more s pherical than early endosomes, being concentrated near the microtubule organizing center and having the appearance of multivesicular bodies (MVBs). They can be differentiated from early endosomes by their lower lumenal pH, different protein and lipid comp ositions and association with different small GTPases of the rab family (104) One of the most distinct features of late endosomes is the presence of internal membranes, hence the reason that late endosomes have also been referred to as m ultivesicular bodies (MVBs) or multivesicular endosomes (MVEs). The accumulation of internal membranes starts at the early endosome and is thought to continue as the
50 endosome `matures' to a late endosome. The lipid composition of late endosomes differs fro m that of earlier endocytic compartments, being enriched in triglycerides, cholesterol esters and selected phospholipids, including BMP (97) BMP is however found to be more heavily enriched in the internal membran es of mammalian late endosomes/MVBs than in the limiting membrane (44) Because each acyl chain of BMP contains an unsaturated site, this lipid may increase the overall bilayer disorder of model and endosomal membra nes due to intermolecular packing constraints (93) Although earlier studies postulated BMP to be highly hydrophobic and cone or i nvertedcone shaped (93, 97, 98) our studies have shown that BMP forms stable, lamellar vesicle structure s, implying a possible function in formation of MVBs (105) Because of its high level of accumulation in late endosomes (106) its unusual structure and stereochemistry, BMP is thought to play important roles in the late endosome, including structural integrity, endosome maturation, and lipid/protein sorting and trafficking (101, 1 06) For instance, because of its unusual structure, BMP is inefficiently degraded by phospholipases, leading to its accumulation in intraluminal vesicles of late endosomes (44) BMP is presumably synthesized in s itu within the acidic organelles of the endocytic pathway (107) Studies have also shown that accumulation of antibodies against BMP in the late endosomes, which are observed in patients suffering from antiphospholipid syndrome, inhibit multivesicular endosome formation, suggesting that BMP does play a role in this process ( 108) BMP may also be involved in cholesterol transport (44) possibly via formation of lateral microdomains. Moreover, BMP has been found to be essential for the activator stimulated hydrolysis of ganglioside GM 1 (109) and GM2 (110) and for the hydrolysis of
51 ceramide by acid ceramidase (111) It is unclear how BMP achieves these functions, but it is likely that the structure and biosynthesis of BMP, coupled to the change in pH during the maturation of the late endosome induces intraendosomal vesicular body formation. Overview of Biophysical Studies of BMP To elucidate the functional role of BMP in late endosomes, a series of studies have already been aimed at obtaining a more detailed understanding of the biophysical characteristics of this lipid. Holopainen et al studied the thermotropic behavior of pure dioleoyl BMP mono and bilayers using Langmuir lipid monolayers, elect ron microscopy, differential scanning calorimetry (DSC), and fluorescence spectroscopy. Holopainens group reported that BMP formed metastable, liquidexpanded monolayers at an air/buffer interface, and its compression isotherms lacked any indication for structural phase transitions. Furthermore, pure BMP formed multilamellar vesicles with no structural transitions or phas e transitions between 10 and 80 C at a pH range of 3.0 7.4 (93) Holopainens group also studied mixed BMP/dipalmitoylphosphatidylcholine (DPPC) bilayers by DSC and fluorescence spectroscopy and found that incorporating increasing amounts of BMP (up to XBMP (molar fraction) = 0.10) decreased the co operativity of the main transition for DPPC, and a decrease in the main phase transition as well as pretransition temperature of DPPC was observed but with no effect on the enthalpy of this transition. 1palmitoyl 2 oleoy l phosphatidylcholine (POPC)/LBPA mixed bilayers were found to be more fluid, and no evidence for lateral phase segregation was observed. These results were confirmed using fluorescence microscopy of Langmuir lipid films composed of POPC and BMP (up to XBM P= 0.50) with no evidence for lateral
52 phase separation. Because late endosomes are eminently acidic, they examined the effect of lowering pH on lateral organization of mixed PC/LBPA bilayers by DSC and fluorescence spectroscopy and reported that even at pH 3.0, there was no evidence of BMP induced microdomain formation at BMP contents found in cellular organelles (93) Kobayashis group reported that BMP can induce the formation of multivesicular liposomes that resemble the multivesicular endosomes where this lipid is found in vivo. They also reported that the formation of the multivesicular liposomes depends on the acyl chain composition of BMP and the position of esterification in the glycerophosphate backbone (98) Hayakawa et al reported that DOBMP membranes form a closely packed multilamellar structure at low pH by adding the hydrophobic amine, D threo 1 phenyl 2 decanoylamino3 morpholino1 propanol (D PDMP) (112) They also showed that this interaction between BMP and D PDMP is a major cause of the structural alteration of the degradative organelles found in D PDMP treated cells and the accumulation of cholesterol in these organelles (112, 113) A series of studies have also demonstrated that agents that perturb BMP induce alterations in the sorting and t rafficking of proteins and/or the receptor in late endosomes accompanied by structural changes of the late endosome organelle(44, 114) along with abnormal accumulation of cholesterol (108) BMP has also been shown to be an antigen in the antiphospholipid syndrome, a condition in which endosomal sorting and multivesicular endosome formation is disrupted (98) The trafficking defects ob served in the cholesterol storage disorder NiemannPick type C can be recapitulated by disruption of LBPA function(115, 116)
53 Sandhoff et al demonstrated that incorporation of BMP into liposomes containing glucos ylceramide greatly facilitates the degradation of the glycosphingolipid by glucosylcerebrosidase and sphingolipid activator protein C (117) BMP also facilitates the degradation of GM2 by hexosaminidase A and GM2a ctivator protein, as well as ceramide by acid ceramidase/SAP B and sphingomyelin by acid sphingomyelinase (46) BMP also functions in cholesterol efflux from late endosomes and lysosomes. If antibodies against BMP are internalized by fluidphase endocytosis, they bind to BMP and accumulate in late endosomes. Under these conditions, cholesterol released from low density lipoprotein (LDL) remains trapped in the late endosomes and cannot be transported out from this organelle as would normally occur if the antibody were absent. The network of membrane tubules and ves icles within the lumen of late endosomes might thus have an important function in sphingolipid degradation and cholesterol distribution in the cell. Accumulation of endocytosed antibodies against BMP also results in the defective sorting/trafficking of proteins that transit via late endosomes, presumably because membrane properties are altered (118) The function and maintenance of the highly curved membrane structures are still poorly understood, but obviously BMP membrane domains do contribute to the selectivity in handling of lipid rafts in the endocytic pathway. Alterations in these processes appear to cause not only accumulation of lipid rafts but also result in disturbances in protein traffic (113) Scope of Dissertation Because of the unique structure and stereochemistr y of BMP, our lab was interested in gaining a better understanding for the important functional roles that this lipid plays in the late endosome. In an effort to provide further insight on the morphology and sizes exhibited by BMP hydrated dispersions and extruded vesicles, dynamic light
54 scattering ( DLS ) and negative staining transmission electron microscopy ( TEM ) experiments were performed at various experimental conditions, including variations in lipid composition, pH conditions, ionic strength and lipi d concentrations. Chapter 1 has introduced a detailed background on lipids, with a focus on glycerophospholipids structure and function. Different model membrane systems were also discussed, with a special interest in lipid vesicles A detailed discussion on the unique structure and stereochemistry of BMP was also given, followed by its role in the late endosome and an overview of previous biophysical studies that have been performed on BMP. Chapter 2 gives a discussion of the major techniques that i utilized in analysis of the lipid dispersions and vesicles studied here with a focus on the particle sizing technique dynamic light scattering ( DLS ) and negative staining transmission electron microscopy ( TEM ) The chapter begins with a detailed discussion o n the theory of DLS followed by an overview of applications of DLS for particle sizing. Theory of transmission electron microscopy and its applications for biological studies is also given. A discussion on FRET, which was utilized for monitoring leakage o f fluorophoreencapsulated vesicles, is also presented in detail, and lastly, a general overview of two chromatographic techniques, TLC and column chromatography are also presented. TLC was used to test for lipid integrity, whereas column chromatography was utilized for the separation of fluorophore encapsulated vesicles from free fluorophore. Chapter 3 discusses the characterization of the morphology and size distribution observed in hydrated lipid dispersions and extruded lipid vesicles of BMP, POPC, POPG and lipid mixtures of POPC:BMP and POPC:POPG using DLS and negative
55 staining TEM To test the analytical capability of DLS, i nitial control experiments were performed to test different populations of POPC vesicles extruded using 30, 100 or 400 nm pore si ze extrusion membranes using DLS To assess the stability of BMP vesicles (stability defined as the ability of the vesicles to maintain the same size distribution over time), assays were performed in which BMP, POPC and POPG vesicles were mechanically pass ed through 30 nm pore size extrusion membranes and the size of the vesicle suspensions (stored at room temperature) were monitored by DLS measurements over a five week period. Experiments were also performed t o confirm that BMP forms lamellar vesicles with an interior volume. To this end, vesicle leakage assays were performed using fluorescence resonance energy transfer (FRET ), and the percent release of the encapsulated fluorophore monitored as a function of increasing concentrations of titrated sodium dodecyl sulfate (SDS) detergent. In Chapter 4, studies on the effect of introducing ganglioside GM1 in BMP dispersions at varying concentrations, under late endosomal pH (5.5) conditions are discussed Dynamic light scattering and negative staining transmission electron microscopy were utilized to monitor the morphological and structural characteristics in hydrated dispersions of BMP: GM1 lipid mixtures. Other investigations in Chapter 4 included the characterization of the morphology and size distribution of BMP hydrated lipid dispersions under different pH conditions, ranging from acidic (pH 4.2) to neutral (pH 7.4) conditions, using negative staining TEM and DLS. S tudies were also performed that monitored the effect of incorporating GM1 and BMP in typical phosphatidylcholine (POPC) membranes. Because POPC is abundantly present in biological cellular membranes, it has been well characterized and studied as a typical
56 model system. By utilizing dynamic light scattering and transmission emission spectroscopy, the morphology and size distribution of POPC:GM1 and POPC:BMP: GM1 hydrated dispersions and extruded vesicles were investigated. The s tudies presented in Chapter 5 summarize the use of dynamic light scattering and negative staining transmission electron micros copy to characterize the vesicle size and macroscopic morphologies observed when cholesterol (CHOL) is incorporated in lipid mixtures of POPC, BMP and ganglioside GM1. These experiments were performed at pH 5.5, in efforts to mimic the late endosome environment. Finally, Chapter 6 provides a summary of the major findings from this dissertation and a brief discussion of some future perspectives including the possibility of using BMP vesicles for pharmaceutical applications .
57 C HAPTER 2 TECHNIQUES UTILIZED IN LIPID ANALYSIS Dynamic Light Scattering (DLS) Theory D ynamic l ight scattering (DLS), is a powerful noninvasive nondestructive light scattering technique used for characterizing the properties of macromolecular particles in suspensions, solutions of coll oids and polymers in solution (119) DLS is also commonly referred to as p hoton correlation s pectroscopy (PCS) or q uasi elastic l ight scattering (QELS) QELS was the first name given to the techni que because when photons are scattered by mobile particles, the process is quasi elastic. QELS measurements yield information on the dynamics of the scatterer, whi ch gave rise to the acronym DLS. T he acronym DLS will be employed throughout this dissertati on because its use has become more prevalent and it presents a logical juxtaposition to static light scattering (SLS) (120) The application of DLS to particle sizing and its commercialization occurred about seven years after the first size measurements were made in 1972, to check the alignment of a multi angle research light scattering system (121) T hrough the 1970s, DLS gained wide acceptance among experts in light scattering and to date it is a widely accepted technique for broad applications in particle sizing The main focus in this section will be on the theory and application of DLS for particle sizing which relates to the translational motion (diffusion) of particles in liquids. By nature, all materials scatter and absorb li ght and since the first light scattering experiments by Tyndall (122) static light scattering experienced major developments in th e first half of the 19th century (123) The dynamic light scattering theory is built upon
58 the earlier foundation of classical light scattering theory, which is based on two light scattering theories: Rayleigh scattering and Mie scattering (120) In Rayleigh scattering, i f the particles are small compared to the wavelength of the laser used ( which is typically less than d = or ~ 60 nm for a He Ne laser), then the scattering from a particle illuminated by a verticall y polarized laser will be essentially isotropic, i.e. equal in all directions. The Rayleigh approximation is represented in Equations 21 and 22 I d6 (2 1 ) 4 (2 2 ) w here is the intensity of scattered light, d is the particl e diameter and is the laser wavelength (119) The d6 term means that a 50 nm particle will scatter 106 or one million times as much light as a 5 nm particle; h ence there is the danger that the lig ht from the larger particles will swamp the scattered light from the smaller ones. This d6 factor also means that it may be difficult with DLS to measure for instance a mixture of 1000 nm and 10 nm particles because the contribution to the total light scat tered by the small particles will be extremely small. The inverse 4 means that a higher scattering intensity is obtained as the wavelength of the laser used decreases (119, 124) In Mie scattering, the size of the particles becomes roughly equivalent to the wavelength of the illuminating light giving the complete solution for spherical particles of any size. The use of experimental DLS is thought to have beg un with the advent of the laser. In the early 1960s Pecora (125) pioneered a new kind of light scattering: time dependent light scat tering. He showed that, by analyzing the frequency distribution of
59 the intensity fluctuations of light scattered from suspensions of macromolecules, information can be obtained about the translational and rotational diffusion coefficients of the macromolec ules (120) Initially DLS was used to measure the diffusion coefficient of macromolecules, from which a hydrodynamic size was calculated. It is thought that a few industrial users tried this technique for submicron particle s izing in an effort to replace transmission electron microscope (TEM) measurements in quality control (QC) applications. During the second half of the 1970s improvement of digital correlators and the introduction of several algorithms for analyzing decay time distributions were realized. Measurements could now be done in shorter times Bertero and group (126, 127) published a fundamental paper in 1984 that derived the limiting conditions for the resolution of the "noisy" sum of an unknown number of exponentials based on information theory. The advent of highly efficient nonlinearly spaced correlators led to an increase in the utility of DLS particle sizing in the mi d 1980s, and the emerging fast computers speeded up data handling (120) Fundamentals of DLS D ynamic light scattering measures Brownian motion and relates it to the size of the particles. Brownian motion is the random movement of particles due to collisions with the solvent molecules that surround them. In DLS, t he larger the particle, the slower the Brownian motion, hence s maller particles are kicked farther by collisions with the solvent molecules and move more rapidly th an larger particles B ecause the viscosity of a liquid is related to its temperature, it is necessary to know the temperature of the liquid. The temperature also needs to be stable in order to minimize convection
60 currents in the sample, which would cause n on random movements that can ruin the correct interpretation of size (119, 124) The velocity of the Brownian motion is defined by a property known as the translational diffusion coefficient given by th e symbol D The size of the particle is calculated from the translational diffusion coefficient using the Stokes Einstein equation in Equation 23. d (H (2 3) w here d ( H) is the hydrodynamic diameter D is the translational diffusion coefficient k is Boltzmanns constant T is the absolute temperature and is the liquid viscosity T he diameter that is measured in DLS is a value that refers to how a particle diffuses within a fluid, so it is referred to as a hydrodynamic diameter. Additionally, t he diameter that is obtained by thi s technique is assumed to be t he diameter of a sphere that has the same translational diffusion coefficient as the particle. Factors that affect the translational diffusion coefficient The translational diffusion coefficient will depend not only on the size of the particle core, but also on any surface structure, the concentration and type of ions in the medium and the shape of the particles (i) Ionic strength of medium The ions in the medium and the total ionic concentratio n can affect the particle diffusion speed by changing the thickness of the electric double layer called the Debye length (K1) (119, 128) This implies that a low conductivity medium will produce an extended double layer of ions around the particle, reducing the diffusion speed and resulting in a larger, apparent hydrodynamic diameter. Conversely, higher conductivity
61 media will suppress the electrical double layer and the measured hydrodynamic diameter will be reduced Suitable polystyrene latex standards are used to verify the performance of a DLS instrument The International Standard on DLS advises that dilution of any polystyrene standard should be made in 10 mM NaCl, so that at this conce ntration of salt, the ele ctrical double layer will be suppressed enough and the hydrodynamic diameter reported will be the same as the hydrodynamic diameter on the certificate or the expected diameter (129) (ii) Surface s tructure Any changes to the surface of a particle that affects the diffusion speed will also change the apparent size of the particle. For instance, an adsorbed polymer layer projecting out into the medium will reduce the diffusion speed more than if the polymer was lying flat on the surface. The nature of the surface and the polymer, as well as the ionic concentration of the medium can affect the polymer conformation, which in turn can change the apparent size by several nanometers (130) (iii) Non spherical particles A n inherent problem experienced with all particle sizing techniques lies in describing the size of non spherical particles. The sphere is the only object whose size can be unambiguously described by a single figure. Different techniques are sensitive to differ ent properties of a particle such as projected area, density, scattering intensity, and in general will produce different mean sizes and size distributions for any given sample. The hydrodynamic diameter of a non spherical particle is the diameter of a sphere that has the same translational diffusion speed as the particle (119, 131) If the shape of a particle changes in a way that affects the diffusion speed, then the
62 hydrodynamic size will change. For example, small changes in the length of a rodshaped particle will directly affect t he size, whereas changes in the rods diameter, which will hardly affect the diffusion speed, will be difficult to detect. The conformation of proteins and macromolecules are usually more dependent on the exact nature of the dispersing medium. As conformat ional changes will usually affect the diffusion speed, DLS is a very sensitive technique for detecting these changes (119, 124, 132) Basis of i n tensity f luctuations in DLS The main concept in DLS is that the diffus ion of th e scatterers causes the phases of the fields scattered from each of them to change with time, so that the total scattered intensity will fluctuate with time owing to constructive and destructive interference (120) E ssentially, t he use of DLS to determine particle s ize is based on the measurement of the diffusion coefficients of suspended particles undergoing Brownian motion via the autocorrelation of the time dependence of the scattered light. This is done by measuri ng the rate at which the intensity of the scattered light fluctuates when detected using a suitable optical arrangement. To determine how these fluctuations in the intensity of scattered light arise, imagine if a cuvette, containing particles which are st ationary, is illuminated by a laser and a frosted glass screen is used to view the sample cell. A classical speckle pattern would be observed, as illustrated in Figure 21
63 Laser Sample cell Incident beam Screen Speckle pattern Figure 21. Schematic representation of a speckle pattern observed in DLS. Adapt ed from Ref. (119) The speckle pattern will be stationary both in speckle size and position because the whole system is stationary. The dark spaces are where the phase additions of the scattered li ght are mutually destructive and cancel each other out (F igure 2 2A), while t he bright blobs of light in the speckle pattern are where the light scattered from the particles arrives with the same phase and interfere constructively to form a bright patch ( F igure 2 2B ).
64 Figure 2 2. A) D estructive interference and B) c onstructive interference observed in a speckle pattern. For a system of particles undergoing Brownian motion, a speckle pattern is observed where the position of each speckle is seen to be in constant motion. This is because the phase addition from the moving particles is constantly evolving and forming new patterns. The rate at which these intensity fluctuations occur will depend on the size of the particles.
65 Large particles Small particles Intensity (au) Intensity (au)B A Time Time Figure 23. Typical intensity fl uctuations for A) large particles and B) small particles. Figure 2 3 schematically illustrates typical intensity fluctuations arising from a dispersion of large particles ( A) and a dispersion of small particles (B) The small particles cause the intensity to fluctuate more rapidly than the large ones. While it may be possible to directly measure the spectrum of frequencies contained in the intensity fluctuations arising from the Brownian motion of particles, it is quite inefficient to do so, and t he best w ay is to use a device called a digital auto correlator.
66 How the digital autocorrelator works A digital correlator is basically a device that compares signal s. It is designed to measure the degree of similarity between two signals, or one signal with itself at varying time intervals. If the intensity of a signal is compared with itself at a particular point in time and at another time much later, then for a randomly fluctuating signal it is obvious that the intensities and correlation between the two signal s are going to vary over time, as shown in Figure 24 Intensity0 t t 2 t 3 t 4 t Time Figure 24. Schematic showing the fluctuation in the intensity of scattered light as a function of time. Adapted from Ref. (119) Knowledge of the initial signal intensity will not necessarily allow the signal intensity at time t = infinity ( ) to be predicted, which is true of any random process such as diffusion. However, if the intensity of the signal at time = t is compared to the
67 intensit y a very short time later ( such as t+ ) there will be a strong relationship or correlation between the intensities of two signals, hence t he two signals are said to be strongly or well correlated. If the signal derived from a random p rocess such as Brown ian motion at t there will still be a reasonable comparison or correlation between the two signals, but it will not be as good as the correlation t, which implies that t he correlation reduc es with time. The period small, on a scale of nanoseconds to microseconds and is called the sample time of the correlator. The t = time maybe on the order of a millisecond or tens of milliseconds. If the signal intensity at t is compared with i tself then there is perfect correlation as the signals are identical. Perfect correlation is indicated by unity (1.00) and lack of any correlation is indicated by zero (0.00). and so on are compared with the signal at t, the correlation of a signal arriving from a random source will decrease with time unti l at some time, when t = there will be no correlation. Additionally, for large particles, the signal changes slowly and the correlation remains strong for a long t ime (F igure 2 5A ) whereas for small particles that are moving rapidly the correlation reduce s more quickly (Figure 25B ).
68 A Time (s)Correlation coefficient B Time (s)Correlation coefficient Figure 25. Correlation sp ectrum for a sample containing A) large particles with long dec ay time and B) small particles with rapi d decay time. Essentially, v iewing the correl ation spectrum from a measurement can give a lot of information about the sample. For instance, t he time at which the correlation starts to significantly decay is an indication of the mean size of the sample, an d t he steeper the line, the more monodisperse the sample while the mor e extended the decay, the greater the sample polydispersity. The auto correlation f unction (ACF) We have so far established that particles in a liquid dispersion are in a constant, rando m Brownian motion, which causes the intensity of scattered light to fluctuate as a function of time. The digital autocorrelator u tilized in a DLS instrument will then construct the correlation function G related to t ime as shown in Equation 24
69 G )> (2 4 ) w h is the time difference (the sample time) of the correlator. For a large number of monodisperse particles in Brownian motion, the correlatio n function (given the symbol ) is an exponential decaying function of the correlator 2 5 G [1 + B exp ( ( 2 5 ) where A is the baseline of the correlation function and B is the intercept of th e correlation 6. 2 ( 2 6 ) wh ere D is the translational diffusion coefficient which can be obtained using the Stokes Einstein relationship in Equation 2 8 q is a factor defined by the relati onship in Equation 27. 0 (2 7) 0 is the wavelength of the laser and the scattering angle. ( 2 8 ) where k is Boltzmanns constant, T is temperature, is viscosity and d (H) is the hydrodynamic diameter of the particles For polydisperse samples, the equat ion for the correlation function can be described as shown in Equation 29. G [1 + B g1 ) 2 (2 9 ) where g1 is the sum of all t he exponential decays contained in the correlation function.
70 The particle s ize and size distribution can be obtained from the correlation function by u tilizing two established algorithms : (1) to fit a single exponential to the correlation function to obtain the mean size (z average diameter) and an estimate of the width of the distribution (polydispersity index) (this is called the Cumulants analysis), or (2) to fit a multiple exponential to the correlation function to obtain the distribution of particle sizes (such as Non negative least squares (NNLS) or CONTIN program) The size distribution obtained is a plot of the relative intensity of light scattered by particles in various size classes and is therefore known as an intensity size distribution. If the distribution by intensity is a single fairly smooth peak, then there is no point in doing the conversion to a volume distribution using the Mie theory. If the optical parameters are correct, this will only provide a slightly different shaped peak. However if the plot shows a substantial tail, or more than one peak, then Mie theory can make use of the input parameter of sample refractive index to convert the intensity distribution to a volume distribution. This will then give a more realistic view of the i mportance of the tail or second peak present. In general terms it will be seen that d (intensity) > d (volume) > d (number) (120) A very simple way of describing the difference between intensity, volume and number distribut ions is to consider two populations of spherical particles of diameter 5 nm and 50 nm present in equal numbers as illustrated in Figure 26
71 Figure 26. Number, volume and intensity distributions of a bimodal mixture of 5 and 50 nm lattices present in equal numbers. A) Number distributions. B) Volume distributions. C) Intensity distributions Modified from Ref. (119) If a number distribution of these two particle populations is plotted, a plot consisting of 2 peaks (positioned at 5 and 50 nm) of a 1 to 1 ratio would be obtained. If this number distribution was converted into volume, then the 2 peaks would change to a 1:103 ratio ( given that (d/2)3). If this was further converted into an intensity distribution, a 1: 106 ratio between the two peaks would be obtained, given that the intensity of scattering is proportional to d6, from Rayleigh s approximati on ). Notably, in DLS, the distribution obtained from a measurement is based on intensity (119, 128) Instrumental design for DLS Generally, a typical DLS system should include four major functional components : a lig ht source, an optical system, a detector system and a digital autocorrelator as indicated in Figure 27
72 Solid state laser Focusing lens Cuvette, with dispersions Coherence optics Detector Autocorrelator Computer readout Figure 27 Typical e xperimental set up in a dynamic light scattering instrument (i) The light s ource Practical requirements for a sufficiently inte nse light source demand a narrow band, polarized, monochromatic, continuous wave ( CW ) laser. Table 21 summarizes the popular options available for light sources (120) The DLS instrument utilized for our studies ( Brookhaven 90Plus Nanoparticle Analyzer) uses a solid state laser, operated at a wavelength of 635 nm Table 21. Types of lasers commonly used in DLS instruments. Laser Type Wavelength Power Size HeNe 632.8 nm 5 35 mW 0.4 1.5 m Laser diodes 635780 nm 5 100 m W 0.05 0.14 m Ar+(air cooled) 488 514.5 nm ~100 mW 1m Ar+(water cooled) 488 514.5 nm ~1.7 W 1.5 2 m DPSS 532 nm 10 mW 4W 0.2 0.5 m DPSS, diode pumped solidstate lasers Adapted from Ref. (120)
73 (ii) Optical s ystem A lens focuses the laser beam into the sample, which is enclosed in an optional temperaturecontrolled scattering cell surrounded by a refractive index matching liquid. The scattered light is focused ont o a photomultiplier tube ( PMT ) by another lens. Such systems a re best constructed on a precision turntable with a stepper motor, and typically allow experiments to be conducted over a 10 160 angular range. (iii) Detector s ystem Photomultiplier tubes ( PMT s) are almost universally used as detectors in DLS instruments. The PMT should have a low dark count and a high gain since most work is done in th e single photon counting regime (iii) The digital autoc orrelator The correlator has become the device of choi ce for generating raw data in a DLS experiment, although earlier experiments mostly employed "wave analyzers" or "spectrum analyzers". The autocorrelation function ( ACF ) is formed by recording the number of photons arriving in each sample time, maintaining a history of this signal over a large range of sample times (time series), multiplying the instantaneous and the delayed signal for a range of time delays td (the "channels") and accumulating these products. The BI 9000AT autocorrelator employed in our st udies is an entirely digital, high speed, signal processor which can be us ed as an auto or c ro ss correlator for dynamic light scattering (DLS) measurements and as a photon counter for static light scattering measurements (SLS). DLS Applications The major ity of applications for DLS in particle sizing include the rapid and routine measurements of mean sizes of particles in quality control and research laboratories
74 Manufacturers of latexes, pigments, emulsions, micelles, liposomes, vesicles, sils and silica can track the consistency of the desired particle sizes rapidly and accurately, independent of different operators and different instruments making DLS very versatile. Samples that tend to coagulate can also be easily tracked by DLS. Although the exact size distribution cannot be determined, the correlation will be very sensitive enough to any changes in the distribution and give a fairly accurate response. DLS based particle sizing is also widely used for biological samples such as bacteria, viruses, proteins, DNA and so on. Many applications have also been performed in crystal growth and polymer research. Whereas there is a currently a huge volume of studies that have employed DLS, some of the more specific earlier examples of DLS applications include t he spontaneous vesicle formation in a biological surfactant (ganglioside GM3) that was investigated by Cantu and group (133) while s tudies of BSA and lysozyme, two very low molecular weight proteins, were conducted by Dh adwal and group in 1993 (134) Studies of pro tein (lysozyme) crystallization (135) s ubmicron emulsion systems (136) and use of a fiber optic backscattering device to observe particleparticle interaction in highly concentrated latex suspension (137) have all utilized DLS A widely used industrial process, hydro metallurgi cal solvent extraction, was investigated by Neuman and group (138) in which they applied the DLS technique to very small sizes of ~ 2 nm. Caldwells group (139) investigated emulsions by employing DLS and sedi mentation field flow fractionation (SFFF) methods in a complementary mode. SFFF provides for a high resolution fractionation of the sample, whereas DLS measures the sizes without the need to know the density of the sample.
75 As any with other particle sizing technique, DLS has advantages and disadvantages which must be kept in mind in trying to use it for different applications. Advantages of DLS Some of the desirable features of the DLS technique include: Fast m easurements from seconds to minutes. technique is absolute, from first principles, and c alibration with a known size distribution is not necessary. sample quantities can be measured (sample volume: ) absorbing, relatively clear and not too viscous. to a broad range of particle sizes from about 2 nm to 6 m. ilable for both research and QC measurements with automation including data analysis. s least ambiguous with a narrow distribution, an effective diameter and polydispersity index are measurable even with broad distributions. Disadvantages of DLS DLS does not produce a highresolution histogram of the size distribution. imaging techniques an equivalent sphere diameter is usually, although not always, assumed of all the particles, and as such information on the shape of the particles cannot be easily obtained.
76 particles in the sample can make measurement and interpretation of data quite difficult (120) The research present ed in this dissertation utilized DLS as the main particle sizing technique, to measure the size and size distribution of liposomes/lipid vesicles suspended in different buffer solutions. The instrument used was a Brookhaven 90PLUS Nanoparticle Analyzer (Brookhaven Instruments Corporation, Holtsville, NY), which has broad range sizing capabilities of 2 nm to 6 m. Transmission Electron Microscopy (TEM) Theory Transmission electron microscopy (TEM) is a n established microscopy technique in which a beam of electrons is transmitted through an ultra thin specimen, interacting with the specimen as it passes through. An image is formed from the interaction of the electrons transmitted through the specimen; the image is then magnified and focused onto an imaging device such as a fluorescent scr een, a layer of photographic film, or detected by a sensor such as a CCD camera. TEM instruments have the desirable capability of imaging at a significantly higher resolution than light microscopes, owing to the small de Broglie wavelength of electrons. Th is makes it possible for the user to examine fine detail s of the specimen, which can be as sma ll as a single column of atoms, making it thousands of times smaller than the smallest resolvable obj ect in a light microscope. TEM is a major analysis method in a range of scientific fields both in the physical and biological sciences, including applications in cancer research, virology, materials science as well as pollution and semiconductor research.
77 At smaller magnifications TEM image contrast is due to absor ption of electrons in the material, due to the thickness and composition of the material. At higher magnifications however, complex wave interactions modulate the intensity of the image, requiring expert analysis of observed images. Alternate modes of use allow for the TEM instrument to observe modulations in chemical identity, crystal orientation, electronic structure and sample induced electron phase shift as well as the regular absorption based imaging (140, 141) The first TEM instrume nt was developed by Max Knoll and Ernst Ruska in 1931, and the same group developed the first TEM with resolving power greater than that of light in 1933, and the first commercial TEM became available in 1939 (142) Theoret ically, the maximum resolution ( d ) that one can obtain with a light microscope i s limited by the wavelength of the photons that are being used to probe the sample, and the numerical aperture (NA) of the system (141) as shown in Equation 2 10. d = / 2 NA ( 2 10) To overcome the limitations of the relatively large wavelength of visible light ( s of 400 700 n m ) early twentieth century scientists theorized the use of electr ons. Like all matter, electrons have both wave and particle properties (as shown by Louis Victor de Broglie), and the wave like properties imply that a beam of electrons can be made to behave like a beam of electromagnetic radiation. The wavelength of elec trons is found by equating the de Broglie equation to the kinetic energy of an electron. An additional correction must be made to account for relativistic effects, such as in a TEM instrument, a n electron's velocity approaching the speed of light, C shown in Equation 211 (143)
78 [ 2m0 E (1+ E/2m0C2 (2 11) w here h is Planck's constant ( 6.626068 1034 m2 kg / s ) m0 is the rest mass of an electron and E is energy of the accelerated electron. Electrons are usually generated in an electron microscope by a process known as thermionic emission from a filament, usually tungsten, in the same manner as a light bulb, or alternatively by field electron emission (144) The electrons are then accelerated by an electric potential that is measured in volts and focused by electrostatic and electromagnetic lenses onto the sample. The transmitted beam contains information about electron density, phase and periodicity and this beam is used to form an image. The general experimental layout of a TEM instrument is illustrated in Figure 2 8. Electron gun Anode Condenser lens Specimen Objective aperture lens Intermediate lens Projector lens Fluorescent screen Figure 28 Layout of major components in a basic transmission electron microscopy ( TEM ) instrument A typical TEM instrument consists of several important components, including an electron emission source for generation of the electron stream (electron gun), a vacuum system in which the electrons travel, a series of electromagnetic lenses and
79 electrostatic plates that allow for guiding and manipulation of the beam as required, imaging devices to create an i mage from the electrons that exit the system and a specimen holder. By connecting the electron gun to a high voltage source (typically ~100300 kV) the gun will, with sufficient current, emit electrons by thermionic emission from tungsten hairpin cathodes, or LaB6 rods or by field emission from pointed tungsten filaments (140) Once extracted, the upper lenses of the TEM allow for the formation of the electron probe to the desired size and location for later interaction with the sample (145) Manipulation of the electron beam can be performed using two physical effects. The interaction of electrons with a magnetic field will cause electrons to move according to the right hand rule, thus allowing for electromagnets to manipulate the electron beam. The use of magnetic fields allows for the formation of a magnetic lens of variable focusing power, in which the lens shape originat es due to the distribution of magne tic flux. Alternatively electrostatic fields can cause the electrons to be deflected through a constant angle. Coupling of two deflections in opposing directions with a small intermediate gap allows for the formation of a shift in the beam path, which is mostly used in TEM for beam shifting (140, 145) The lenses in a TEM allow for beam convergence. T he angle of convergence can be varied, which gives the TEM the ability to change magnification simply by modifying t he amount of current that flows through the coil, quadrupole or hexapole lenses. The quadrupole lens is an arrangement of electromagnetic coils at the vertices of the square, enabling the generation of lensing magnetic fields, while the hexapole configurat ion enhances the lens symmetry by using six, rather than four coils. Typically
80 the optics in a TEM con sist of a threestage lens system: the condense r lenses, the objective lenses, and the projector lenses as shown in Figure 28 The condense r lenses are responsible for primary beam formation, while the objective lenses focus the beam down onto the sample itself. The projector lenses are used to expand the beam onto the phosphor screen or other imaging device, such as film. The magnifications attainable i n a TEM are due to the ratio of the distances between the specimen and the objective lens' image plane (140) Additional quad or h exapole lenses allow for the correction of asymmetrical beam distortions, known as astigmatism. It is worth noting that TEM optical configurations differ sig nificantly with implementation and different manufacturers using custom lens configurations, such as in spherical ab erration corrected instruments, or TEMs utilizing energy filtering to correct electron chromatic aberration (145) Apertures are annular metallic plates, through which electrons that are further than a fixed distance from the optic al axis may be excluded. These ap ertures consist of a small metallic disc that is sufficiently thick to prevent electrons from passing through the disc, while permitting axial electrons. A pertures are important because they decrease the beam intensity as electrons are fi ltered from the be am, which is particularly desirable f or beam sensitive samples. The filtering process by apertures also removes electrons tha t are scattered to high angles due to unwanted processes such as spherical or chromatic aberration, or due to diffraction from interaction within the sample (146) Apertures devices can either be fixed within the column, such as at the condenser lens, or are a movable aperture, which can be inserted, withdrawn from the beam path, or moved in the plane perpendicular to the beam path.
81 Imaging systems in a TEM consist of a phosphor screen, which may be made of fine (10ct observation by the operator, or an image recording system such as film based or doped Yttrium aluminum garnet (YAG, Y3Al5O12) screen coupled CCDs (147) T hese devices can be removed or inserted into the beam path by the operator as required. Contrast formation in the TEM depends greatly on the mode of operation. Complex imaging techniques, which utilize the unique ability to change lens strength or to deactivate a lens, allow for many operating modes. The most common mode of operation for a TEM is the bright field imaging mode, in which the contrast formation, when considered classically, is formed directly by occlusion and absorption of electrons in the sample. Thicker regions of the sample or regions with a higher atomic number will appear dark, while regions with no sample in th e beam path will appear bright, hence the term "bright field". The image is effect ive ly assumed to be a simple two dimensional projection of the sample down the optic al axis, and to a f irst approximation may be model ed via Beer's law (144) The need for a vacuum system in TEM To increase the mean free path of the electrongas interaction, a standard TEM is evacuated to low pressures, typically on the order of 10 Pascal (147) which serves two purposes. F irst this allow s for the voltage diff erence between the cathode and the ground without generating an arc, and secondly it reduce s the collision frequency of electrons with gas atoms to negligible levels, an effect that is characterized by the mean free path. For these reasons, TEM specimen holders and film cartridges are routinely inserted or replaced to allow for a system that has the ability to reevacuate on a regular basis. Consequently, TEMs are equipped with multiple pumping systems and airlocks and are not permanently vacuum sealed (148) High voltage TEMs require ultra high
82 vacuums on the range of 10 to 10 Pa to prevent generation of an electrical arc, particularly at the TEM cathode (149) Poor vacuum in a TEM can result in several problems, including deposition of gas inside the TEM onto the specimen as it is being viewed through a process known as electron beam induced deposition, or in more severe cases damage to the cathode from an electrical discharge (149) TEM specimen stage designs include airlocks to allow for insertion of the specimen holder into the vacuum with minimal increase in pressure in other areas of the microscope. The specimen holders are adapted to hold a standard size of grid upon which the sample is placed or a standard size of self supporting specimen. Standard TEM grid s h ave are 3.05 mm diameter ring, with a thickness and mesh size ranging sample is placed onto the inner meshed area with a diameter of ~ 2.5 mm. Ideal grid materials are copper, molybdenum, gold or platinum. Th e grid is placed into the sample holder which is paired with the specimen stage (140, 147) Once inserted into a TEM, the sample often has to be manipulated to present the region of interest to the beam, such as in single grain diffraction, in a specific orientation. To accommodate this, the TEM stage allows for the translation of the sample in the XY plane of the sample, for Z hei ght adjustment of the sample holder, and usually for at least one rotation degree of freedom for the sample. Thus a TEM stage may provide four degrees of freedom for the motion of the specimen. Most modern TEMs provide the ability for two orthogonal rotati on angles of movement (140) with specialized holder designs called doubletilt sample holders. The design criteria of TEM
83 stages a re complex, owing to the simultaneous requirements of mechanical and electronoptical constraints and have thus generated many unique implementations. Sample preparation methods in TEM Sample preparation for TEM specimens can pose several challenges. Ideal ly, a ny specimen preparation techniques for TEM must avoid the collapse or loss of specimen structures during preparation and observation, since the specimens are viewed in a vacuum (150) Additionally, biological specimens are extremely sensitive to bombardment by electrons, which is a significant factor in the high noise levels of electron micrographs (151, 152) The incident electrons lose large amounts of energy by inelastic collisions, forming highly reactive ions and free radicals, which may disrupt bonds and fragment molecules in the specimen Incident electrons can also directly transfer their momentum to atomic nuclei in the structure, resulting in atom displacement. T he atoms that are present in biological molecules ( such as C, H, O, N, etc.) scatter electrons weakly, produci ng images with low intrinsic contrast, and so in order to increase the attainable resolution, electronbeam damage to the specimen should be minimized whereas image contrast should be maximized (140) TEM specimens are also required to be only a few hundreds of nanometers thick. High quality samples will have a thickness that is comparable to the mean free path of the electrons t hat travel through the samples, which may be only a few tens of nanometers. Consequently, preparation of TEM specimens is specific to the material under analysis and the desired information needed from the specimen, hence many generic techniques have been used for the preparation of the required thin sections (141)
84 Material s that have dimensions small enough to be electron transparent, such as powders or nanotubes, can be quickly prepared by the deposition of a dilute sample containing the specimen onto support grids or films. For biological science applications, biological specimens can be fixated using either a negative staining material such as uranyl acetate or by plastic embedding in order to withstand the instrument vacuum and facilitate handling S amples may also be fast frozen and held at liquid nitrogen temperatures after embedding in vitreous ice (153) In material science and metallurgy the specimens tend to be naturally resistant to vacuum, but still must be prepared as a thin foil, or etched so that some portion of the specimen is thin enough for the beam to penetrate. Constraints on the thickness of the material may be limited by the scattering crosssection of the atoms from which the material is comprised (150) Negative staining The TEM studies performed in this dissertation employed negative staining as a preparative method for lipid ve sicles. Just like in light microscopy where samples can be enhance d by stains that absorb light, TEM samples of biological tissues can also utilize heavy metals with high atomic numbers as stains to enhance contrast. The stain absorbs electrons or scatters part of the electron beam which otherwise is projected onto the imaging system. Compounds of heavy metals such as osmium, lead, or uranium have been used for negative staining TEM studies to selectively deposit electrondense atoms in or on the sample in desired cellular or protein regions. Although negative staining is technically simple to perform, provides high contrast and has a low sensitivity to the electron beam (150, 151) it still suffers from several disadvantages. For instance, i t only shows surface detail, with th e distribution of the heavy metal atoms rather than the density of the specimen being revealed. (154)
85 N egative staining TEM also has limited resolution of 1 2 nm because of stain movement during imaging (151, 155) To overcome some of these limitations, negative staining is sometimes complemented with electron cryo microscopy ( or cryo electron microscopy, cryo EM ) a technique that has revolutionized the analysis of m acromolecular structure by electron microscopy and 3D reconstruction studies (150, 152, 156) In cryo EM, samples are embedded in vitreous ice on a holey carbon grid and maintained at low temperatures (100 113 K) whil e under the electron beam (156) Vitreous ice is a super cooled liquid that is produced when water is very rapidly cooled below 273 K (at about 105 Ks1) (150) which avoids damage to the specimen by ice crystal formation. Vitreous ice forms a structureless medium in which th e molecules are hydrated, despite the requirement for vacuum conditions T he low temperatures result in a very slow rate of sublimation of the ice, and t he specimen is therefore imaged under conditions that are a lot like those in its native environment. The low temperature and the use of low doses of electrons minimize beam damage (157) TEM Applications Transmission electron microscopy is a well establi shed imaging technique for applications in both the physical and biological sciences due to its capabilities for high magnification and resolution. In the biomedical field, TEM has proved rewarding as a diagnostic tool, especially in basic and clinical vi rology. Since it was first utilized in the rapid diagnosis of smallpox, TEM developed to a diagnostic routine in the early 1960s, utiliz ing negative staining for sample preparation (158) A routine procedure for negative staining was first developed for the ex amination of virus particles (159, 160) and was
86 subsequently applied to sub cellular fractions (161) oth er cell membrane preparati ons (162) and artificial membrane systems (163) TEM has since been versatile in providing structural and morphological information of biomembranes such as proteins and lipids. For instance, electron microscopy is very useful as an assay for the purity and homogeneity of preparations of oligomeric proteins because it permits one to distinguish between different assembly forms and nonspecific aggregates that all may be present within the same preparation, and which may be difficult to discriminate by other means (164) More importantly, negative staining TEM has provided a means by which aq ueous suspensions of phospholipids in a liposomal state can be characterized as unilamellar and multi lamellar systems of varying dimensions (55) It has been demonstrated that TEM applied to negatively stained or freeze fractured liposomes constitutes an appropriate method to study the formation and morphology of liposome structures and their interaction with biological tissue (165) These techniques have also been shown to be very convenient in the characteriz ation of the intermediate aggregates formed during the interaction of different surfactants with liposomes (166 168) Negative staining TEM imaging was utilized in the studies presented in this dissertation, to char acterize the structural morphology and size analysis of hydrated dispersions and extruded vesicles of phospholipid membranes. Negative staining t ransmission electron microscopy (TEM) images were obtained using a Hitachi H 7000 transmission electron microsc ope operated at 75100 kV with a SoftImaging System MegaViewIII with AnalySIS digital camera (Lakewood, CO). Further details of the TEM
87 instrument and specific sa mple preparation procedures using negative staining are presented in the experimental section o f Chapters 3, 4 and 5. Fluorescence Resonance Energy Transfer (FRET) Theory Fluorescence or F a fluorophore (the donor) in an excited state transfers its energy to a neighboring molecule (the acceptor) by nonradiative dipoledipole interactions, and although not necessary, in most cases the acceptor is also a fluorescent dye (169, 170) FRET can be an accurate measurement of molecular proximity at angstrom distances (10 100 ), and highly efficient if the donor and acceptor are positioned within the F (the distance at which half the excitation energy of the donor is transferred to the acceptor, typically 3 6 nm). Consequently, FRET measurements can be utilized as an effective molecular ruler for determining distances between biomolecules labeled with an appropriate donor and acceptor fluoro phore when they are within 10 nanometers of each other (171, 172) The theory behind energy transfer is based on the concept of treating an excited fluorophore as an oscillating dipole that can undergo an energy exchange with a second dipole having a similar resonance frequency. Consequently, resonance energy transfer is analogous to the behavior of coupled oscillators, such as a pair of tuning forks vibrating at the same frequency. In contrast, radiative energy transfer requires emission and reabsorption of a photon and depends on the physical dimensions and optical properties of the specimen, as well as the geometry of the container and the wavefront
88 pathways. Unlike radiative mechanisms, resonance energy transfer can yield a significant amount of structural information concerning the donor acceptor pair (171) Principles of FRET The process of res onance energy transfer (RET) can take place when a donor fluorophore in an electronically excited state transfers its excitation energy to a nearby chromophore, referred to as the acceptor. Typically, if the fluorescence emission spectrum of the donor molecule overlaps the absorption spectrum of the acceptor molecule, and the two are within a minimal spatial radius, the donor can directly transfer its excitation energy to the acceptor through long range dipoledipole intermolecular coupling (169, 171) A theory proposed by Theodor F described the molecular interactions involved in resonance energy transfer, and Frsters equation (Equation 212) defines the relationship between the transfer rate, inter chromophore distance, and spectral properties of the involved chromophores (169, 173) Because resonance energy transfer is a nonradiative quantum mechanical process, it does not require a collision or involve production of heat. When energy transfer occurs, the acceptor molecule quenches the donor molecule fluorescence, and if the acceptor is itself a fluorophore, increased or sensitized fluorescence emission is observed, as illustrated in Figure 2 9 of the FRET Jablonski diagram. The FRET phenomenon can be o bserved by exciting a specimen containing both donor and acceptor molecules with light of wavelengths corresponding to the absorption maximum of the donor fluorophore, and detecting light emitted at wavelengths centered near the emission maximum of the acc eptor. An alternative detection method is to measure the
89 fluorescence lifetime of the donor fluorophore in the presence and absence of the acceptor (171, 174) S1S1S0S0Donor energy transfer Donor excited state transitions Acceptor excited state transitions Coupled transitions Donor a bsorption (Excitation) Donor f luorescence emission Resonance e nergy transfer Non Radiative d onor e nergy transfer Non Radiative a cceptor excitation A cceptor emission Vibrational relaxation Figure 29. A Jablonski diagram illustrating the cou pled transitions involved between the donor emission and acceptor absorbance in FRET. In the FRET Jablonski diagram in Figure 29, a bsorption and emission of the donor molecule are represented by straight purple and green vertical arrows respectively whi le vibrational relaxation is indicated by wavy yellow arrows. In the presence of a suitable acceptor, the donor fluorophore can transfer excited state energy directly to the acce ptor without emitting a photon, as illustrated by a black straight arrow
90 in Figure 29 The resulting sensitized fluorescence emission has characteristics similar to the emission spectrum of the acceptor. In summary, in order for resonance energy transfer to occur, three specific conditions must be fulfilled: (i) The emission spect rum of the donor fluorophore must overlap the acceptor molecules absorption spectrum as illustrated in Figure 210, and although the acceptor can be (and often is) a fluorophore, this is not always a requirement for FRET. Wavelength (nm) FRET Wavelength (nm) No FRET Donor emission Acceptor absorption A B Figure 210. A) Overlap of the emission spectrum of the donor and acceptor absorption spectrum results in FRET. B) Lack of overlap of the spectra means no FRET observed. (ii) The donor and acceptor molecules must be positioned within a range of 1 to 10 nanometers of each other. Equation 212 derived by Frster shows that the energy transfer efficiency between donor and acceptor molecules decreases as the sixth power of the distance separating the two (175) Henc e, the abi lity of the donor fluorophore t o
91 transfer its excitation energy to the acceptor by nonradiative interaction decreases sharply with increasing distance between the molecules, limiting the FRET phenomenon to a maximum donor acceptor separation radius of approximately 10 nanometers. The distance dependence of the resonance energy transfer process is the primary basis for its utility in investigation of molecular interactions. In living cell studies involving molecules labeled with donor and acceptor fluorophores, resonance energy transfer will occur only between molecules that are close enough to interact biologically with one another (171, 173) (iii) T he fluorescence lifetime of the donor molecule must be of sufficient duration to permit the event to occur. Both the rate (KT) and the efficiency (ET) of energy transfer are directly related to the lifetime of the donor fluorophore in the presenc e and absence of the acceptor according to Frster's theory describ ed by E quation 212. KT = (1/ D0/r 6 (2 12) w here R0 is the Frster critical distance, D is the donor lifetime in the absence of the acceptor and r is the distance separating the donor and acceptor molecules The Frster critical distance R0 is defined as the acceptor donor separation radius for which the transfer rate equals the rate of donor decay (deexcitation) in the absence of acceptor. In essence, when the donor and acceptor radius (r) equals the Frster distance, then the transfer efficiency is 50 percent. At this separation radius, half of the donor excitation energy is transferred to the acceptor via resonance energy transfer, while the other half is dissipated through a combination of all the other available processes, including fluorescence emission (171, 173)
92 Ideally, the Frster critical distance is the maximal separation length between donor and acceptor molecules under which resonance energy transfer will still occur. The critical dis tance value typically falls within a range of 2 to 6 nanometers, which is on the order of many protein molecular dimensions. T he critical distance range also corresponds to several other biologically significant dimensions, such as cell membrane thickness and the distance separating sites on proteins having multiple subunits (171) The value of R0 (in nanometers) may be calculated as shown in Equation 213. R0 = 2.11 x 102 k2 J ( 4 D 1 /6 (2 13) w here k2 is a factor describing the relative orientation in space between the transition dipoles of the donor and acceptor, J ( is the overlap integral in the region of the donor emission and acceptor absorbance spectra ( the wavelength is expressed in n m ), represents the refractive index of the medium, and QD is the quantum yield of the donor molecule (171, 175) The efficiency of energy transfer ET, is a measure of the fraction o f photons absorbed by the donor that are transferred to the acceptor, and is related to the donor acceptor separation distance, r, by E quation s 2 14 and 215 r = R0 1/ET 1 1/6 (2 14) ET = 1 ( DA/ D) (2 15) DA is the donor l ifetime in the presence of the acceptor and D is the donor lifetime in the absence of the acceptor. So by measuring the donor fluorescence lifetime in the presence and absence of an acceptor (which is indicative of the extent of donor quenching due to the acceptor), it is possible to determine the distance separating donor and acceptor molecules (175) T he rate of energy transfer therefore depends
93 upon the extent of spectral overlap between the donor emission and acceptor absorption spectra, the quantum yield of the donor, the relative orientation of the donor and acceptor transition dipole moments, and the distance separating the donor and acceptor molecules (171) Methods for m easurement of FRET Methods that have been used for measuring FRET can be divided into four fundamental categories (176) : (i) methods that monitor changes in donor fluorescence (ii) methods that examine changes in acceptor fluorescence (iii) methods that simultaneously measure changes in both donor and acceptor fluorescence using spectral imaging and (iv) methods that monitor changes in the orientation of the fluorophores The most direct methods for measuring FRET efficiencies are based on monitoring changes in donor fluorescence (either lifetime or intensity) in the presence and absence of acceptor. The two most commonly used approach es are fluorescent lifetime imaging (FLIMFRET) and acceptor bleaching. In FLIM FRET, FRET is measured by monitoring changes in a donors fluorescent lifetime, in terms of how rapidly a population of fluorophores emits light a fter a short excitation pulse) (177179) A cceptor bleaching is an approach that measures the intensity of donor fluorescence before and after photobleaching of acceptor molecules (180 182) The fluorescent lifetime of a donor should reduce if FRET is occurring hence by comparing the lifetime of the donor in the presence and absence of acceptors, the FRET efficiency can be measured. Measuring the fluorescence intensity of a donor before and after photobleaching acceptors is also equivalent to measuring the intensity of the donor in the presence and
94 absence of acceptors. Bleaching the acceptor should produce an increase in the donors fluorescent emission if FRET had been occurring An advantage of acceptor bleaching is that it can be perform ed on normal widefield microscopes (176) The most popular methods used for measuring FRET involve monitoring acceptor emission as a result of donor excitation. The main advantage of this approach is that, like acc eptor bleaching, it can be performed on simple widefield fluorescent microscopes that are widely used in cell biology laboratories (183) Microscope configurational parameters for fluorescence resonance energy transfer investigations vary with the requirements of the fluorophores, specimen, and imaging mode(s), although any upright or inverted microscope can be retrofitted for FRET microscopy. In general, the microscope should be equipped with a highresolution (12bit) cooled and intensified CCD camera system coupled to quality interference filters having low levels of crosstalk and band pass regions corresponding closely to the fluorophore spectra. The detector sensitivity determines how narrow the filter band pass can be and still enable data acquisition to proceed at acceptable speeds with a minimum of spectral bleedthrough noise. In most cases, a single dichromatic mirror coupled to excitation and emission filter wheels or sliders should be used to acquire image s in order to minimize or eliminate image shifts. Biological Applications of FRET In biological investigations, the most common applications of fluorescence resonance energy transfer are the measurement of distances between two sites on a macromolecule, such as a protein, nucleic acid or lipid or the examination of in vivo interaction between biomolecular entities. Proteins can be labeled with synthetic fluorophores or immunofluorescent fluorophores to serve as the donor and acceptor,
95 although advances in fluorescent protein genetics now enable label ing of specific target proteins with a variety of biological fluorophores having differing spectral characteristics. In many cases, the amino acid tryptophan is used as an intrinsic donor fluorophore, which can be coupled to any number of extrinsic probes serving as an acceptor (171, 174) Advances in fluorescent probe development have produced smaller and more stable molecules with new mechanisms of attachment to biologic al targets. Fluorophores have also been developed with a wide range of intrinsic excited state lifetimes, and a significant effort is being placed on development of a greater diversity in genetic variations of fluorescent proteins. Entirely new classes of fluorescent materials, many of which are smaller than previous fluorophores and allow evaluation of molecular interactions at lower separation distances, promise to improve the versatility of labeling and lead to new applications of the FRET technique (171) The simplicity and sensitivity of f luorescence assays has also made them highly useful for membrane fusion and vesicle contents leakage applications For instance, the NBD Rh odamine assa y has been use d in monitoring the fusion of cationic liposomes or their DNA complexes with cells (184) the fusion of secretory granules with liposomes (185) and virus fusion with liposomes (186, 187) FRET has also been used to test the hypothesis that raft proteins or lipids are enriched in domains with submicron dimensions (173, 188191) ). For proteins and lipids in membranes, FRET can occur by chance if the concentration of donors and acceptors is high enough. However, it is possible to distinguish this type of nonspecific FRET from FRET that occurs as the
96 result of clustering of proteins within lipid rafts, as well as to test various models of domain organization, with the aid of mathematical modeling (181, 192) Based on FRET criteria, some raft proteins have show n little evidence for clustering in FRET studies (181, 193) while others appear to be clustered in submicron domains, as predicted by the lipid raft model (194196) In addition to providing information about the size and area fraction of lipid rafts, FRET also holds great potential for reveal ing transient interactions between r aft associated molecules This w as illustrated by a recent FRET study of dynamic events that occur during B cell signaling (197) For FRET studies monitoring vesicle fusion or vesicle leakage, several self quenching fluorophores or FRET pairs have been utilized. For instance, the release of aqueous contents that may accompany divalent cationinduced vesicle fusion has been monitored by using liposomes containing carboxyfluorescein (198 201) The intracellular fate of liposomes and the intracytoplasmic delivery of liposome contents were assessed with either carboxyfluorescein (202) which is permeable through liposome and endosome membranes at low pH (203, 204) or calcein, which is retained in liposomes at low pH (205208) These fluorophores have also been used in monitoring the channel forming properties of bacterial and other toxins (209, 210) membrane destabilization by viral proteins (211) immune complex mediated lysis of liposomes (212) the stability of liposomes containing ar chaeal bolaform lipids (213) and the pH sensitivity of liposomes (207, 208)
97 To monitor the leakage of aqueous contents of liposomes at neutral pH carboxyfluorescein or calcein is encapsulated at self quenching concentrations inside liposomes. Their leakage into the external medium, and hence dilution, results in a decrease of self quenching and in an increase in the fluorescence signal (202204) .The FRET assay for l eakage of ANTS DPX from liposomes at acidic pH results in an increase in fluorescence and was used initially to examine the destabilization of liposomes at low pH (214) The assay has also been employed to as sess the interaction with liposomal membranes of surfactant associated proteins (215) and pe ptides derived from viral fusion proteins (216218) The FRET assay was utilized in this dissertation to monit or the leakage of vesicle dye content s encapsulated in BMP liposomes. For studies at neutral pH 7.4, calc ein was used whereas the FRET pair, ANTS DPX was used for experiments under acidic pH 4.5. Details of liposome preparation and performance of the assay are discussed in the experimental section of Chapter 3, under the FRET vesicle leakage assays subsection. Chromatography Separations Chromatography refers to a set of laboratory techniques utilized for the separation of mixtures. Generally, i t involves passing a mixture that is dissolved in a mobile phase through a stationary phase, which separates the analyte to be measured from other molecules in the mixture based on differential partitioning between the mobile and stationary phases. D ifferences in the compounds partition coefficient results in differential retention on the stationary phase, hence separation.
98 Chromatography may be preparativ e or analytical: preparative chromatography is used mainly to separate the components of a mixture for further use, so it is also a form of purification whereas analytical chromatography is done usually with smaller a mounts of material and is used for measuring the relative proportions of analytes in a mixture. Two types of chromatography were utilized in our studies; thin layer chromatography (TLC) and column chromatography. TLC was used to determine the integrity of our lipid dispersions and vesicles. C olumn chromatography was used t o separate vesicleencapsulated fluorophore from the free fluorophore, and the leakage of encapsulated fluorophore into the bulk solution was later monitored by FRET. Thin Layer Chromatogr aphy (TLC) Thin layer chromatography (TLC) was the earliest chromatographic method used to assess phospholipids, and is still frequently used today TLC is performed on a sheet of gl ass, plastic, or aluminum foil that is coated with a thin layer of adsorbe nt material, mostly silica gel, aluminum oxide, or cellulose. The adsorbent material is known as the stationary phase (219) Once the sample is applied on the plate, a solvent or solvent mixture, known as the mobile phase, is drawn up the plate via capillary action, an d b ecause different analytes ascend the TLC plate at different rates or speeds separation is achieved (220) TLC plates are usually prepared by mixing the adsorbent, such as silica gel, with a small amount of inert binder like calcium sulfate (gypsum) and water. This mixture is spread as thick slurry on an unreactive carrier sheet, usually glass, thick aluminum foil, or plastic. The resultant plate is dried and activated by heating in an oven for half an hour at 110 C. The thickness of the adsorbent layer is typically ~ around 0.1 0.25 mm for analytical purposes and ~ 0.5 2.0 mm for preparative TLC (220)
99 To perform TLC, a small spot of solution containing the sample is applied to a pla te, about one centimeter from the base. The plate is then dipped in to a suitable solvent, such as hexane or ethyl acetate, and placed in a sealed container. The solvent moves up the plate by capillary action and meets the sample mixture, which is dissolved and carried up the plate by the solvent. Different compounds in the sample mixture elute at different speeds due to the differences in their interaction with the stationary phase, and differences in solubility in the solvent. By changing the solvent, or using a mixture of solvents the separation of components (measured by the retention factor, Rf value) can be adjusted (219, 220) Separation of compounds in TLC is based on the competition of the solute and the mobile phase for binding with the stationary phase. For instance, if normal phase silica gel is used as the stationary phase it can be considered polar. In a situation with two compounds th at differ in polarity, the most polar compound would have a stronger interaction with the silica and would therefore bind more strongly with the stationary phase. Consequently, the less polar compound moves higher up the plate, resulting in a higher Rf val ue. On the other hand, i f the mobile phase is changed to a more polar solvent or mixture of solvents, it would be more capable of displacing solutes from the silica binding places and all compounds on the TLC plate will move higher up the plate. For instance if a mixture of ethyl acetate and heptane are used as the mobile phase, adding more ethyl acetate results in higher Rf values for all compounds on the TLC plate, and c hanging the polarity of the mobile phase will not result in reversed order of elution of the compounds on the TLC plate (219, 221)
100 In most cases, the chemicals being separated are colorless; hence several methods exist to visualize the spots. A small amount of a fluorescent compound, usually mangan ese activated zinc silicate, can be added to the adsorbent, a llowing the visualization of spots under a blacklight (UV254), hence t he adsorbent layer will fluoresce light green by itself, but spots of analyte quench this fluorescence. Another approach is the use of Iodine vapors as a general unspecific color reagent into which the TLC plate is dipped or sprayed onto the plate. For lipid analysis the chromatogram may be transferred to a Polyvinylidene Fluoride ( PVDF ) membrane and then subjected to further analysis, for example mass spectrometry. Once visible, the Rf value, or Retention factor of each spot can be determined by dividing the distance traveled by the product by the total distance traveled by the solvent (the solvent front). These values depend on the solvent used, and the type of TLC plate (222, 223) Because of its speed better separation efficiency than paper chromatography, and the ability to cho ose between different stationary phases, TLC has found versatile applications including assaying the radiochemical purity of radiopharmaceuticals, determination of plant pigments, detection of pesticides or insecticides in food, analyzing the dye composition of fibers in forensics, or identifying compounds present in a given substance, monitoring organic reactions and for the qualitative analysis of reaction products (221, 224, 225) Column Chromatography In column chromatography, the stationary bed is within a tube. The particles of the solid stationary phase or the support coated with a liquid stationary phase may fill the entire inside volume of the tube (packed column) or be concentrated on or along the
101 inside tube wall leaving an open, unrestricted path for the mobile phase in the middle part of the tube (opentubular column) (226, 227) The classical preparative chromatography column is a glass tube with a diameter from 50 mm and a height of about 50 cm to 1 m with a tap at the bottom. To prepare a column, the dry method or the wet method can be employed. In the dry method, the column is first filled with the dry stationary phase powder, followed by the addition of mobile phase, which is flushed through the column until it is completely wet, and then henceforth the column is never allowed to run dry. For the wet method, slurry is prepared of the eluent with the stationary phase powder and then carefully poured into the column, avoiding any formation of air bubbles. An organic solvent is pipetted on top of the stationary phase, and t his layer is usually topped with a small layer of sand, cotton or glass wool to protect the shape of the organic layer from the velocity of newly added eluent. Eluent is slowly passed through the column to advance the organic material. Often a spherical eluent reservoir or an eluent filled and stoppered separating funnel is put on top of the column (226) The individual analytes are retained by the stationary phase depending on how they interact with it, and separate from each other while they are el uting at different rates through the column, arriving a t the end of the column one at a time. During the elution process the eluent is collected in a series of fractions. The composition of the eluent flow can be monitored and each fraction is analyzed for dissolved compounds using detection methods such as analytical chromatography, UV absorption, or f luorescence. Colored compounds or fluorescent compou nds with the aid of an UV lamp can be easily seen through the glass wall as moving bands (228)
102 The stationary phase or adsorbent in column chromatography is a solid. The most common sol id stationary phase s for column chromatography are silica gel and alumina although c ellulose powder has also been used. The mobile phase or eluent can be either a pure organic solvent or a mixture of different solvents. The mobile phase is chosen so that the retention factor value (Rf) of the compound of interest is approximately 0.2 0.3 in order to minimize the time and amount of eluent used to run the chromatography. The eluent is also chosen in such a way that different compounds can be separated eff ectively (219) A faster flow rate for the eluent minimizes the time required to run a column and therefore minimizes diffusion, resulting in a better separation (229) as illustrated by Van Deemter's equation in Equation 216. H = A + B/u + C.u (2 16) w here A is Eddy diffusion, B is l ongitudinal di ffusion, C is the m ass transfer kinetics of the analyte between mobile and stationary phase s and u is the linear v elocity. A is equivalent to the multiple paths taken by the chemical compound, and in open tubular capillaries this term will be zero as there are no multiple paths. The multiple paths occur in packed columns where several routes through the column packing are possible, which results in band spreading. B/u is equal to the longitudinal diffusion o f the particles of the compound, whereas C u is equal to the equilibration point. In a column, there is an interaction between the mobile and stationary phases, Cu accounts for this (229) A simple laboratory column runs by gravity flow, and t he flow rate of such a column can be increased by extending the fresh eluent filled column above the top of
103 the stationary phase or decreased by the tap controls. Better flow rates can be achieved by using a pump or by using compressed gas such as air, nitrogen, or argon, to push the solvent through the column ( referred to as flash column chromatography) (219, 228) Column chromatography has been used for both analytical and preparative separations for well over four decades. It has now largely replaced planar chromatography, which was most widely used in the 1940s 1960s, owing to the fact that column chromatography is much easier to instrumentalize than planar chromatography and that analytes are far easier to detect and quantitate in a stream of solution than in a layer of particles (226) For studies in this dissertation, Sephadex G 5 0 Fine column ( 5 cm internal diameter, 100 cm height) from GE Healthcare was used to separate fluorophoreloaded BMP vesicles from free or unencapsulated fluorophore, and the eluted fractions were analyzed by fluorescence resonance energy transfer (FRET).
104 C HAPTER 3 INVESTIGATION OF BMP VESICLE SIZE AND MOR PHOLOGY IN MODEL MEMBRANES Introduction Bis(monoacylglycero)phosphate (BMP) is a negatively charged phospholipid found in elevated concentrations in the late endosome. BMP represents less than 1 mol % o f the total phospholipid mass in most tissues and cell types (45, 46) although in the late endosome, the concentration of BMP is about 15 mol % of the total lipid content of the organelle (44, 94, 95) and can comprise as much as 70 mol % of the lipid composition of the intraendosomal vesicles (94) Endosomes are intracellular organelles that act as a staging area for sorting endocytosed material either back to the plasma membrane for recycling or to specialized organelles such as lysosomes for degradation (46, 230) Endosomes can be identified by the presence of vesicular bodies (VBs) inside the lumen of a limiting membrane or by lipid composition (44, 97) Early endosomes have a limiting membrane with a lipid composition very similar to that of the plasma membrane, whereas late endosomes are characterized by an absence of a significant amount of cholesterol and a rel atively high concentration of BMP (97) During endosome maturation, the internal pH undergoes a series of changes from that of the neutral cytosol to that of the acidic lysosome. It is within the late endosome intra luminal membranes that BMP is first found in relatively higher concentrations and is thought to be partly responsible for the formation of the intraendosomal VBs (98) BMP has an unusual structure that differs from that of typical phospholipids such as POPC, (1 Palmitoyl 2 Oleoyl sn Glycero 3 Phosphocholine) and POPG, (1Palmitoyl2 Oleoyl sn Glycero 3 Phosphorac 1 Glycerol) as shown in Figure 31.
105 O O O O H P O-O O N+O B Na+O O O H O H H P O-O O O O O C N H4 +O O H P O O O O H O-H O H O O Asn3 sn2 sn1 sn3 sn2 sn1 sn3 sn2 sn1 s n1 s n2 s n3 Figure 31. Chemical s tructures of A) BMP B ) POPC and C ) POPG A comparison of the chemical structures BMP, POPC and POPG is shown in Figure 3 1. The unusual structure and stereochemistry of BMP is different from that of typical phospholipids in two aspects. Firstly, BMP has two glycero components (Figure 3 1A) each w ith a single acyl chain, unlike POPC (Figure 3 1B) and POPG (Figure 3 1C) both of which have one glycero component that is esterified to two hydrophobic acyl chains. The structures in Figure 31 depict the two glycero groups in BMP and one glycero group i n POPC and POPG.
106 Secondly, the structure of BMP isolated from biological sources differs from the typical sn 3 glycerophosphate structures exhibited by most other glycerophospholipids (2, 8, 46) in that it possesse s an unusual sn 1 glycerophosphosn 1' glycerol (sn1:sn1') stereoconfiguration (47, 48, 50, 99, 100, 231) The sn 3: sn 1' configuration has been reported for BMP isolated from baby hamster kidney (BHK) cells, although it is postulated that this configuration is an intermediate in the synthesis of the sn 1: sn 1' BMP (48) The unusual structure and stereochemistry of BMP are thought to be responsible for important r oles in the endosome, including structural integrity, endosome maturation, and lipid/protein sorting and trafficking (101, 106) Although it is unclear how this occurs, it is possible that the structure and biosynth esis of BMP, together with the change in pH during the maturation of the endosome induces intraendosomal vesicular body formation. This chapter presents results of the characterization of the morphology and size distribution observed in hydrated lipid disp ersions and extruded lipid vesicles of BMP, POPC, POPG and lipid mixtures of POPC:BMP and POPC:POPG. Data and results were obtained by utilizing dynamic light scattering (DLS) and negative staining transmission electron microscopy (TEM) imaging to provide information on the vesicle sizes, size distribution and macroscopic morphology of hydrated lipid dispersions and extruded vesicles. Initial DLS control experiments were performed to test different populations of POPC vesicles extruded using 30, 100 or 400 nm pore size extrusion membranes. Vesicle mixtures in various v/v ratios were then manually mixed as 30 nm with 400 nm and 100 nm with 400 nm extruded vesicles and the resulting vesicle populations measured with DLS. The aim of these experiments was to validate the
107 analytical capability of dynamic light scattering as a particle sizing technique in measuring and discriminating between different known vesicle populations. To assess the stability of BMP vesicles (stability defined as the ability of the ves icles to maintain the same size distribution over time), assays were performed in which BMP, POPC and POPG vesicles were mechanically passed through 30 nm pore size extrusion membranes and the size of the vesicle suspensions (stored at room temperature) were monitored by DLS measurements over a five week period. Results from this assay clearly indicate that BMP 30 nm vesicles are smaller and more stable than either POPC or POPG. This is an interesting finding that may go a step further in elucidating the si gnificant role of BMP in the formation of intraendosomal vesicular bodies whose vesicle diameters (~200 nm) are in agreement with BMP vesicle sizes extruded with 400 nm membranes. The smaller, stable BMP vesicles can also be further explored for drug deli very applications. To confirm that BMP forms lamellar vesicles with an interior volume, vesicle leakage assays were performed using fluorescence resonance energy transfer (FRET). Vesicles mechanically passed through 400 nm polycarbonate extrusion membranes were encapsulated with either calcein at neutral pH 7.4 or the FRET pair DPX/ANTS at acidic pH 4.2, and the percent release of the encapsulated fluorophore monitored as a function of increasing concentrations of titrated sodium dodecyl sulfate (SDS) deter gent. DLS and TEM results presented in this chapter also illustrate the effect of pH and ionic strength on BMP vesicle size and morphology; experiments were performed at both neutral and acidic pH, and in the presence and absence of 100 mM NaCl salt in th e buffer solution used to hydrate the vesicles. Finally, DLS and negative staining TEM
108 studies were performed to determine the change in vesicle sizes and shapes of lipid mixtures POPC: BMP and POP: POPG at both acidic and neutral pH and the results compar ed to those observed in pure BMP, POPC and POPG. Experimental section Materials and Reagents Used All of the following lipids, dissolved in chloroform, were purchased from Avanti Polar Lipids (Alabaster, AL), stored at 20C and used without further purifi cation. BMP18:1, ((S,R Isomer) sn (3 Oleoyl 2 H ydroxy) Glycerol 1 Phosphosn (1' Oleoyl 2' Hydroxy) Glycerol, ammonium Salt)); POPC, (1Palmitoyl2 Oleoyl sn Glycero 3 Phosphocholine) and POPG, ( 1Palmitoyl 2 Oleoyl sn Glycero 3 (Phosphorac (1 Glycerol), sodium salt)). C30H26N2O13, (Calcein); ANTS, (8 amino napthalene1, 3, 6 trisulfonic acid) and DPX, (pxylenebispyridinium bromide) were purchased from Molecular Probes (Invitrogen, Carlsbad, CA). SDS, (sodium dodecyl sulfate); HEPES, (4 (2 hydroxyethyl ,) 1 piperazineethanesulfonic acid, C8H18N2O4S); NaOAc, (sodium Acetate); EDTA, (ethylenediamine tetraacetic acid, C10H16N2O8) and NaCl, (sodium chloride) were purchased from Fisher Biotech (Pittsburgh, PA). CH3Cl, (chloroform); MeOH, (methanol); CH3CH2OH (ethanol); C6H12, (cyclohexane); NH4OH, (ammonium hydroxide); HCl, (hydrochloric acid) and NaOH, (sodium hydroxide) were obtained from Fisher Scientific (Pittsburgh, PA). UO2 (CH3COO) 2. 2H2O, (uranyl acetate) and 400mesh Formvar coated copper grids were purchased from Ted Pella (Redding, CA). Single sealed 50to 1000 mL disposable cuvettes (10mm path length) were obtained from Eppendorf (Westbury, NY). 30nm, 100nm, and 400 nm polycarbonate extrusion membranes and filter supports were purchased from Avanti Polar Lipids
109 (Alabaster, AL). Silica coated aluminum thin layer chromatography (TLC) plates were purchased from Whatman (Florham Park, New Jersey). Hydrated Dispersions and Extruded Vesicle P reparation All the glassware, syringes and vials used for vesicle sample preparation were cleaned and rinsed in a series of ethanol, chloroform, ethanol and copious amounts of water or buffer solutions before use. Before vesicle sample preparation, the lipid stock bottles, previously stored in the freezer at 20 C, were allowed to reach room temperature before opening. For dynamic light scattering (DLS) and negativestaining transmission electron microscopy (TEM) measurements, the desired amount of stock lipid (238 L of BMP (5mg/mL), 57L of POPC (10mg/mL), 116 L of POPG (10mg/mL), 49 L POPC and 17 L POPG for POPC:POPG ( 85:15) and 49 L POPC and 36 L BMP for POPC:BMP ( 85:15)) dissolved in chloroform, was dried under a gentle nitrogen stream for approximately 10 minutes or until all the solvent was evaporated, resulting in a dry, thin lipid film at the base of the test tube. Each sample was then further dried under vacuum in a desiccator for at least 12 hours to remove residual solvent. The thin lipid film was hydrated with 2 mL of either HEPES (4(2 hydroxyet hyl,) 1 piperazineethanesulfonic acid) buffer, pH 7.4 (5 mM HEPES, 100 mM NaCl (sodium chloride), 0.1 mM EDTA (ethylenediamine tetraacetic acid)) or NaOAc (sodium acetate) buffer, pH 4.2 (5mM sodium acetate, 100 mM NaCl, 0.1mM EDTA) to obtain a final lipid concentration of 0.75 mM. Additional samples were hydrated in the absence of 100 mM NaCl salt, in HEPES buffer, pH 7.4 (5 mM HEPES, 0.1 mM EDTA) and in NaOAc buffer, pH 4.2 (5 mM NaOAc, 0.1 mM EDTA). Hydrated lipid dispersions were then vortex mixed 5 10 times,
110 freeze thawed five times in liquid nitrogen and water respectively, and incubated at room temperature for at least 12 hours before extrusion or measurement by DLS and TEM. The extended incubation period of the hydrated dispersions allowed for compl ete lipid vesicle hydration. To form unilamellar vesicles, (either as small unilamellar vesicles (SUVs) or large unilamellar vesicles (LUVs), the multilamellar hydrated dispersions were mechanically passed 31 times through 30, 100, or 400 nm polycarbonate extrusion membranes. Unilamellar vesicles samples were then immediately used for DLS measurements. Phospholipid integrity was verified by thin layer chromatography (TLC). Approximately 10 L of sample was spotted on silica coated aluminum plates. Plates w ere placed in a chamber containing a CH3Cl:MeOH: NH4OH (65:25:10) mobile phase. The TLC plates were developed in an iodine chamber and visualized by eye. S ample Pr eparation for Vesicleleakage A ssays For fluorophoreloaded vesicle assays (232) 200L of stock BMP (5mg/mL), dissolved in chloroform, was placed in a medium sized roundbottomed test tube and dried under a stream of nitrogen gas for approximately 10 minutes or until all the solvent was evaporated, forming a dry thin lipid film at the base of the test tube. The sample was subsequently placed in a vacuum desiccator overnight to remove any residual solvent. The dry lipid film was hydrated with 2 mL of either 5 mM HEPES buffer, pH 7.4 containing 70 mM calcein fluorescent dye or 5 mM NaOAc buffer (pH 4.2) containing 25 mM ANTS and 90 mM DPX fluorescent dyes. Hydrated lipid dispersions were passed through 400nm extrusion membranes as described above to form large unilamellar vesicles.
111 Fluorophoreloaded unilamellar vesicles were separated from free fluorophore on a Sephadex G 50 Fine column (GE Healthcare, Piscataway, NJ). For this assay, 10 L of the lipid sus pension was diluted to 300 L with either 5 mM HEPES or 5 mM NaOAc buffer in a 4mm light path quartz cuvette (Starna, Atascadero, CA). Sodium dodecyl sulfate (SDS) detergent solubilization of calceinloaded vesicles was monitored by fluorescence spectrosc opy with excitation at 490 nm and detection of emission intensity at 520 nm. The ANTS/DPX leakage assay utilized an excitation of 360 nm and emission at 530 nm and used wider slit widths (10 20 nm). Concentrated SDS detergent (10% w/w) was titrated into the diluted lipid suspension, with a subsequent 10min incubation period at 27 C, with stirring, before obtaining the emission intensity after each SDS titration step. Percentage fluorophore release was calculated as shown in Equation 31. % Fluorophore rel ease = (I I0)/ (I I0) .100 (3 1) where I is the emission intensity after addition of SDS, I0 is the emission intensit y in the absence of SDS, and I is the emission intensity after the addition of a 5 mL (20% w/w) SDS. All emission intensities wer e corrected for dilution factor from titrations and each assay was performed in triplicate. Instrumentation Used Dynamic light s cattering (DLS) Dynamic light scattering size distribution and characterization measurements of lipid vesicles were performed us ing a Brookhaven 90Plus/BI MAS ZetaPALS spectrometer operated at a wavelength of 659 nm and at 25 C. The instrument uses a BI 9000AT digital autocorrelator and 9KDLSW data acquisition software. A 100mL sample volume in a disposable cuvette was used for each measurement. For each sample, 3 runs were performed with each run lasting 3 minutes. Raw Data and
112 histograms were further analyzed and converted into B spline plots using OriginPro 8 software. Negative staining t ran smission electron m icroscopy Negati ve staining t ransmission electron microscopy (TEM) images were obtained using a Hitachi H 7000 transmission electron microscope operated at 75100 kV with a Soft Imaging System MegaViewIII with AnalySIS digital camera (Lakewood, CO). The microscope has a m aximum resolution at 0.2 nm with a magnification range of 110 to 600,000. Before TEM measurements, samples were further prepared by negative staining. Briefly, for all samples, a drop of the lipid vesicle sample was spread on a 400mesh Formvar coated c opper grid and incubated for 2 minutes. Excess sample was gently dabbed away with filter paper, and the grid was allowed to dry for 2 minutes. A drop of deionized water was optionally added to the grid to remove any excess salt from the buffer solution use d in vesicle preparation. One drop of 2 % uranyl acetate was then added to the grid and allowed to stain for 2 minutes, after which any excess uranyl acetate was wiped away, and the sample was allowed to dry for another 2 minutes before being placed in the electron microscope specimen holder for imaging. Fluorescence measurements Fluorescence spectra were acquired with a FluoroMax 3 fluorometer (Jobin Yvon Horiba, Edison, NJ) with a temperaturecontrolled sample cell and a Haake K20 temperature controller ( Thermo Electron, Waltham, MA). Measurements were made using a 4mm light path quartz cuvette (Starna, Atascadero, CA). All spectra were collected at 27 C, with both the excitation and emission slits set to 5 nm and with excitation and emission polarizers set to 90 and 0 respectively.
113 Results and Discussion Evaluation of Mixing Different Sizes of Neat POPC V esicle P opulations T o validate the analytical capability of dynamic light scattering as a particle sizing technique, control experiments were designed to measure the size distribution of POPC hydrated dispersions (unextruded), and vesicles extruded using polycarbonate membranes with varying pore sizes of 30, 100 or 400 nm. POPC vesicles of known size distributions were then manually mixed; 30 with 400nm and 100 with 400nm extruded vesicles in specific v olume to v olume ratios with 10 L increments to give a total of 100 L for each sample. T he resulting physical mixtures of vesicles were measured by dynamic light scattering to determine the ability of the DLS technique to distinguish between manually mixed vesicle populations For each sample, DLS measurements were taken in triplicates, averaged and the data further analyzed and converted into B spline plots using OriginPro 8 software. A B 0 50 100 150 200 0 20 40 60 80 100 Intensity (au)Vesicle Diameter (nm) 30 nm pores 100 nm pores 0 300 600 900 1200 1500 1800 2100 0 20 40 60 80 100 Intensity (au)Vesicle Diameter (nm) 400 nm pores Hydrated Dispersions Figure 32. DLS histograms of size distributions of POPC vesicles extruded with polycarbonate membranes of varying pore sizes A) Black line, 30 nm A) Red line, 100 nm B) G reen line 400 nm B) B lue line, hydrated unextruded dispersions
114 Table 31. Summary of POPC vesicle diameters extruded with polycarbonate membranes of varying pore sizes. Membrane pore size Average vesicle diameter (nm) 30 nm 50 30 100 nm 100 20 400 nm 400 100 Unextruded dispersions 1300 300 Figure 32 re presents the dynamic light scattering histograms of POPC vesicles that were mechanically passed through polycarbonate extrusion membranes with pore sizes of 30 nm, 100 nm, 400 nm and hydrated, unextruded dispersions that are also referred to as multilamellar vesicles (MLVs). Table 31 is a sum mary of the POPC average vesicle diameters shown in Figure 32, obtained by taking an average of three independent DLS traces for each sample. The error bars were calculated as standard deviation of the average vesicle diameter in each case. The data in F igure 32 and Table 31 show that the 3 0 nm extruded vesicles (Figure 3 2 A, black solid line) have the smallest diameter range, with an average vesicle size at ~ 50 nm wh ereas the vesicles extruded with 100 nm polycarbonate membranes (Figure 3 2 A, red sol id line) have a slightly larger but narrower vesicle size distribution that is c entered at ~ 100 nm. In Figure 32 B, the green solid line represents the vesicle size distribution of vesicles extruded with 400 nm pore sizes, with the intensity centered at a vesicle diameter of ~ 400 nm. The blue solid line in Figure 32 B is representative of the size distribution obtained for unextruded, hydrated dispersions of POPC, showing large vesicle diameters of up to ~1300 nm. Taken together, these data indicate that dynamic light scattering can accurately measure and report the vesicle size distributions of vesicles that have been mechanically extruded with polycarbonate extrusion membranes of varying pore sizes.
115 0 200 400 600 800 1000 1200 0 20 40 60 80 100 10L, 30nm + 90L, 400 nm Intensity (au)Vesicle diameter (nm) 0 200 400 600 800 1000 1200 0 20 40 60 80 100 0L, 30nm + 100L, 400 nm Intensity (au)Vesicle diameter (nm) 0 200 400 600 800 1000 1200 0 20 40 60 80 100 20L, 30nm + 80L, 400 nm Intensity (au)Vesicle diameter (nm) 0 200 400 600 800 1000 1200 0 20 40 60 80 100 30L, 30nm + 70L, 400 nm Intensity (au)Vesicle diameter (nm) 0 200 400 600 800 1000 1200 0 20 40 60 80 100 40L, 30nm + 60L, 400 nm Intensity (au)Vesicle diameter (nm) 0 200 400 600 800 1000 1200 0 20 40 60 80 100 50L, 30nm + 50L, 400 nm Intensity (au)Vesicle diameter (nm) 0 200 400 600 800 1000 1200 0 20 40 60 80 100 60L, 30nm + 40L, 400 nm Intensity (au)Vesicle diameter (nm) 0 200 400 600 800 1000 1200 0 20 40 60 80 100 70L, 30nm + 30L, 400 nm Intensity (au)Vesicle diameter (nm) 0 200 400 600 800 1000 12000 20 40 60 80 100 100L, 30nm + 0L, 400 nmIntensity (au)Vesicle diameter (nm) 0 200 400 600 800 1000 12000 20 40 60 80 100 90L, 30nm + 10L, 400 nmIntensity (au)Vesicle diameter (nm) 0 200 400 600 800 1000 12000 20 40 60 80 100 80L, 30nm + 20L, 400 nmIntensity (au)Vesicle diameter (nm) a j i h g f e d c b k 0 200 400 600 800 1000 1200 0 20 40 60 80 100 a b c d e f g h i j k Intensity (au)Vesicle diameter (nm) l Figure 33 DLS histograms of populations of manually mixed POPC vesicle s extruded with 30 nm and 400 nm pore size membranes and mixed in various volume/volume ratios in 5 mM HEPES buffer
116 Following the accurate measurement of various populations of POPC vesicle sizes by DLS, experiments were carried out to determine the ability of DLS to differentiate between manually mixed vesicle populations of different sizes Figure 33 a l is an illustration of the DLS vesicles size distributions obtained for POPC vesicles populations that were manually mixed at various v: v ratios after extrusion through 30 and 400 nm polycarbonate membranes, respectively. Each vesicle mixture consisted of 100 L total volume, starting at 0 L of 30 nm extruded vesicles mixed with 100 L of 400 nm extruded vesicles in Figure 33 a There fore, the sample in Figure 33a was made up of only 400 nm extruded vesicles, which is consistent with the DLS histogram obtained, with a unimodal vesicle size distribution that is centered at ~ 35 0 nm. Figure 33b illustrates the vesicle size distributi on obtained for 10 L of 30 nm extruded vesicles mixed with 90 L of 400 nm extruded vesicles. As would be expected, two vesicle populations can be seen; a less intense size distribution centered at 70 nm that is representative of the 30 nm extruded vesic le population, and a more intense size distribution with an average diameter of ~ 400 nm, representing the 400 nm extruded vesicles. This trend is observed in all of the subsequent samples represented by Figures 33c j, with all samples exhibiting two dis tinct peaks that are representative of the two premixed vesicle populations. Figure 33k shows 100 L of 30 nm extruded vesicles mixed with 0 L of 400 nm extruded vesicles, hence this sample contained vesicles that were only extruded with a 30 nm pore siz e, and as expected, the DLS histogram has only one vesicle population that with an average vesicle size at ~ 70 nm. Figure 33l is a summary of all the DLS histograms for all the vesicle mixtures shown in
117 Figures 33ak, showing a bimodal size distribution that represents two distinct vesicle populations in all instances. It is noteworthy to mention that in almost all cases of the physically mixed vesicle populations, the peaks representing the smaller vesicle sizes that were extruded with 30 nm pore sizes are less intense than the larger sized vesicle peaks, even when higher concentrations of smaller vesicles are mixed with lower concentrations of larger vesicles ( for instance in Figure 33h, 70 L of 30 nm extruded vesicles were mixed with 30 L of 400 nm extruded vesicles, but the smaller volume of larger vesicles results in a more intense peak than the larger volume of smaller vesicles ). This observation is attributed to the fact that in dynamic light scattering technique, larger particles scatter more light than smaller particles, which results in more intense peaks for observed larger particles, even when there might be a larger volume of small vesicles than large vesicles. Similar experiments were carried out by physically mixing 100 nm extruded vesi cles with 400 nm extruded vesicles, then measuring the resulting size distributions of the vesicle mixtures with dynamic light scattering. Results from these experiments are illustrated in Figures 34al. In Figure 34a, 0 L of 100 nm extruded vesicles were mixed with 100 L of 400 nm extruded vesicles, and as expected, the DLS histogram indicates one vesicle population that is centered at ~ 400 nm since this sample contained only vesicles extruded with the 400 nm pores Figure 34b for 10 L of 100 nm ex truded vesicles mixed with 90 L of 400 nm extruded vesicles exhibits two vesicle populations, with a small, less intense peak at ~ 100 nm and a more intense peak at ~ 400 nm. Likewise, the subsequent samples represented by Figures 34c h all show two dist inct vesicle peaks repres entative of the two mixed vesicle populations.
118 0 200 400 600 800 1000 12000 20 40 60 80 100 0 L, 100 nm + 100L, 400 nm Intensity (au)Vesicle diameter (nm) 0 200 400 600 800 1000 12000 20 40 60 80 100 10 L, 100 nm + 90L, 400 nm Intensity (au)Vesicle diameter (nm) 0 200 400 600 800 1000 12000 20 40 60 80 100 20 L, 100 nm + 80L, 400 nm Intensity (au)Vesicle diameter (nm) 0 200 400 600 800 1000 12000 20 40 60 80 100 30 L, 100 nm + 70L, 400 nm Intensity (au)Vesicle diameter (nm) 0 200 400 600 800 1000 12000 20 40 60 80 100 40 L, 100 nm + 60L, 400 nm Intensity (au)Vesicle diameter (nm) 0 200 400 600 800 1000 12000 20 40 60 80 100 50 L, 100 nm + 50L, 400 nm Intensity (au)Vesicle diameter (nm) 0 200 400 600 800 1000 12000 20 40 60 80 100 60 L, 100 nm + 40L, 400 nm Intensity (au)Vesicle diameter (nm) 0 200 400 600 800 1000 12000 20 40 60 80 100 70 L, 100 nm + 30L, 400 nm Intensity (au)Vesicle diameter (nm) 0 200 400 600 800 1000 12000 20 40 60 80 100 80 L, 100 nm + 20L, 400 nm Intensity (au)Vesicle diameter (nm) 0 200 400 600 800 1000 12000 20 40 60 80 100 90 L, 100 nm + 10L, 400 nm Intensity (au)Vesicle diameter (nm) 0 200 400 600 800 1000 12000 20 40 60 80 100 100 L, 100 nm + 0L, 400 nm Intensity (au)Vesicle diameter (nm) a d k j i h g f e c b 0 200 400 600 800 1000 1200 0 20 40 60 80 100 Intensity (au)Vesicle diameter (nm) a b c d e f g h i j k l Figure 34 DLS histograms showing populations of manually mixed POPC vesicles extruded with 10 0 nm and 400 nm pore size membranes in various volume/volume ratios
119 Figure 34i for 80 L of 100 nm extruded vesicles mixed with 20 L of 400 nm extruded vesicles has only one size distribution centered at ~ 150 nm. Likewise, Figure 3 4j also show s only one size distributions centered at ~ 180 nm although it represents 90 L of 100 nm extr uded vesicles mixed with 10 L of 400 nm extruded vesicles The unimodal size distributions observed in Figures 34i j are however broader than those seen in the previous bimodal size distributions, hence it can be assumed that at a certain increased volum e of ~ 80 L of 100 nm extruded vesicles, the vesicle populations start mixing so that DLS can no longer distinguish between them and reports them as one broad vesicle size distribution. Figure 34j for 100 L of 100 nm extruded vesicles also shows one nar row intense peak with an average vesicle diameter at ~ 120nm, whereas Figur e 34k summarizes the DLS histog rams observed for all the vesicle mi x tures, exhibiting two distinct vesicle populations in all cases. Taken together, the data observed i n Figures 33ak and Figures 34a k indicate that the dynamic light scattering technique has the general capability to accurately measure and differentiate between manually mixed vesicle populations shown as two vesicle peaks in the DLS histograms. Additional exper iment s were performed to determine the measured average vesicle diameters of BMP, POPC and POPG as a function of extrusion membrane pore diameter. In these experiment s, samples of BMP, POPC and POPG lipid dispersions that were each hydrated in buffer containing 5 m M HEPES, 100 m M NaCl, and 0.1 mM EDTA were mechanically passed 31 times through 30, 100, and 400 nm polycarbonate extrusion membranes then measured by dynamic light scattering to determine the vesicle size d istributions. Results of these experiment s are summarized in Table 32.
120 Table 32. Summary of BMP, POPC and POPG average vesicle diameter as a function of extrusion membrane pore diameter s. Extrusion m embrane pore diameter Measured vesicle diameter s (nm) BMP POPG POPC 30 nm 50 2 0 80 10 80 20 100 nm 100 20 130 20 120 10 400 nm 230 3 0 300 100 400 180 Error bars are standard deviations of the average vesicle diameters, which were obtained from the average of three independent DLS traces. Lipids were hydrated in 5 mM HEPES, 100 mM NaCl and 0.1 mM EDTA, pH 7.4. Table 32 is a summary of the measured vesicle diameters obtained for BMP, POPC and POPG lipid dispersions that were extruded with 30, 100 and 400 nm polycarbonate membranes. From Table 3 2, BMP vesicl es extruded with 30 nm pore membranes have an average vesicle diameter of 50 nm while POPG and POPC both have larger average vesicle diameters of 80 nm. For the 100 nm extrusion membrane, both POPC and POPG form vesicles with average diameters of 120 and 130 nm respectively, whereas BMP vesicles have a smaller average vesicle diameter of 100 nm. More interestingly, BMP vesicles extruded with 400 nm pores have much smaller vesicle diameters of only 230 nm, compared to the larger vesicle diameters of 300 and 400 nm for POPG and POPC respectively. The data in Table 32 show conclusively that perhaps because of its unique structure, BMP prefers to form smaller sized vesicles compared to the vesicles formed by typical phospholipids POPC and POPG. Comparison of Vesicle S tability : BMP v ersus POPC Vesicles After demonstrating that BMP forms smaller vesicles compared to either POPC or POPG vesicles when extruded with different sizes of polycarbonate membranes it was of interest to determine the stability of BMP vesicles compared to POPC vesicles. Stability here is defined as the ability of the vesicles to maintain the same size distribution over time, si nce small vesicle structures are known to generally fuse and
121 form larger vesicles over time. DLS experiments were performed on BMP and POPC vesicles extruded with 30 nm pore membranes in buffer containing 5 mM HEPES, 100 mM NaCl and 0.1 mM EDTA, at neutral pH 7.4. The average vesicle diameters of the 30nm extruded vesicles of BMP and POPC under neutral were measured and monitored over a five week period to observe any changes in the vesicles size distributions. The samples were stored in 5mL vials at room temperature. F igure 35 shows results from DLS measurements performed on the 30nm extruded vesicles of BMP and POPC over a five week peri od. (A) BMP (B) POPC 0 200 400 600 800 1000 1200 0 20 40 60 80 100 Intensity (au)Vesicle Diameter (nm) Week 1 Week 2 Week 3 Week 4 Week 5 0 20 40 60 80 100 Intensity (au) Week 1 Week 2 Week 3 Week 4 Week 5 Figure 35 Dynamic light scattering size distributions of A) 30nm extruded BMP and B) POPC vesicles monitored over a five week period. Lipids were hydrated in 5 mM HEPES, 100 mM NaCl and 0.1 mM EDTA, at neutral pH 7.4.
122 Because BMP hydrolyzes rapidly under acidic conditions, the stability data were collected only for neutral pH 7.4. From the data in Figure 35 it can clearly be see n that the POPC vesicles fuse to form larger structures as early as two weeks after extrusion. On the other hand, although the size distribution of the BMP 30 nm extruded vesicles broadens the average vesicle size is still near 50 nm, and the distribution tails off near 100 nm. Combined, t he DLS results from these experiments in dicate that BMP prefers to form small, stable vesicular structures. BMP Vesicle Leakage A ssay s under Acidic and N eutral pH Conditions To verify tha t BMP forms lip osomes that contain an interior volume, fluorescence leakage assays were performed using calcein fluorescent dye for experiments at neutral pH, and the fluorescence resonance energy transfer (FRET) pair of ANTS/DPX ( ANTS, 8 amino nap h t halene1, 3, 6 trisul fonic acid and DPX, p xylene bispyridinium bromide) for vesicles at acidic pH Because the quantum yield of calcein is too low at acidic pH, the ANTS/DPX pair was used instead, where DPX quenches ANTS Lipid vesicle leakage assays were developed by loading vesicles with the appropriate fluorescent dye, and monitoring the change in fluorescence intensity as the vesicle contents leak ed out into the bulk solution after solubilization with the sodium dodecyl sulfate (SDS) detergent. Dyeloaded vesicles were obtained by including the fluorophore in the lipid hydration buffer, and then once the vesicles were formed, the free dye was separated from the encapsulated dye by running the lipid dispersions down a Sephadex column.
123 A) Calcein B) ANTS C) DPX D) SDS Figure 36. Chemical structures of A ) calcein, B) ANTS, C) DPX and D) SDS Figure 37 Schematic illustration of SDS detergent solubilizing the liposome and causing leakage of the vesicle contents Figure adapted from Tom Frederick.
124 Figure 37 is a schematic illustration of vesicle content s leaking into the bulk solution after solubilization of the liposome by the sodium dodecyl sulfate (SDS) detergent. For fluorescence exp eriments at neutral pH the calcein dye was excited at 490 nm and emission was set at 520 nm whereas for the ANTS/DPX pair at acidic pH the excitation wavelength was at 360 nm and the emission wavelength set at 530 nm The % fluorophore release is given by Equation 32 % Fluorophore Release = 100 ( F F0) / (Ft F0) (3 2 ) F = Fluorescence intensity wit h addition of SDS detergent F0 = Fluorescence intensity without any SDS Ft = Fluorescence intensity after addition of a high concentration of SDS ( 5 L of 20 % w/w SDS) A 0.0 0.1 0.2 0.3 0.4 0.5 0 20 40 60 80 100 % Leakage B Figure 38 Vesicle leakage assay for BMP vesicles. A) N eutral pH 7.4 B) A cidic p H 4.2 Figure 38 A plots the percentag e of calcein release for 400 nm extruded BMP vesicles loaded with calcein as a function of titrated SDS concentration. Because calcein enclosed within a large unilamellar vesicle ( LUV ) has low fluorescence emission as a result of collisional quenching, an increase in fluorescence intensity on SDS
125 titration is interpreted as release of the fluorophore with SDS partitioning into the BMP LUV and eventual complete micellization. As a control ( data not shown), the fluorescen ce intensity of calceinloaded BMP vesicles was monitored over a two hour period and showed less than 2 % fluorescence intensity change over this time period. Hence, from these resul ts, it is clear that extruded BMP vesicles contain an interior volume th at can trap the calcein fluorophore. The data from an analogous experiment under acidic conditions are shown in Figure 38 B Because the quantum yield of calcein is very low under acidic conditions, the FRET pair of ANTS/DPX was utilized, where ANTS and D PX were co entrapped within the vesicle. Again, control experiments over two hours without addition of SDS show less than 2 % increase in fluorescence intensity, indicating that the vesicles are stable during the time course of the titration experiment. On addition of SDS, liposome contents are released into the bulk solution and diluted, producing a dequenching of ANTS fluorescence. These results demonstrate that under acidic conditions, 400 nm extruded BMP unilamellar vesicles are stable and contain an interior volume that can entrap molecules. It is noteworthy to mention that the two graphs in Figure 38 differ in shape because of two main reasons; for neutral pH in A, the vesicles used had a much higher concentration (~10 mM) than that at acidic pH in B, hence more SDS was needed to solubilize the vesicles in A than in B. Additionally, the ca l cein experiment in A utilizes fluorescence measurements, whereas the DPX/ANTS assay utilizes FRET, hence fundamental differences between the two techniques may resu lt in the different shapes obtained for the two graphs. Combined, the vesicle leakage assay experiments at both neutral and acidic pH demonstrate that BMP forms unilamellar vesicles that are
126 stable and have an interior volume that can be utilized to encaps ulate aqueous molecules. Characterization of BMP and POPC Hydrated Dispersions and U nilamellar V esicles. Effect of pH Dynamic light scattering and negative staining transmission electron microscopy imaging experiments were performed on BMP and POPC hydrated dispersions and vesicles extruded through 400 nm pore diameters under both neutral and acidic pH conditions to determine the vesicle diameter size distributions and vesicle morphology f or the two lipids under similar conditions. The use of 400 nm poresized membranes for vesicle extrusion resulted in formation of large unilamellar vesicles with a large interior volume, which has the potential for studies involving encapsulation of aqueous molecules. B 0 200 400 600 800 1000 0 20 40 60 80 100 Intensity (au)Vesicle diameter (nm) BMP POPC A 0 500 1000 1500 2000 0 20 40 60 80 100 BMP POPCIntensity (au)Vesicle diameter (nm) Figure 39 DLS vesicle size distributions of BMP an d POPC vesicles, under neutral pH 7.4 A) H ydrated dispersions B) 400 nm E xtruded vesicles Table 33 Summary of average vesicle diameters of BMP and POPC dispersions and extruded vesicles at neutral pH 7.4 Average vesicle diameter (nm) Lipid Type Hydra ted dispersions 400 nm Extruded vesicles BMP 500 250 200 30 POPC 1400 200 400 100
127 DLS is a viable technique for providing information on the size distribution of particles in solution whereas negative staining TEM is useful for providing i nformation on morphological organization as well as vesicle size. Taken together, the two techniques provide valuable information on the morphology and size distribution of macromolecules. Figure 39 illustrates the DLS measurements of BMP and POPC hydrated dispersions and extruded unilamellar vesicles at neutral pH 7.4, while Table 33 provides a summary of the average vesicle diameters for BMP and POPC lipid dispersions and extruded vesicles, observed in the DLS histograms The hydrated dispersions data in Figure 3 9A black solid line show that BMP forms vesicles with an average diameter of ~ 500 nm, while the POPC hydrated dispersions (Figure 3 9A, black dotted line) have their diameter centered at approximately 1.4 m. In Figure 39B, the black solid l ine represents BMP vesicles that were mechanically passed through 400 nm pore sizes membranes have a narrow size distribution, with the highest vesicle intensity observed at ~ 200 nm, while the POPC vesicles have a broader size distribution centered at ~ 400 nm. These data interestingly suggest that under neutral pH, BMP forms smaller vesicles than POPC vesicles. Figure 310. Negative staining TEM images of BMP and POPC under neutral pH conditions. A) BMP hydrated dispersions B) POPC hydrated dispersions. C) BMP 400 nm extruded vesicles D) POPC 400 nm extruded vesicles.
128 TEM images of hydrated dispersions and extruded vesicles of BMP and POPC lipids under neutral pH conditions are presented in Figure 310A D. Under neutral pH BMP hydrated dispersions (Figure 3 10A) form cauliflower like highly structured, nonspherical vesicular morphologies that appear aggregated in clusters of smaller vesicles possibly containing small budlike protrusions. The red arrow in Figure 310A points to a possible side vi ew of the vesicle, indicating that the clustering seen from the top down views is likely not a vesicle inside a vesicle effect but rather the fused tubular structure of smaller vesicle shapes. A size analysis of TEM images of the BMP hydrated dispersions g ives a distribution between 200 and 800 nm for vesicle diameter, centered at 400 nm, which is in agreement with results obtained from DLS measurements. It is interesting to note that when the BMP dispersions are passed through 400nm pore size extrusion m embranes to produce the unilamellar vesicles represented in Figure 310C the clustered structures disappear and well rounded, spherical structures with an average diameter of 200 nm are formed. The size is also consistent with that observed from DLS meas urements. The fact that BMP 400 nm extruded vesicles produce 200 nm vesicles can be understood by considering that the clustered dispersions may have their smaller budlike protrusions sheared off when passed through the extrusion membranes. In comparis on, we found that POPC hydrated dispersions (Figure 310B) form uniform spherical structures that are much larger in size (~2000 nm) than the BMP dispersions, and analysis of the TEM images of POPC 400 nm extruded vesicles reveals the expected spherical structures with vesicle diameter near 500 nm.
129 The clustered macroscopic structure of the BMP dispersions was found to vary with pH. S imilar dynamic light scattering and transmission electron microscopy experiments were performed on the hydrated dispersions and extruded unilamellar vesicles of the BMP and POPC at acidic pH 4.5. 0 1000 2000 3000 4000 0 20 40 60 80 100 Intensity (au)Vesicle diameter (nm) BMP POPC 0 200 400 600 800 1000 1200 1400 0 20 40 60 80 100 Intensity (au)Vesicle diameter (nm) BMP POPC A B Figure 3 11. DLS measurements of BMP and POPC A) hydrated dispersions and B) 400nm extruded large unilamellar vesicles at acidic pH 4.2 Table 34 Average vesicle diameters of BM P and POPC hydrated dispersions and 400nm extruded vesicles at acidic pH 4.2 Average vesicle diameter (nm) Lipid Type Hydra ted dispersions 400 nm Extr uded vesicles BMP 1000 400 250 50 POPC 2400 700 750 250 Figure 311 illustrates the vesicle diameters of hydrated dispersions of BMP (A black solid line) and POPC (A, black dotted line), and 400 nm extruded vesicles of BMP (B, black solid line) and POPC (B black dotted line). The DLS average vesicle diameters data for BMP a nd POPC at acidic pH is also summarized in Table 34. Regardless of pH, BMP dispersions have average vesicle diameters smaller than those of POPC Dispersions obtained when samples are hydrated with sodium acetate buffer ( at pH 4.5) are larger in size for both BMP (~1000 nm) and POPC (~24 00 nm)
130 than under neutral conditions, where vesicles of BMP and POPC hav e average diameters near 500 nm and 1400 nm, respectively. However, the size of the BMP extruded vesicles does not change significantly when pH is al tered. As shown in Figure 3 11B, (black solid line), the BMP vesicles have averag e diameters near 250 nm at pH 4.5 and near 23 0 nm for pH 7.4 (Figure 310B, black solid line) which are both smaller than the diameters of the 400nm pores through which the dispersions were mechanically pass ed. POPC extruded vesicles at acidic pH 4.5 have average vesicle diameters at ~ 750, which is larger than that observed at pH 7.4 (~400 nm) In addition, the extruded BMP vesicles have a much narrower size distribution than that of POPC. A 1000nm B 500nm 1000nm C D 1000nm Figure 312. Negative staining TEM images of BMP and POPC hydrated dispersions and 400nm extruded vesicles at acidic pH. A) BMP hydrated dispersions. B) BMP extruded vesicles. C) POPC hydrated dispersions. D) POPC extruded vesicles.
131 TEM data also shows that the morphology of BMP dispersions varies with pH, forming highly structured, small vesicle clusters at neutral pH, whereas uniformly spherical vesicles are observed at acidic pH. POPC forms uniform, spherical vesicles at both acidic a nd neutral pH conditions. Figure 312 shows TEM images of BMP hydrated dispersions (A) and 400 nm extruded vesicles (B) at pH 4.5 A clear distinction can be seen between the BMP vesicle structures formed at the two pHs with samples at pH 7.4 exhibiting t he clustered, budding structures and those at pH 4.5 forming uniformly nonstructured, spherical shapes ranging from 400 to 800 nm again much smaller in size than POPC dispersions (Figure 312C ). BMP and POPC e xtruded vesicles (Figures 3 12B & D, respecti vely) form spherical vesicles as expected, although again BMP vesicles are relatively smaller in size (~ 250 nm) than POPC vesicles (~ 500 nm). The pH dependence of BMP vesicle morphology may have significant implications in the process of endosome maturation Effects of Ionic strength In addition, the clustered morphology observed in BMP dispersions is dependent on salt. A previous report in the literature provides TEM images of BMP hydrated in 5 mM HEPES buffer pH 7.4 with a spherical morphology (93, 101, 106) We obtained consistent results when using buffer lacking 100 mM NaCl, and these results are shown in Figure 313A On ex trusion through 400 n m membranes, spherically shaped vesicles near 200 nm in diameter are obtained for neutral pH in the absence of NaCl ( Figure 313B ).
132 1000nm 1000nm A 1000nm 5 00nm B Figure 313. Negative staining TEM images of BMP A) hydrated dispersions and B) 400 nm extruded vesicles Vesicles were hydrated in buffer lacking NaCl salt. B A Figure 314. DLS histogra ms of BMP A) hydrated dispersions and B) 400 nm extruded vesicles that were hydrated in the absence of NaCl. Figure 314 shows DLS size distributions of BMP hydrated dispersions and extruded vesicles that were hydrated at neutral pH with buffer that lacked NaCl salt Table 35 Summary of average vesicle diameters of BMP vesicles hydrated without NaCl in the buffer. Average vesicle diameter (nm) BMP vesicles Hydrated dispersions 500 200 400 nm Extruded vesicles 200 40
133 Dynamic light scattering measurements were als o performed on BMP dispersions and extruded vesicles in the absence of salt, at neutral pH 7.4, and the results presented in Figure 3 14 and summarized in Table 35 are consistent with TEM data. BMP hydrated dispersions have average diameters near 500 nm, whereas the 400 nm extruded vesicles are ~ 200 nm in diameter. Combined, the DLS and TEM data presented in this section demonstrate clearly that BMP forms small stable lamellar vesicle structures that have an interior volume, and the vesicle morphology of BMP dispersions vary with pH and ionic strength. Characterization of Hydrated Dispersions and Extruded Vesicles of POPC Mix ed With BMP and POPG Following the detailed characterization of BMP and POPC vesicle structures as a function of pH, ionic strength and polycarbonate extrusion pore diameters, we were interested in determining the effect of BMP when incorporated in typical phospholipid membranes such as POPC and POPG at certain concentrations Because BMP is found at elevated concentrations of up to 1 5 mol% in the late endosome (233) DLS and negative staining TEM experiments were performed on POPC:BMP (85:15) mixtures To study the effect of charge, s imilar DLS and negative staining TEM experiments were performed on POPC: POPG (80:20) mixture since POPG carries a negatively charged like BMP and is also a structural isoform of BMP In order to mimic our previous studies on BMP and POPC lipids, experiments on the POPC: BMP and POPC: POPG lipid mixtures were performed on both their hydrated dispersions and 400 nm extruded vesicles, and the lipids hydrated in a neutral pH 7.4 buffer containing 5 m M HEPES, 0.1 m M EDTA and 100 m M NaCl.
134 0 500 1000 1500 0 20 40 60 80 100 Intensity (au)Vesicle diameter (nm) 0 200 400 600 800 1000 0 20 40 60 80 100 Intensity (au)Vesicle diameter (nm) A B Figure 315. DLS size distribution of POPC: BMP (85:15) A) hydrated dispersions and B) 400nm extruded vesicles at neutral pH. Table 36 Summary of the average vesicle diameters of POPC: BMP (85:15) hydrated dispersions and 400 nm extruded vesicles at neutral pH. Dispersion type Average vesicle diam eter s (nm) Hydrated dispersions 900 200 400 nm Extruded vesicles 350 170 The DLS aver age vesicle diameters for P OPC: BMP hydrated dispersions and 400 nm extruded vesicles are summarized in Figure 3 15 and Table 36. POPC: BMP hydrated dispersions (A) form vesicles with diameters near 900 nm, which is much smaller than that of POPC dispersions at neutral pH (~1400 nm), but slightly larger than the diameters observed in BMP dispersions (~500 nm) at the same pH The 400 nm extruded POPC: BMP vesicles also have relatively smaller diameters (~350 nm) than POPC (~400 nm) but still larger than BMP vesicle diameters (~230 nm). 2000 nm A 2000 nm B Figure 316. Negative staining TEM images of POPC: BMP (85:15) A) hydrated dispersions and B) 400 nm extruded vesicles at neutral pH.
135 POPC: BMP (85:15) dispersions form vesicles with morphologies that differ from those observed for both BMP and POPC dispersions at neutral pH conditions. From the TEM images in Figure 316A, POPC: BMP hydrated dispersions form heterogeneous vesicle structures that are composed of both spherical and nonspherical vesicles. The apparent heterogeneity can likely be attributed to some degree of immiscibility between BMP and POPC lipids, with BMP forming most of the observed nonspherical vesicle structures while POPC forms the uniformly spherical vesicles. A size analysis of the negative staining TEM ima ges also reveals that the PO PC: BMP vesicle structures have diameters over a wide range of 200 1800 nm. The extruded POPC:BMP vesicles (Figure 3 16B) are uniformly spherical and nonstructured as a result of being mechanically passed 31 times through 400 nm extrusion membranes. 0 500 1000 1500 2000 2500 3000 0 20 40 60 80 100 Intensity (au)Vesicle diameter (nm) POPC: POPG POPG 0 100 200 300 400 500 0 20 40 60 80 100 Intensity (au)Vesicle diameter (nm) POPC: POPG POPG A B Fi gure 317. DLS measurements of POPG and POPC: POPG (80:20) hydrated dispersions and extruded vesicles at neutral pH. Table 37 Summary of average vesicle diameters of POPG and POPC: POPG (80:20) vesicles Average vesicle diameter (nm) Lipid Type Hydrated dispersions 400 nm Extruded vesicles POPG 1500 500 230 90 POPC: POPG 1100 300 250 60
136 Results of DLS experiments performed on POPG and POPC:POPG hydrated dispersions and extruded vesicles are presented in Figure 317 and the average vesicle diameters are s ummarized in Table 37. Average diameters of POPG dispersions (~ 1500 nm) differ only slightly from POPC: POPG dispersions ( ~1100 nm), although both are relatively similar to the diameters previously observed for POPC dispersions (~1400 nm), but still smaller than BMP dispersions(~ 500 nm) under neutral pH conditions. Interestingly, POPG and POPC: POPG extruded vesicle diameters (230 and 250 nm, respectively) are close in size to BMP extruded vesicle diameters (~ 230 nm), although POPG and POPC: POPG extrude d vesicles have more broadened size distributions. Taken together the characterization of POPC:BMP and POPC: POPG lipid mixtures at neutral pH reveals that BMP induces small vesicle formation when incorporated in typical POPC membranes at certain concentr ations. BMP also alters the morphology of POPC hydrated dispersions, forming heterogeneous sph erical and nonspherical vesicle structures POPG does not have a significant effect on the vesicle diameter of POPC hydrated dispersions, although it results in f ormation of much smaller vesicles when extruded with 400 nm pore membranes, with diameters resembling those observed for BMP vesicles. Conclusions The analytical capability of the dynamic light scattering (DLS) technique was successfully demonstrated through the accurate measurement of manually mixed vesicle populations. DLS and negative staining transmission electron microscopy (TEM) were utilized to characterize the size of hydrated and extruded BMP and POPC sam ples, revealing that when BMP is hydrated from a dry lipid film, it forms lipid
137 dispersions that have diameters much smaller than those of POPC dispersions regardless of pH. The non spherical clustered vesicle morphology observed in the TEM images of BMP hydrated under neutral pH in the presenc e of 100 mM NaCl differs from what was reported earlier for this lipid. However, the data show that the absence of NaCl can account for the different morphology observed. It is interesting that spherical vesicle shapes were observed under acidic conditions In a 2004 Science report, the Gruenberg group (98) showed that intravesicular structures will spontaneously develop for lipid mixtures containing BMP when a pH gradient was established across the liposome with the interior compartment being acidic. The work reported here has also demonstrated, using vesicle leakage assays and vesicle stability assays, that BMP forms small, stable lamellar vesicles with an interior volume that can encapsulate molecules. Followin g the characterization of POPC: BMP and POPC: POPG lipid mixtures, it was shown that BMP can induce the formation of small vesicles when incorporated in typical POPC membranes at certain concentrations. Taken together, these data suggest that the physical ch emical properties of BMP, dictated by its unique dual glycerol structure and orientation of the phosphate group, may provide a mechanism for stabilization of small vesicle structures in the maturing endosome as the lumen acidifies, shedding further light on the potential functional role of this lipid in the late endosome.
138 CHAPTER 4 ANALYSIS OF CHANGES IN BMP VESICLE SIZE AND MORPHOLOGY IN THE PRESENCE OF GANGLIOS IDE GM1 AT LATE ENDO SOMAL PH 5.5 Introduction Bis(monoacylglycero)phosphate (BMP) is a characteristic lipid of the endocytic degradative pathway that is found in the late endosome luminal membranes in concentrations of approximately 15 mole percent (44) sn1 sn2 sn3 sn1 sn2 sn3 s n3 s n2 s n1 sn glycerol 3phosphate stereoconfiguration in phosphatidylcholine sn 1 glycerophosphosn 1 glycerol stereoconfiguration in BMP A B Figure 41. Chemical structure of A) Phosphatidylch oline and B) BMP The chemical structure of BMP, shown in Figure 41 B differs from that of other glycerophospholipids in that BMP contains two glycero components, each with a single acyl chain (2, 8, 46) Addition ally, BMP has a n sn 1 glycerophospho sn 1' glycerol (sn1:sn1') stereoconfiguration that differs from the typical sn 3 glycerophosphate stereoconfiguration found in other glycerophospholipids as illustrated by the red circles in Figure 41 (47 50)
139 Due to its increased concentration in the late endosomes, BMP is thought to play important structural and functional roles in this organelle (106) Several in vivo investigations have demonstrated that antibodies and chemicals that interact with BMP lead to changes in the sorting and trafficking of proteins and lipids in late endosomes, resulting in an altered struc ture of the late endosome (44, 114) and abnormal accumulation of cholesterol (108) BMP is also essential for lysosomal catabolism processes (46) such as the activator stimulated hydrolysis of gangliosides GM1 (109) and GM2 (110) and the hydrolysis of ceramide by acid ceramidase (111) Gangliosides are sialic acidcontaining glycosphingolipids found in the cell membranes of vertebrates and are particularly high in abundance in the plasma membrane of neuronal cells (234) Glycos phingolipid degradation proceeds via endocytosis from the plasma membrane and subsequent transportation to the endosomes, followed by intraendosomal vesicle formation and final trafficking as intralysosomal vesicles for degradation (235) Deficiencies in the ca tabolism of gangliosides result in lysosomal glycosphingolipid accumulation, leading to clinical disorders known as sphingolipid storage diseases that mainly affect neuronal cells within the brain (236) Much insight into the process of membrane digestion has been realized through the investigation of glycosphingolipid catabolism (46) The lysosomal degradation of glycosphingolipids is a sequential pathway of reactions that are catalyzed by exohydrolases with acidic pH optima. These enzymes are assisted by small glycoprotein cofactors, known as the sphingolipid act ivator proteins (SAPs), and lipid composition has been shown to alter the in vitro degradation kinetics; optimum activity
140 is obtained with low cholesterol content and the presence of the negatively charged lipid BMP (46) Work presented previously in Chapter 3 discussed in detail the characterization of the morphology and molecul ar organization of sn3 sn 1 BMP (BMP) under both acidic and neutral pH conditions I t was showed that when hydrated, BMP forms small, stable lamellar vesicles with interior volumes and with acyl chain dynamics and packing similar to other glyce rophospholipids (105) Given the unique chemical structure of BMP, this vesicle morphology was surprising as it had been previously assumed that BMP would f orm either micellar structures similar to detergent or inverted hexagonal morphologies, as is seen with phosphatidylethanolamine lipids (237) T he main objective of the work reported in this chapter was to study the effect of adding ganglioside GM1 to BMP membranes at varying concentrations, under late endosomal pH conditions. Dynamic lig ht scattering (DLS) and negative staining transmission electron microscopy (TEM) were utilized to monitor the size, morphological and structural changes in hydrated dispersions and extruded vesicles of BMP: GM1 mixtures. Results presented in this chapter al so include the characterization of the morphology and size distribution of BMP hydrated lipid dispersions under different pH conditions ranging from acidic (pH 4.2) to neutral (pH 7.4) conditions, using TEM and DLS Finally, studies were performed that mo nitored the effect of incorporating GM1 and BMP in typical phosphatidylcholine (POPC) membranes POPC is abundantly present in biological cellular membranes and has been well characterized as a model system; hence it made for a good lipid choice for typica l model system studies By utilizing dynamic light scattering and transmission emission spectroscopy, the
141 morphology and size distribution of POPC: GM1 and POPC: BMP: GM1 hydrated dispersions and extruded vesicles were investigated. Experimental Section Ma terials Used BMP18:1, ((S, R Isomer) sn (3 Oleoyl 2 Hydroxy) Glycerol 1 Phosphosn 3' (1' Oleoyl 2' Hydroxy) Glycerol, ammonium Salt)), in chloroform and ganglioside GM1 powder, were purchased from Avanti Polar Lipids (Alabaster, AL) and used without furth er purification. HEPES, (4(2 hydroxyethyl,) 1 piperazineethanesulfonic acid), C8H18N2O4S)); NaOAc, (sodium Acetate); EDTA, (ethylenediamine tetraacetic acid, C10H16N2O8) and NaCl, (sodium chloride) were purchased from Fisher Biotech (Pittsburgh, PA). CH3C l, (chloroform); MeOH, (methanol); CH3CH2OH (ethanol); C6H12, (cyclohexane); NH4OH, (ammonium hydroxide); HCl, (hydrochloric acid) and NaOH, (sodium hydroxide) were obtained from Fisher Scientific (Pittsburgh, PA). UO2 (CH3COO) 2.2H2O, (uranyl acetate) and 400 mesh Formvar coated copper grids were purchased from Ted Pella (Redding, CA). Singlesealed 50to 1000 mL disposable cuvettes (10mm path length) were obtained from Eppendorf (Westbury, NY). 400nm polycarbonate extrusion membranes and filter support s were purchased from Avanti Polar Lipids (Alabaster, AL). Silica coated aluminum thin layer chromatography (TLC) plates were purchased from Whatman (Florham Park, New Jersey). Preparation of H yd rated Lipid Dispersions and E xtruded Unilamellar V esicles The desired amount of stock lipid (5 mg/mL BMP in chloroform, or 1mg/mL GM1 in chloroform methanol mixture ), was dried under a gentle nitrogen stream for about 10 minutes or until the solvent evaporated, forming a dry, thin lipid film. The sample was then further dried under vacuum in a desiccator for
142 solvent. Dry lipid films of 100% BMP or BMP:GM1 lipid mixtures were hydrated with either 2 mL of 5 mM NaOAc buffer for pH 4.2, 5.5 and 6.1, or 2 mL 5m M HEPES buffer for pH 7.4. All buffers contained 100 mM NaCl and 0.1 mM EDTA, and the final lipid concentration was approximately 0.75 mM. Hydrated BMP:GM1 lipid mixtures were freeze thawed in liquid N2 five times. All hydrated dispersions were incubated at room temperature for ap proximately 12 hours before extrusion or measurement by dynamic light scattering (DLS) and negative staining transmission electron microscopy (TEM). To form large unilamellar vesicles (LUVs), hydrated lipid dispersions were extruded by passing 31 times through 400 nm polycarbonate extrusion membranes. Phospholipid integrity was verified by thin layer chromatography (TLC), where approximately 10 L of lipid sample was spotted on silica coated aluminum plates. Plates were placed in a chamber containing a CH3Cl: MeOH: NH4OH (65:25:10) mobile phase. The TLC plates were developed in an iodine chamber and visualized by eye. Instrumentation Dynamic light s cattering (DLS) Size distribution measurements of hydrated dispersions and extruded unilamellar lipid vesicles were performed with a Brookhaven 90Plus/BI MAS ZetaPALS spectrometer operated at a wavelength of 659 nm and at 25 C. The instrument uses a BI 9000AT digital autocorrelator and 9KDLSW data acquisition software. A 100L sample volume in a disposable cuvet te was used for each measurement. For each sample, 3 runs were performed with each run lasting 3 minutes. Data and histograms were further analyzed and converted into B spline plots using OriginPro 8 software. DLS data were reported as an average of 3 runs for each sample, and errors calculated as a standard deviations of the mean diameter.
143 Negative staining transmission electron m icroscopy (TEM) TEM images were obtained using a Hitachi H 7000 transmission electron microscope operated at 75100 kV with a S oft Imaging System MegaViewIII with AnalySIS digital camera (Lakewood, CO). The microscope has a maximum resolution at 0.2 nm with a magnification range of 110 to 600,000. Prior to TEM measurements, samples were further prepared by negative staining. Bri efly, for all samples, using a disposable pipette, a drop of the lipid vesicle sample was spread on a 400mesh Formvar coated copper grid held by tweezers and incubated for 2 minutes. Excess lipid sample was gently dabbed away with filter paper, and the gr id was allowed to dry for 2 minutes. In some instances a drop of deionized water was added to the grid to remove any excess salt from the buffer solution used in vesicle preparation. One drop of 2% uranyl acetate was then added to the grid and allowed to s tain for 2 minutes, after which any excess uranyl acetate was wiped away, and the sample was allowed to dry for 2 minutes before being placed in the electron microscope specimen holder for image analysis and collection. Results and Discussion Characterizat ion of BMP Hydrated Lipid Dispersions as a Function of pH Because the pH varies in different sub compartments of the endocytic pathway, with acidification increasing progressively from the endocytic carrier vesicles and early endosomes to late endosomes, and eventually lysosomes (238) BMP hydrated dispersions were prepared at four different pH conditions; pHs 4.2, 5.5, 6.1 and 7.4, indicative of the in vivo pH in the lysosome, late endosome, early endosome and cytosol, respectively.
144 pH 4.2 1000 nm pH 5.5 500 nm pH 6.1 500 nm 200 nm pH 7.4 Figure 42 Negative staining TEM images of BMP lipid dispersions as a f unction of pH. Figure 42 exhibits negative staining TEM images of BMP lipid dispersions as a function of pH. These images show that BMP dispersions exhibit different morphologies as the pH is varied; progressing from fully spherical, nonstructured vesic les at acidic pH 4.2 to nonspherical, highly structured vesicle clusters at neutral pH 7.4. In addition, the sizes of the BMP dispersions are found to vary as a function of pH. At pH 4.2, BMP lipid dispersions are homogenously spherical, nonstructured, and have an average size of ~ 1 m. This is in contrast to the vesicle morphology of BMP dispersions seen at late endosomal pH 5.5, which though spherical, show some slight structuring and are also significantly smaller, with a size distribution of 0.5 1 m. BMP vesicles at pH 6.1 exhibit extensive structural deviations from a spherical shape appearing like aggregations of a number of smaller vesicles. The vesicles at early
145 endosomal pH 6.1 are also smaller in size than those at both pH 4.2 and pH 5.5, wi th a size distribution of 0.5 0.8 m. At pH 7.4, BMP dispersions have a highly budded and protruding non spherical shape appearing highly clustered, with an average vesicle size of 500 nm. 0 500 1000 1500 2000 2500 0 20 40 60 80 100 Intensity (au)Vesicle Diameter (nm) a, pH 4.2 b, pH 5.5 c, pH 6.1 d, pH 7.4 a c d b F igure 4 3. Dynamic light scattering size distributions of BMP hydrated dispersions as a function of pH. Table 41 Summary of the average diameter of BMP hydrated dispersions at specific pH conditions pH 4.2 5.5 6.1 7.4 BMP Vesicle Diameter 950 310 390 150 670 330 500 250 The size distributions of BMP hy drated dispersions at various pH conditions were also determined by utilizing dynamic light scattering, and the r esults are shown in Figure 4 3, and summarized in Table 41 These data were obtained by averaging three independent DLS histograms and the err or calculated as standard deviation from the
146 average size. The DLS data shows increasing vesicle diameter with increasing acidity, except endosomal pH 5.5 which exhibits the lowest vesicle diameter at ~ 400 nm At acidic pH 4.2, the vesicles have the largest average diameter of ~ 1 m while at pH 6.1 the vesicles exhibit diameters in the range of ~ 700 nm. Neutral pH 7.4 shows vesicles with diameters of ~ 500 nm and vesicles at endosomal pH 5.5 differ from this general trend by exhibiting the smallest vesic le diameter of ~ 400 nm. The variation in vesicle diameter at pH 5.5 could be attributed to the possible formation of intraendosomal vesicular bodies that are significantly smaller in size and this finding is consistent with the TEM data discussed previously Taken together, results from these experiments showed that the morphology of the hydrated BMP dispersions vary with pH, further suggesting a role for BMP in intraendosomal vesicular body formation, which is triggered in the late endosome by the biosynthesis of BMP and a drop in endosomal lumen pH (105) C haracter ization of BMP: GM1 Hydrated Dispersions and Extruded V e sicles at Specific Concentrations Based on their individual molecular geometries and chemical structures (Figure 44) BMP and GM1 independently exhibit different polymorphisms when hydrated. The ganglioside GM1 is a surfactant molecule with a bulky sugar headgroup that forms micelles rather than lamellar liposomes (239) On the other hand, BMP adopts a lamellar bilayer structure in an aqueous environment (105) We investigated the different macroscopic shapes and sizes in mixtures of BMP and GM1 under endosomal pH.
147 A: BMP B: GM1 F igure 44. C hemica l structures of A) BMP and B) ganglioside GM1. Figure 44 is an illustration of the chemical structures of BMP and ganglioside GM1. By preparing samples with varying concentrations of BMP: GM1 mixtures, DLS and TEM investigations were performed to determine what lipid polymorphism would dominate as the relative mole percentages of each lipid were varied. Hydrated lipid dispersions and 400nm extruded vesicles of 100% BMP, and BMP: GM1 lipid mixtures in 10 mole% GM1 increments at 90:10, 80:20, 70:30, 60:40 and 50:50 were prepared as discussed in the experimental section. DLS measurements were performed on both hydrated dispersions and extruded vesicles, whereas only the hydrated dispersions of BMP and BMP: GM1 were imaged by TEM, because it is expected that the extruded vesicles would form uniformly spherical morphologies as a result of being mechanically forced through 400 nm pore membranes as observed in previous investigations discussed in C hapter 3.
148 A 100:0 1m B 90:10 1m 1m C 80:20 1m D 70:30 1m E 60:40 1m F 50:50 F igure 45. Negative staining TEM images of hydrated BM P:GM1 dispersions at specific molar ratios at pH 5.5. 500 nm 1000 nm A B Figure 46 TEM images of 70:30 mol % BMP: GM1 lipid mixture, showing A) small, ag gregated homogenous vesicles and B) magnified images of the same vesicles. Figure 4 5 show the negative staining TE M images of BMP : GM1 hydrated dispersions where the mol % of GM1 is varied from 0 % to 50 % in 10 % increments. Figure 46A shows the apparent aggregated morphology of 30% GM1 in 70% BMP
149 hydrated dispersions, and the magnified images of the same sample is shown in Figure 4 6B. The 100 % BMP dispersions (Fig ure 45A, 100:0) are spherical in shape with diameters ranging between ~ 200 500 nm Upon mixing with 10 % GM1, (Figure 45B, 90:10), the vesicle morphology changes to nonspherical heterogeneous structures mixed with some small spherical vesicles observed. The average diameter for this mixture is between ~ 300 60 0 nm, with sizes larger than 1 m detected. Note the presence of the smaller, more spherical vesicles, which may correspond to predominant BMP vesicles, suggesting immiscibility of the two lipids at this composition. For BMP:G M1 mole ratios of 80:20 ( Figure 45C) and 70:30 (Figure 45D ), spherical vesicle structures are obtained. The vesicles formed from the 80:20 BMP:GM1 mixture have a spherical shape, with average diameters ranging from ~ 300 500 nm. On the other hand, BMP:GM1 (70:30 ) produced nearly homogeneous spherica l vesicles of diameter ~100 nm, that appeared aggregated in the TEM images. Figure 47 shows dynamic light scattering si ze distribution histograms of BMP: GM1 hydrated dispersions (A) and extruded unilamellar vesicles (B) at specific mol %, whereas Table 42 displays a summary of the average vesicle diameters and error bars of the same samples in Figure 4 7, obtained from c alculating the average of three independent DLS histograms. The error bars were obtained as standard deviations of the most probable vesicle diameter for each sample.
150 A B Figure 47 DLS s ize distribution histograms of BMP: GM1 A) Hydrated dispersions and B) 400 nm E xtruded unilamellar v esicles at specific concentrations. Table 42 S ummary of DLS average vesicle s izes of BMP:GM1 hydrated lipid d ispersions and 400 nm extruded un ilamellar vesicles at specific concentrations. Lipid composition 100% BMP 90:10 BMP:GM1 80:20 BMP:GM1 70:30 BMP:GM1 60:40 BMP:GM1 50:50 BMP:GM1 Hydrated D ispersions (nm) 390 150 420 150 380 70 430 250 540 110 650 110 Extruded V esicles (nm) 230 40 230 70 180 20 230 30 230 50 240 50 Error bars were calculated as standard deviations of the most probable average vesicle size obtained from averaging three independent DLS histograms.
151 Results from DLS measurements of 30 mol % GM1 sample shown in Table 4 2 reveal a size distribution of 200 600 nm, with the average vesicle size observed at 430 nm. The increased average vesicle size seen with DLS is likely due to the aggregation of the smaller vesicles that are discernable in the TEM images. This difference in size can be interpreted to arise from the fact that DLS reports the average spherical shape of the aggregate hydrodynamic diameter of the aggregations, and not that of the individual vesicles. Perhaps the relatively low laser power (35 mW) and detector (photo multiplier tube, PMT) of the particular DLS instrument that was used may also limit detection of the smaller sized particles. It is noteworthy that the formation of homogeneous vesicles with 20 30 mol % GM1 in BMP membranes is consistent with previous work by Lee and coworkers (239) using Langmuir monolayer preparat ions of GM1 mixtures with dipalmitoylphosphatidylcholine (DPPC) to show that GM1 has a condensing effect on DPPC lipids over the concentration range of 20 30 mol %. Taken together, these findings indicate a biologically relevant concentration range for GM1 incorporation into lipid raft domains or being trafficked to ganglioside enriched endosomal vesicles in the lysosome. For concentrations eterogeneous nonspherical structures are aga in obtained. At this r atio (Figure 45E ), both small spherical vesicles and irregularly shaped larger structures are observed. This heterogeneity in size and structure gives a distribution of diameters ranging f rom 400 700 nm. The presence of two distinct macroscopic morphologies may indicate immiscibility of the two lipids above the 30 mol
152 % ratio. Similar findings are seen for the 50:50 BMP: GM1 sample (Figure 45F ), where the size distribution increases to 5 00 800 nm. In general, the vesicle diameters determ ined from DLS measurements of the hydrated dispersions (Figure 4 7 A ) are consistent with results from TEM images discussed above. The DLS measurements of the unilamellar vesicles extruded with 400 nm polycarbonate membranes (Figure 4 7B) reveal vesicle diameters that are consistently centered at ~ 230 nm for all mol % ratios as would be expected, except for the 80:20 BMP:GM1 ratio, which has an average diameter at ~180 nm However as mentioned previousl y, for the 70:30 BMP:GM1 ratio, the DLS measurements of the hydrated dispersions give a larger size than seen with TEM, likely because of aggregation of the smaller vesicles. In earlier studies, Kobayashi and coworkers examined the membrane structure of v arious BMP: ganglioside mixtures under neutral and acidic pH conditions and found that at pH 8.5 6.5, the BMP:GM1 (1:1 mol/mol) mixture formed small vesicular aggregates, whereas the mixture formed closely packed lamellar structures under acidic conditio ns (pH 5.54.6) (101) The apparent miscibility of GM1 with BMP over the 2030 mol % r atio s, as evidenced by the spontaneous formation of small homogenously shaped vesicles with narrow diameter size distribution, suggests that optimum interactions between molecules may occur for a given molecular ratio of 3 BMP molecules for 1 GM1 molecule to form a lipid complex. This hypothesis is drawn from the analysis of Langmuir monolayer studies, which showed that the pressurearea isotherms of GM1 and DPPC mixtures follow ideal mixing behavior when the two
153 species were considered to be a 3:1 DPPC:G M1 complex interacting with excess DPPC (239) Effect of GM1 and BMP Mixing with POPC Membranes Eukaryotic cellular membranes are predominantly composed of a high conc entration of phospholipids (2, 8) especially phosphatidylcholine (POPC) lipid moieties, making POPC one of the most well studied and characterized phospholipids. In Chapter 3, 100 % POPC vesicle size and morphology were characterized by dynamic light scattering ( DLS ) and transmission electron microscopy (TEM) under neutral, physiological pH 7.4. 0 500 1000 1500 2000 0 20 40 60 80 100 Intensity (au)Vesicle Diameter (nm) Hydrated dispersions Extruded vesicles A 200nmB Figure 48. Dynamic light scattering average vesicle diameters of POPC A ) black solid line, h ydrated dispersions, A ) red solid line, unilamellar vesicles extruded with 4 00 nm polycarbonate membranes and B) TEM image of POPC hydrated dispersions F igure 48 re presents results of 100 % POPC characterization under neutral pH conditions in which POPC hydrated dispersions (A, black solid line) were demonstrated
154 to have average vesicle diameters of ~ 1.3 m, while unilamellar vesicles extruded with 400 nm pore membranes ( A, red solid line) ha d ~ 400 nm vesicl e diameters TEM imaging of POPC hydrated dispersions reveals spherical vesicle structures with a size range (1 2 m) that is consistent with DLS analysis. In order to monitor the effect of GM1 and B MP on typical phospholipid macroscopic morphology, structure and size distribution, DLS and TEM experiments were performed that incorporated 20 mol % GM1 in 80 mol % POPC, then 15 mol % GM1 and 15 mol % BMP in 70 mol % POPC at late endosomal pH 5.5. B ASize distribution (nm) A: Hydrated dispersions 2100 800 B: Extruded vesicles 380 100 C: TEM size range 5003000 500nmC 0 1000 2000 3000 4000 5000 0 20 40 60 80 100 Intensity (au)Vesicle diameter (nm) 0 100 200 300 400 500 600 700 800 0 20 40 60 80 100 Intensity (au)Vesicle diameter (nm) F igure 49 Dynamic light scattering average vesicle diameters of POPC : GM1 (80:20) A) hydrated dispersions B) 400 nm extruded vesicles and C) TEM images of POPC : GM1 hydrated dispersions at pH 5.5.
155 Figure 49 is an illustration of the DLS average vesicle di ameters obtained for POPC : GM1 hydrated dispersions (A), extruded vesicles (B) and the TEM images of POPC : GM1 hydrated dispersions (C) The DLS data reveal larger vesicle diameters for the POPC : GM1 hydrated dispersions (Figure 4 9 A), with average vesicle di ameters of ~ 2.1 m compared to the vesicle diameters observed for POPC hydrated dispersions (~1.4 m ) Th e increased size is also supported by TEM analysis, where images of the POPC : GM1 hydrated dispersions (Figure 49 C) have vesicle diameters of up to 3 .0 m The POPC:GM1 vesicle morphology is also different from that observed for 100 % POPC, displaying structured, bud ding like vesicles. The structured morphology and i ncreased vesicle sizes of POPC: GM1 hydrated dispersions could be as a result of the pre sence of ganglioside GM1, whose bulky sugar head groups contribute favorably to the formation of larger, lipid vesicles (239) POPC:GM1 unilamellar vesicles that were e xtruded with 400 nm polycarbonate membranes (Figure 4 9B) were also measured by dynamic light scattering, showing average vesicle diameters of ~ 380 nm, as would be expected. 1000nm Size distribution (nm) A : Hydrated dispersions 500 230 B : Extruded vesicles 230 50 C : TEM size range 500 1000 0 100 200 300 400 500 600 0 20 40 60 80 100 Intensity (au)Vesicle diameter (nm) A B C 0 200 400 600 800 1000 1200 1400 0 20 40 60 80 100 Intensity (au)Vesicle diameter (nm) Figure 410. Ave rage vesicle diameters of POPC:BMP: GM1 (70:15:15) A) hydrated dispersions B) 400 nm extruded vesicles and C) TEM images of POPC: BMP: GM1 hydrated dispersions.
156 The incorporation of 15 mol % BMP in POPC a n d GM1 lipid membranes mimics the percent concentration of BMP in the late endosome, where it is found in concentr ations of up to 15 mol %. Figure 410 is an illustration of the average vesicle diameters of POPC:BMP: GM1 (70:15:15) hydrated dispersions, unilamellar vesicles extruded with 400 nm polycarbonate membranes and TEM images of POPC:BMP: GM1 hydrated dispersions The average vesicle diameters were obtained by calculating the average of three independent DLS histograms, and all experiments were performed under pH 5.5. Interestingly, the vesicle diameters of the POPC BMP: GM1 hydrated dispersions are much smaller ( ~ 500 nm ) than those observed for the POPC : GM1 dispersions ( ~ 2100 nm) discussed previously.TEM analysis also reveals highly heterogeneous vesicles formed by the POPC :BMP: GM1 dispersions (Figure 410C), displaying vesicles of varied sizes, ranging from 500 1000 nm. Some of the vesicle structures are non spherical and clustered, while others are uniformly spherical and nonclustered, indicating possible immiscibility among the POPC, BMP and GM1 lipids. The unilamellar extruded vesicles have average diameter s at ~230 nm, which is smaller than that obtained for POPC:GM1 extruded vesicles(~380 nm), but similar to the vesicle diameter obtained for 100 % BMP vesicles extruded with 400 nm polycarbonate membranes. The significantly reduced vesicle diameters for t he POPC BMP: GM1 hydrated dispersions and extruded vesicles could be attributed to the presence of BMP in the lipid mixture, which perhaps because of unique structure and stereochemistry in it s phosphate headgroup allows for the ene rgy required for the st abilization of smaller vesicle formation (105) The heterogeneous formation of vesicles with varied
157 morphologies could also be as a result of the ganglioside GM1 in the lipid mixture which hinders proper mixing and packing because of increased steric hindrance in the bulky sugar headgroups of the ganglioside (239) It might be interesting to perform studies that can enable tracking of the vesicles in such a lipid mixture, such as the use of secondary Au antibody labeling, to determine the preference of each individual lipid in forming either smaller or larger vesicles. Conclusions The results in this chapter summarize the effect of pH, concentration and lipid composition on the structural morphology and vesicle diameters of phospholipid membranes. Results show that the BMP vesicle morphology and size vary with pH, progressing from highly st ructured, nonspherical vesicle clusters at neutral pH 7.4 to uniformly spherical nonstructured vesicles at acidic pH 4.2. BMP vesicle sizes also increase with increasing acidity, although pH 5.5 f orms unusually smaller vesicles indicative of the small intraendosomal vesicular bodies found in the late endosome. These findings might give more insight in to the functions of this unique lipid in the late endosome. The results on GM1 interactions with B MP demonstrate that GM1 mixes with BMP to form small (~100 nm) spherical shaped vesicles with a narrow size distribution at similar concentrations that were seen to condense the unsaturated DPPC lipid and form a specific complex. The apparent miscibility of GM1 with BMP over the 2030 mol % ratio s, as evidenced by the spontaneous formation of small homogenously shaped vesicles with narrow diameter size distribution, suggests that optimum interactions between molecules may occur for a given molecular ratio o f 3 BMP molecules for 1 GM1 molecule to form a lipid complex. This hypothesis is drawn from the analysis of
158 Langmuir monolayer studies, which showed that the pressurearea isotherms of GM1 and dipalmitoyl phosphatidylcholine ( DPPC ) mixtures follow ideal mixing behavior when the two species were considered to be a 3:1 DPPC:GM1 complex interacting with excess DPPC (239) This specific mixture of GM1 with BMP may be impor tant for in vivo vesicular trafficking and lipid sorting in the endosome/lysosome pathways. Finally, w hen incorporated in typical POPC membranes at specific concentrations ganglioside GM1 and BMP alter the morphology and size of the dispersions and vesicl es that are formed. Larger vesicle diameters are formed in the presence of 20 mol % GM1 in 80 mol % POPC, whereas smaller, structured vesicles are obtained when 15 mol % BMP and 15 mol % GM1 lipids are mixed with 70 mol % POPC .Further studies on this model lipid system using assays such as secondary antibody labeling may shed more light on the lipid distributions in the vesicle formation, which would provide further understanding on the role of BMP and GM1 in the endocytic pathway.
159 CHAPTER 5 EFFECT OF CHO LESTEROL MIXING WITH GM1 AND BMP ON PHOSPHOLIPID MODEL MEMBRANES AT LATE ENDOSOMAL PH 5.5 Introduction Biological membranes obtained from mammalian cells are complex structures that contain a wide variety of lipids, including phospholipids and cholesterol. Cholesterol (Figure 5 1) is known to play a structural as well as a regulatory role in biomembranes, and is nonhomogeneously distributed in cell membranes, with a high concentration in the plasma membrane and a low concentration in the membranes of intrac ellular organelles such as the late endosomes (240) A major characteristic feature of late endosomes is a com plex system of internal membranes within the lumen (44, 97) I t is thought that l ate endosomes are the most complex organelles of the endocytic pathway due to their ability to communicate with other organelles, such as in inter organellar trafficking with Golgi, lysosomes, or e ndoplasmic reticulum (ER) and also due to the communication between the intralumenal vesicles and the limiting membrane, in intra organellar trafficking In essence, b oth proteins and lipids are responsible for generating and maintaining the compartmentalization and proper functioning of the late endosome (241) Kobayashis group (108) demonstrated in 1999 that the internal membrane network of late endosomes contains high amounts of the unique, poorly degradable phospholipid bis(monoacylglycero)phosphate (BMP), which is erroneous ly also termed as lysobisphosphatidic acid (LBPA) as shown in Figure 51 BMP also forms a specialized membrane domain within endosomes (44)
160 Studies show that BMP plays a function in cholesterol efflux from late endosomes and lysosomes. In one study, it was shown that i f antibodies against BMP are internalized by fluidphase endocytosis, they bind to BMP and accumulate in late endosomes. Under these conditions, cholesterol released from low density lipoprotein (L DL) remains trapped in the late endosomes and cannot be transported out from this organelle as would normally occur if the antibody were absent (241, 242) C holesterol accumulation in late endosomes is therefore cau sed by highly specific perturbations of internal membranes with a monoclonal antibody against BMP or with human antibodies which recognize BMP (108) Additionally, t he network of membrane tubules and vesicles withi n the lumen of late endosomes might have an important function in sphingolipid degradation and cholesterol distribution in the cell. Hence, c holesterol transport depends not only on its intrinsic physical properties but also on the physicochemical and dyna mic properties of B MP rich internal membranes. T he a ccumulation of cholesterol within BMP rich internal membranes is thus predicted to alter m embrane properties even further (243) Accumulation of endocytosed antibodies against BMP also results in the defective sorting/trafficking of proteins that transit via late endosomes, presumably because membrane properties are altered (118) P athological accumulation of specific lipids, especially cholesterol, in the late endosome is also associated with a s pecial class of diseases referred to as lipid storage diseases which are subsequently thought to trigger membrane traff icking defects (244, 245) The consequences of the accumulation of a given lipid on membrane organization, compartmentalization and function are generally still poorly understood,
161 although a great number of studies have been directed at better understanding the role of cholesterol in lipid storage diseases For instance, Sobos group (241) st udied the consequences of late endosomal cholesterol accumulation as encountered in the Niemann Pick Type C disease (NPC) (245) NPC is a fatal, autosomal recessive neurodegen erative disease due to mutations in the NPC1 or NPC2 genes (246) and t he main biochemical manifestation in NPC is elevated late endosomal accumulation of free cholesterol (247249) followed by an increase in sphingolipids (250) It has been proposed that endosomal accumulation of cholesterol and sphingolipids would lead to an over load of cholesterol rich raft like membrane domains which means an increase in raft to nonraft membrane ratio, caus ing a general jam in traffic through the endosomal compartment (237) B ecause of the complexity of biomembranes, and ultimately late endosomes, studies of the nature and consequences of the interaction of cholesterol with phospholipids have generally been car ried out on model membranes composed of well defined phospholipids to which cholesterol is added. In a study by Mobius and group, it was indicated by immunoelectron microscopical examination of human B lymphocytes (251) that about 80 % of the cholesterol detected in the endocytic pathway is present in the recycling compartments and in internal membranes of early and late endosom es, whereas it is nearly completely absent in inner lysosomal membranes. Studies on the differential lipid composition of the internal endo or lysosomal membranes have also been obtained with the aid of exogenous addition of ganglioside GM1 derivatives bearing a photo affinity label and fluorescenceor biotinlabels to cultured cells and subsequent monitoring of endocytosis by fluorescence microscopy (252, 253)
162 A number of lipid mixtures have been used to mimic the biophysical characteristics of lipid domains in membrane model systems, and some studies have confirmed the importance of using natural raft mixtures rich in sphingomyelin ( SM ) CHOL and small amounts of glycosphingolipids, such as gangliosides, instead of mixtures containing model raft lipids such as dipalmitoyl phosphatidylcholine ( DPPC ) (254256) Essentially, the experimental mixtures designed to mimic putative coexisting raft and nonraft domains are usually composed of ternary mixtures of unsaturated CHOL and in some cases small amounts of the ganglioside G M1 with all components present in the lipid mix ture at specific molar fractions. The st udies reported in this chapter utilized the particle sizing technique of dynamic light scattering (DLS), and negative staining transmission electron microscopy (TEM) to characterize vesicle size and macroscopic morphologies observed when cholesterol is inc orporated in phospholipid membranes containing BMP and ganglioside GM1. In efforts to mimic the late endosome environment, all experiments were performed at pH 5.5. DLS and TEM experiments were performed on hydrated lipid dispersions and 400 nm extruded v esicles of (1) POPC: CHOL, (2) BMP: CHOL, (3) POPC:BMP: CHOL, (4) POPC:GM1:CHOL and (5) POPC:GM1:BMP:CHOL that varied in lipid concentrations Extruded vesicles were produced by manually passing the hydrated dispersions of each lipid mixture through 400 nm p ore membranes, for a total of 31 times, to ensure a uniform size distribution of vesicles. F indings from these studies may be helpful in
163 further understanding the role that cholesterol /BMP interaction may play i n lipid lipid interactions and formation of l ipid rafts in the cell, specifically in the endocytic pathway. Experimental Details Materials Used BMP18:1, ((S, R Isomer) sn (3 Oleoyl 2 Hydroxy) Glycerol 1 Phosphosn 3' (1' Oleoyl 2' Hydroxy) Glycerol, ammonium Salt)), POPC, (1 Palmitoyl2 Oleoyl sn G lycero 3 Phosphocholine) both in chloroform and Cholesterol ( C27H46O, CHOL) and ganglioside GM1 powder s, were purchased from Avanti Polar Lipids (Alabaster, AL) and used without further purification. NaOAc, (sodium Acetate); EDTA, (ethylenediamine tetraac etic acid, C10H16N2O8) and NaCl, (sodium chloride) were purchased from Fisher Biotech (Pittsburgh, PA). CH3Cl, (chloroform); MeOH, (methanol); CH3CH2OH (ethanol); C6H12, (cyclohexane); NH4OH, (ammonium hydroxide); HC l (hydrochloric acid) and NaOH, (sodium hydroxide) were obtained from Fisher Scientific (Pittsburgh, PA). UO2 (CH3COO) 2.2H2O, (uranyl acetate) and 400mesh Formvar coated copper grids were purchased from Ted Pella (Redding, CA). Singlesealed 50to 1000 mL disposable cuvettes (10mm path leng th) were obtained from Eppendorf (Westbury, NY). 400nm polycarbonate extrusion membranes and filter supports were purchased from Avanti Polar Lipids (Alabaster, AL). Silica coated aluminum thin layer chromatography (TLC) plates were purchased from Whatman (Florham Park, New Jersey). Hydrated Lipid Dispersions and Extruded Vesicle Preparation The desired amount of stock lipid (5 mg/mL BMP and 20 mg/mL POPC in chloroform or 1 mg/mL GM1 and 5 mg/ mL cholesterol in chloroform methanol mixture (2:1, v:v) ) was co llected and dried under a gentle nitrogen stream for about 10 minutes or until the sol vent evaporated, forming a dry thin lipid film. The sample was
164 further dried under in a vacuum desiccator for solvent. Dry lipid films of BMP: CHOL, POPC: CHOL, POPC: GM1: CHOL, POPC: BMP: CHOL and POPC: GM1 BMP: CHOL lipid mixtures at specific mol % ratios were hydrated with 2 mL of 5 mM NaOAc buffer containing 100 mM NaCl and 0.1 mM EDTA, at pH 5.5, forming hydrated lipid dispersions with a final lipid concentration of approximately 0.75 mM. The hydrated lipid dispersions were freezethawed in liquid N2 five times, then incubated at room temperature for approximately 12 hours before extrusion or measurement by dynamic light scattering (DLS) and transmission electron microscopy (TEM). To form extruded large unilamellar vesicles (LUVs), the hydrated lipid dispersions were extruded by passing 31 times through 400 nm polycarbonate extrusion membranes. Phospholipid integrity was verified by thin layer chromatography (TLC), where approximately 10 L of lipid sample was spotted on silica coated aluminum plates. Plates were placed in a chamber containing a CH3Cl: MeOH: NH4OH ( 65:25:10) mobile phase. The TLC plates were developed in an iodine ch amber and visualized by eye. Instrumentation Dynamic light scattering (DLS) Dynamic light scattering size distribution and characterization measurements of hydrated dispersions and extruded unilamellar lipid vesicles were performed with a Brookhaven 90Plus /BIMAS ZetaPALS spectrometer operated at a wavelength of 659 nm and at 25 C. The instrument uses a BI 9000AT digital autocorrelator and 9KDLSW data acquisition software. A 100L sample volume in a disposable cuvette was used for each measurement. For each sample, 3 runs were performed with each run lasting 3 minutes. Data and histograms were further analyzed and converted into B spline plots
165 using OriginPro 8 software. DLS data were reported as an average of three independent histograms for each sample, and errors calculated as a standard deviation from the average vesicle diameter. Negative staining transmission electron m icroscopy (TEM) Transmission electron microscopy images were obtained using a Hitachi H 7000 transmission electron microscope operated at 75100 kV with a Soft Imaging System MegaViewIII with AnalySIS digital camera (Lakewood, CO). The microscope has a maximum resolution at 0.2 nm with a magnification range of 110 to 600,000. Prior to TEM measurements, samples were further prepared by negative staining. Briefly, for all samples, using a disposable pipette, a drop of the lipid vesicle sample was spread on a 400mesh Formvar coated copper grid held by tweezers and incubated for 2 minutes. Excess lipid sample was gently dabbed away with f ilter paper, and the grid was allowed to dry for 2 minutes. In some instances a drop of deionized water was added to the grid to remove any excess salt from the buffer solution used in vesicle preparation. One drop of 2 % uranyl acetate was then added to t he grid and allowed to stain for 2 minutes, after which any excess uranyl acetate was wiped away, and the sample was allowed to dry for 2 minutes before being placed in the electron microscope specimen holder for image analysis and collection. Results an d Discussion Characterization of BMP Vesicle Size and M orphology in the Presence of C holesterol Whereas numerous studies have focused on the phase behavior of cholesterol incorporated phospholipid membranes, few studies address the morphological and size d istribution of vesicle structures observed when CHOL is added to different
166 phospholipid mixtures at specific molar fractions. To study the effect of CHOL on BMP vesicle size and morphological structure, d ynamic light scattering measurements and transmissio n electron microscopy im agi ng experiments were performed on hydrated dispersions and extruded vesicles of BMP: CHOL in 70: 30 mol % fraction s, considering that in the late endosome, BMP is found in higher concentrations than cholesterol. Chemical structures of BMP and cholesterol are shown in Figure 51. B) BMP C) GM1 A) Cholesterol D) POPC Figu re 5 1. Chemical structures of A) Cholesterol, B) BMP, C) GM1 and D) POPC.
167 A B 0 100 200 300 400 0 20 40 60 80 100 Intensity (au)Vesicle diameter (nm) 0 100 200 300 400 500 600 700 0 20 40 60 80 100 Intensity (au)Vesicle diameter (nm) Figure 52 Dynamic ligh t scattering histograms of BMP: CHOL (7:3) A) hydrated dispersions and B) 400nm extruded unilamellar vesicles at pH 5.5. Table 51. Average v esicle of BMP: CHOL (7: 3 ) hydrated dispersions and ex truded Vesicles. Lipid c omposition Ves icle size d istribution s (nm) BMP : CHOL A: Hydrated Dispersions 130 170 460 70 B: 400 nm Extruded Vesicles 200 30 In the previous studies discussed in Chapters 3 and 4, we have demonstrated that BMP hydrated dispersions form vesicles with diameters of ~ 500 nm, and extruded BMP vesicle diameters are near 230 nm, much smaller than the 400 nm pore membranes used for their extrusion. H ere, Figure 52 and Table 1 summarize the dynamic light scattering vesicle diameters observed for the BMP: CHOL (7:3) hydrated dispersions (A) and extruded vesicles (B). These data show that the hydrated dispersions formed by the BMP: CHOL (7:3) lipid mixture have diameters that are similar (~ 50 0 nm) to those observed for BMP, although instead of a single size distribution like that observed from neat BMP, the BMP: CHOL (7:3) mixture forms with sizes in a bimodal distribution, with a less intense population of vesicles with a size range of 130 17 0 nm likely as an effect
168 of cholesterol T he extruded vesicle diameters are also relatively the same (200 nm) as those observed for BMP. 500nm 1000nm Figure 53 Negative staining TEM images of BMP: CHOL (7:3) hydrated dispersions. Figure 53 shows representative negative staining TEM images observed f or t he hydrated dispersions of BMP: CHOL (7:3). Compared to neat BMP, the BMP:CHOL (7:3) dispersions have a different morphology, forming both large and small structures that appear more spherical that those observed i n neat BMP. These vesicles also vary in size, although most are ~ 450 nm in diameter which is consistent with the sizes obtained using DLS. Clearly, the presence of cholesterol alters the morphology of BMP: CHOL (7:3) dispersions from that observed in neat BMP S tudies reported in Chapter 4 showed that BMP and ganglioside GM1interact in a concentrationdependent manner, hence i t might also be interest ing to find out the size and morphology of vesicles formed when cholesterol is mixed with BMP membranes at v arying molar concentrations Investigation of Vesicle Size and M orphology in POPC:CHOL: BMP Mixtures After characterization of the BMP: CHOL membranes, it was also of interest to determine the effect of choles terol on both BMP and POPC membranes. DLS and TE M experiments were utilized to measure the vesicle diameters and observed morphologies
169 of hydrated dispersions and extruded vesicles of POPC: CHOL (80: 20) and POPC: BMP: CHOL (65: 15: 20) in 5 mM NaOAc, 100 mM NaCl, 0.1 mM EDTA at pH 5.5. When c holesterol is added to phosphatidylcholine mixtures it typically aid s in larger vesicle formation and stabilization (241, 257) hence it was expected that these samples would show larger vesicle structures than either neat P OPC or BMP vesicles Figure 54 and Table 5 2 give a summary of the vesicle diameters obtained for hydrated dispersions and extruded vesicles of POPC: CHOL and POPC:BMP: CHOL samples. In previous characterization of POPC, it was demonstrated that POPC disper sions have large vesicles diameters (~1400 nm), and when BMP was introduced in the POPC membranes at 15 mol %, the vesicle diameter s of the POPC: BMP dispersions were significantly reduced to ~ 900 nm. H ere it is found that POPC:CHOL (8:2) membranes form dispersions (Figure 54A) with a broad size distribution, with an average vesicle diameters of ~ 2100 nm, significantly larger than that observed for neat POPC dispersions (1400 nm) which can be attributed to the presence of cholesterol. 0 500 1000 1500 2000 2500 0 20 40 60 80 100 Intensity (au)Vesicle diameter (nm) 0 1000 2000 3000 4000 5000 6000 0 20 40 60 80 100 Intentisty (au)Vesicle diameter (nm) A B 0 500 1000 1500 2000 2500 0 20 40 60 80 100 Intensity (au)Vesicle Diameter (nm) 0 100 200 300 400 500 0 20 40 60 80 100 Intensity (au) Vesicle Diameter (nm) C D Figure 54 Dy namic light scattering histograms of POPC: CHOL (8:2) A) hydrated dispersions B) 400 nm extruded vesicles POC:BMP: CHOL (65:15:20) C) hydrated dispersions and D) 400 nm extruded vesicles Not e the different scales on the x axes.
170 Table 52. Average v esicle d iameters of POPC: CHO L (8:2) and POPC:BMP: CHOL (65:15:20) hydrated dispersions and extruded v esicles Lipid Composition Vesicle Size Distribution s (nm) POPC: CHOL A: Hydrated Dispersions 2100 840 B: 400 nm Extruded Vesicles 1200 410 POPC:BMP: CHOL C: Hydrated Dispersions 1250 360 D: 400 nm Extruded Vesicl es 310 60 Interestingly, the average vesicle diameters of POPC: CHOL extruded vesi cles (Figure 5 4B) are much larger (~ 1200 nm) than those seen for neat POPC extruded vesicles (~400 nm), and triple the size of the 400 nm pore membranes used for the extrusion of these vesicles. This is a clear indication that adding cholesterol at specific concentrations increases the cross sectional diameter of certain phospholipid bilayers, such as POPC. In comparison, POPC:BMP: CHO L hydrated dispersions (Figure 54C) have average diameters of 1250 nm, much s maller than those seen in POPC: CHOL dispersions (2100 nm) and nearly the size of neat POPC dispersions (1400 nm). The POPC:BMP: CHOL extruded vesicles (Figure 5 4D) are also found to have smaller average diameters (~ 310 nm) than POPC: CHOL (1250 nm) but comparable to POPC extruded vesicles (~400 nm) Again, a consistent trend is seen: BMP incorporated into other lipid vesicles results in a drastic decrease in the average vesicle diameters as observed in the vesicles formed by the POPC:BMP: CHOL mixture It is possible that BMP, which has been found to prefer formation of small, stable lamellar vesicles as an attribute to its unique structure and stereoconfiguration, counteracts the cholesterol effect
171 TEM images ob tained for POPC: CHOL disp ersions (Figure 55A) and POPC:BMP: CHOL dispersions (Figure 55B) show sizes and shapes that are consistent with the DLS results obtained for these lipid mixtures. POPC: CHOL dispersions form high ly structured, multilamellar onionlike vesicles with diameters of ~ 1500 nm, whereas in the POPC: BMP: CHOL sample clustered, vesicle structures that appear like budding events are observed, with diameters of ~ 1000 nm. It can be recall ed that in Chapter 3, BMP dispersions at neutral pH were observed to form highly structured nonspherical vesicle clusters. 500nm 500nm A 1000nm 1000nm B Figure 55 TEM images of A) POPC: CHOL (8:2) and B) POPC:BMP: CHOL (65:15:20) hydrated dispersions. TEM analysis shows different vesicle morphologi es for POPC:CHOL and POPC:BMP: CHOL dispersions as shown in Figure 55 The vesicle morphology observed in these phospholipid mixtures is a direct effect of the incorporation of both
172 cholesterol and BMP which seem to have opposing effects on phospholipid m embranes. Addition of 15 mol % BMP and 20 mol % CHOL in the POPC:BMP: CHOL mixture results in formation of spherical vesicles of various sizes and an overall decrease in vesicle diameter (compared to POPC:CHOL), while addition of 20 mol % CHOL in POPC membr anes leads to formation of multilamellar vesicle structures with increased diameters. DLS and T E M e xp eriments were also carried out on POPC lipid mixtures incorporating ganglioside GM1, cholesterol and BMP. Experiments were performed to measure the average vesicle diameter and macroscopic morphology of POPC:GM1:CHOL (70: 10: 20), and POPC:GM1: BMP: CHOL (50:15:15: 20) hydrated dispersions and vesicles extruded with 400 nm pore membranes. All samples were hydrated in 5 mM NaOAc, 100 mM NaCl, 0.1 mM EDTA at pH 5.5. 0 500 1000 1500 2000 2500 0 20 40 60 80 100 Intensity (au)Vesicle diameter (nm) 0 100 200 300 400 0 20 40 60 80 100 Intensity (au)Vesicle diameter (nm) A B 0 200 400 600 800 1000 1200 0 20 40 60 80 100 Intensity (nm)Vesicle diameter (nm) 0 100 200 300 400 500 0 20 40 60 80 100 Intensity (au)Vesicle diameter (nm) D C Figure 56 Dynamic light scattering histograms of A) hydrated dispersions of POPC:GM1:CHOL (70: 10: 20), B) 400 nm extruded vesicles of POC: GM1: CHOL C) hydrated dispersions of POPC:GM1:BMP: CHOL (50:15:15:20) and D) 400 nm extruded vesicles of POPC:GM1:BMP: CHOL Note the different scales on the x axes.
173 Table 53. Vesicle size dist ributions of POPC:GM1:CHOL (70: 10: 20) and POPC:GM1:BMP: CHOL h ydrated dispersions and 400 nm e xtruded Vesicles. Lipid Composition Vesicle Size Distribution s (nm) POPC:GM1: CHOL A: Hydrated Dispersions 1 2 00 300 B: 400nm Extruded Vesicles 220 50 POPC:GM1:BMP: CHOL C: Hydrated Dispersions 80 160 430 150 D: 400nm Extruded Vesicles 280 130 The data presented in F igure 55 and Table 5 3 summarizes the average vesic le diameters obtained for POPC:GM1: CHOL and POPC:GM1: BMP: CHOL hydrated dispersions and extruded vesicles at the late endosomal pH 5.5. Interestingly, th e hydrated dispersions of POPC:GM1:CHOL ( A) have diameters of ~ 1200 nm, but when 15 mol % BMP is introduced in the lipid mixture, the average diameters of POPC:GM1:BMP: CHOL hydrated dispersions (C) are drastically reduced to ~430 nm in diameter and a second, less intense vesicle population with small diameters between 80 160 nm is also observed. Again, the formation of small vesicles in the presence of BMP might be attributed to the ability of BMP to contribute favorably to smaller vesicle formation. In both lipid mixtures, th e vesicles that were extruded with 400 nm pore membranes had small average diameters of 220 nm for POPC:GM1:CHOL and 280 nm for POPC:GM1: BMP: CHOL.
174 A 1000nm 500nm 1000nm 1000nm B Figure 57 Negative staining TEM images of A) POPC:GM1:CHOL (70: 10: 20) and B) POPC:GM1:BMP: CHOL (50:15:15::20) h ydrated dispersions. When the hydrated dispersions of both of these lipid mixtures were imaged by TEM, they both displayed highly heterogeneous nonspherical structures with diameters in the range of 500 1000 nm as observed in Figure 46 A B Th e POPC:GM1:BMP: CHOL dispersions also featured some dispersions that appeared like clusters of small vesicle structures. It may be interesting to study these lipid mixtures further as ideal model systems for tracking individual lipid components in vesicles of lipid mixtures using assays such as secondary antibody Au labeling and negative staining transmission electron microscopy It might also be interesting to investigate whether the small cl usters of vesicles in the POPC:GM1:BMP: CHOL dispersions are
175 actual phaseseparated lipid vesicles due to the cholesterol effect or if they are related to incomplete lipid mixing during the vesicle preparation procedure. Conclusions The studies reported in this chapter have yielded interesting, and potentially significant findings on the effects of incorporating cholesterol, ganglioside GM1 and BMP in typical POPC membranes. It has been demonstrated clearly that in almost all instances, the addition of 20 mol % cholesterol to POPC mixtures results in increased vesicle diam eters. Cholesterol also alters the vesicle morphology of the lipid dispersions, leading to formation of structured, nonspherical vesicles. This chapter also reiterates the findings in chapters 3 and 4, namely that the addition of small amounts of BMP (15 mol %) in all POPC mixtures consistently induces the formation of smaller vesicle structures, as demonstrated by the dynamic light scattering and TEM data. Together, t hese findings can be explored further for a better understanding of the occurrence and interaction of different lipids in the late endosome, and how the physico chemical properties of cholesterol and ganglioside GM1 may affect the functional role of BMP in the endocytic pathway.
176 CHAPTER 6 SUMMARY AND FUTURE P ERSPECTIVES BMP F orms Small Stable Lamellar Vesicle Structures and Induces Formation of Small Vesicles when mixed with POPC The analytical capability of the dynamic light scattering (DLS) technique was successfully demonstrated through the accurate measurement of manually mixed vesicle populations. DLS and transmission electron microscopy (TEM) were utilized to characterize the size of hydrated and extruded BMP and POPC sam ples, revealing that when BMP is hydrated from a dry lipid film, it forms lipid dispersions that have diameters much sma ller than those of POPC dispersions, regardless of pH. I also demonstrated using vesicle leakage assays and vesicle stability assays, that BMP forms small stable lamellar vesicles with an interior volume that can encapsulate molecules. Following the c haracterization of POPC: BMP (85:15) and POPC: POPG (80:20) lipid mixtures, this work showed that BMP can induce the formation of small vesicles when incorporated in typical phospholipid membranes at certain concentrations. Taken together, these data suggest that the physical properties of BMP, dictated by its unique di glycerol structure and stereoconfiguration, provide a mechanism for stabilization of small vesicle structures in the maturing endosome as the lumen acidifies, shedding further light on the pote ntial functional role of this lipid in the late endosome. Gan glioside GM1 Leads to Formation of Small Homogenous Vesicles When Mixed with BMP The results on GM1 interactions with BMP provided morphological and size distribution information that demonstrat ed that GM1 mixes with BMP to form small (~100 nm) spherical shaped vesicles with a narrow size distribution at similar
177 concentrations to those seen to condense DPPC and form a specific complex. The apparent miscibility of GM1 with BMP over the 2030 mol % ratios, as evidenced by the spontaneous formation of small homogenously shaped vesicles with narrow diameter size distribution, suggests that optimum interactions between molecules may occur for a given molecular ratio of 3 BMP molecules for 1 GM1 molecul e to form a lipid complex. This hypothesis is drawn from the analysis of Langmuir monolayer studies, which showed that the pressurearea isotherms of GM1 and DPPC mixtures follow ideal mixing behavior when the two species were considered to be a 3:1 DPPC :GM1 complex interacting with excess DPPC (239) This specific mixture of GM1 with BMP may be important for in vivo vesicular trafficking and lipid sorting in the endos ome/lysosome pathways. Finally, when incorporated in typical POPC membranes at specific concentrations, ganglioside GM1 and BMP alter the morphology and size of the dispersions and vesicles that are formed. Larger vesicle diameters are formed in the presence of 20 mol % GM1 in 80 mol % POPC, whereas smaller, heterogeneous and structured vesicles are obtained when 15 mol % BMP and 15 mol % GM1 lipids are mixed with 70 mol % POPC BMP Counteracts the Cholesterol Effect when Mixed with GM1 and POPC The studi es presented in C hapter 5 have show n interesting and potentially significant findings on the effects of incorporating cholesterol, ganglioside GM1 and BMP in POPC membranes. This work has demonstrated clearly that in almost all instances, the addit ion of 20 mol % cholesterol to POPC mixtures results in increased vesicle diameters. Cholesterol also changes the vesicle morphology of the lipid dispersions, leading to formation of structured, nonspherical vesicles The work in
178 C hapter 5 also reiterates our previous findings in C hapters 3 and 4, namely that the addition of small a mounts of BMP (15 mol %) in POPC and GM1 mixtures consistently induces the formation of smaller vesicles structures, even in the presence of cholesterol, as demonstrated by the dynamic light scattering and negative staining TEM data. Together, these findings can be explored further for a better understanding of the occurrence and interaction of different lipids in the late endosome, and how the physico chemical properties of cholesterol and ganglioside GM1 may affect the functional role of BMP in the endocytic pathway. Future Perspectives One way in which the applicability of BMP vesicles can be explored is through investigation of their potential as drug delivery vehicles. Because l ipo somes or lipid vesicles resemble biological ce ll membranes in their structure and composition, they can be engineered from natural, biodegradable and nontoxic lipid molecules and can encapsulate or bind a variety of drug molecules into or onto their membra nes. As a consequence all these properties make vesicles attractive candidates fo r use as drug delivery vehicles (258) Additionally, liposome applic ations in drug delivery depend on their physicochemical and colloidal characteristics such as composition, size, loading efficiency and the stability of the carrier, as well as their biological interactions with the cells (258) An added advantage of the use of encapsulated drug molecules for drug delivery is the decreased toxicity of liposomal formulations, because liposomeassociated drug molecules cannot normally spill to organs such as the heart, brain and kidneys. This work has characterized the size and morphology of BMP vesicles, and
179 found that BMP forms small, lamellar vesicle structures with an interior volume. These properties of BMP can be utilized further for the possibility of encapsulating hydrophilic drug molecules in the aqueous core of BMP vesicles, and studied for their potential drug delivery applications. Following TEM and DL S studies on BMP mixed with CHOL, POPC and GM1 lipids f urther studies can be carried out on the POPC/BMP/GM1/CHOL model lipid system using assays such as secondary antibody labeling Such experiments may shed more light on the lipid distributions in the h eterogeneous vesicle formations observed for lipid mixtures which would provide further understanding of the role of each of these lipids in the endocytic pathway and in lipid raft formation.
180 APPENDIX A STEP BY STEP ANALYSI S OF DYNAMIC LIGHT SCATTERING DATA 1 2 3 Figure A 1. DLS raw data histograms (1, 2, and 3) of a sample of 100 nm extruded BMP vesicles in 5 mM HEPES, 100 mM NaCl and 0.1 mM EDTA, pH 7.4. Each of the three traces /histograms were collected for 3 minutes.
181 T he data in Figure A 1 were saved as spreadsheets and later imported into Origin Pro 8 or Microsoft Excel soft ware f or further analysis. Table A 1. OriginPro 8 spreadsheet of the imported DLS raw data of 100 nm extruded BMP vesicles D 1 I 1 D 2 I 2 D 3 I 3 Mean Diameter Mean Intensity 59.90698 0 59.48459 0 55.76741 0 58.38633 0 63.73537 0 63.41274 0 59.40784 0 62.18532 0 67.80842 0 67.60029 0 63.28592 0 66.23154 0 72.14175 0 72.06436 0 67.41715 0 70.54109 0 76.75201 0 76.82323 0 71.81806 0 75.1311 0 81.65689 0 81.89636 15.52 76.50626 0 80.01984 5.17 86.87522 34.29 87.3045 55.73 81.50050 21.87 85.22674 37.30 92.42702 68. 89 93.06978 94.33 86.82000 61.75 90.77252 75.0 0 98.33363 100.00 99.21577 100.00 92.48831 100.00 96.67924 100.00 104.6177 67.23 105.7676 59.97 98.52584 91.01 102.97038 7 2.74 111.3033 32.61 112.7521 21.34 104.9575 51.33 109.67097 35.10 118.4163 0 120.1979 0 111.809 13.08 116.80773 4.36 125.9837 0 128.1353 0 119.1078 0 124.40893 0 134.0348 0 136.5969 0 126.883 0 132.5049 0 142.6003 0 145.6173 0 135.1657 0 141.12777 0 151.7133 0 155.2333 0 143.9892 0 150.31193 0 161.4086 0 165.4844 0 153.3886 0 160.09387 0 171.7235 0 176.4124 0 163.4017 0 170.51253 0 182.6976 0 188.062 0 174.0683 0 181.6093 0 194.373 0 200.4809 0 185.4313 0 193.4284 0 206.7945 0 213.72 0 197.5361 0 206.01687 0 220.0098 0 227.8333 0 210.431 0 219.4247 0 234.0697 0 242.8785 0 224.1677 0 233.7053 0 249.0281 0 258.9174 0 238.8011 0 248.91553 0 264.9424 0 276.0154 0 254.3897 0 265.11583 0 281.8737 0 294.2424 0 270.996 0 282.3707 0 299.8869 0 313.6731 0 288.6863 0 300.74877 0 319.0514 0 334.3869 0 307.5314 0 320.32323 0 339.4405 0 356.4687 0 327.6067 0 341.17197 0 361.1327 0 380.0086 0 348.9925 0 363.37793 0 384.2111 0 405.1029 0 371.7743 0 387.02943 0 408.7643 0 431.8545 0 396.0433 0 412.2207 0 The spreadsheet shows the individual diameters (D 1, D 2, and D 3 in nm) and their corresponding intensities (I 1, I2, and I 3) t he Mean Diameter (nm), and Mean Intensity of the three traces. Once imported into OriginPro 8, the Diameter (d) and Intensity (G (d)) values were utilized for calculation of mean diameter and mean intensity values of the three histograms, as illustrated in Table A 2. The average vesicle diameter was obtained from the mean diameter that had the maximum (peak) mean intensity. For this particular example, the average vesicle diameter for the 100nm extruded BMP vesicles is
182 96.67924 nm (in bold red), which can be approximated to 100 nm. The standard deviation ( ) can also be calculated from all the mean diameter values that have nonzero intensities, and in this case was found to be 20. The mean diameter and mean intensity values in Table A 1 were plotted in OriginPro 8 as a B Spline line to obtain a Gaussia n distribution as illustrated in Figure A 2. 0 50 100 150 200 0 20 40 60 80 100 Intensity (au)Vesicle diameter (nm) Average vesicle diameter ~ 100 nm Figure A 2. Average vesicle diameter for 100 nm extruded BMP vesicles.
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204 BIOGRAPHICAL SKETCH Janetricks Nanja la Chebukati (Rhodah) was born o n July 30th 1976, in the Western province of Kenya, in a small r ural township called Bungoma that is situated close to the foothills of Mt. Elgon. She is t he fourth born of ten children to Mr. Gerishom Muchungi Chebukati, a retired primary school teacher, and Mrs. Rita h Nekesa Muchungi, a housewife. From an early age, Janetricks quickly learn t the value of an education through her parents guidance and direction. She started high school in 1991, and graduated in the top 5 % of her class from the prestigious Lugulu Girls High School in 1994. Janetricks joined Jomo Keny atta University of Agriculture and Technology (JKUAT) in Nairobi, Kenya in March 1997 and graduated in April 2001 with honors in BSc (Chemistry). In August 2001, Janetricks started work as a pharmaceutical sales representative for Ranbaxy Laboratories Ltd, and relocated to the lakeside city of Kisumu, Kenya. She remain ed in this position for two years, before enrolling for graduate school in the Department of Chemistry at the University of Florida (UF) in fall 2003. At UF, she briefly worked on Laser Ablation Inductively Coupled Plasma Mass Spectrometry ( LA ICPMS ) in Jim Winefordners group, before eventually joining the Fanucci research group in the summer of 2006. Janetricks spent the next three and half years in the Fanucci group working on the characterization of the physico chemical properties of lipids using analytical techniques such as electron microscopy ( EM ) and dynamic light scattering ( DLS ) She also earned a dual Masters degree in Forensic Drug Chemistry from the UF School of Pharmacy in Decemb er 2008. Janetricks recently accepted a postdoctoral research associate position in Dr. Dale Benos l ab in the Department of Biophysics and Physiology, in the School of Medicine at
205 the University of Alabama, Birmingham, to commence in January, 2010. Janetr icks is married to Dr. George Odhiambo Okeyo, and they are blessed with two daughters, Lavender (13 years old) and Subi (2 years old).