Liposomes Part A

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Introduction......Page 6
Extrusion and Extrusion Devices......Page 7
Mechanism of Extrusion and Vesicle Morphology......Page 9
Formation of Unilamellar Vesicles......Page 11
Effect of Lipid Composition on Extrusion......Page 13
Applications......Page 17
Introduction......Page 18
Giant Unilamellar Vesicle Electroformation Chamber......Page 19
Deposition of Lipids......Page 20
Microinjection......Page 22
Aspects to Be Aware of......Page 24
Preparation of Small Unilamellar Liposomes......Page 27
Preparation of Large Unilamellar Liposomes by Reverse-Phase Evaporation......Page 29
Phosphate Assay to Determine Phospholipid Concentration of Liposomes......Page 30
Introduction......Page 32
Purification of Bacteriorhodopsin......Page 33
Principle......Page 34
Methods......Page 35
Results......Page 37
Method......Page 39
Results......Page 40
Principle......Page 42
Results......Page 43
Principle......Page 45
Results......Page 46
Method......Page 47
Results......Page 48
Acknowledgments......Page 50
Introduction......Page 51
Critical Micellar Concentration of Detergents......Page 53
Mixed Micelles......Page 55
Detergent-Rich Mixed Vesicles......Page 59
Detergent-Poor Mixed Vesicles and Detergent-Free Vesicles......Page 61
Initial Ratio of Detergent to Lipid......Page 62
Lipid Species, Their Phase Transition Temperature, and Charge......Page 63
pH of Aqueous Media......Page 67
Preparation of Mixed Micelle Solutions......Page 68
Gel Chromatography......Page 69
Dialysis Using Flat Membranes......Page 70
Dialysis Using Hollow Fibers......Page 71
Filtration......Page 72
Others......Page 73
Size, Lamellarity, and Shape of Liposomes......Page 74
Encapsulation Efficiency......Page 75
Introduction......Page 76
Materials......Page 79
Solutions......Page 80
Preparation of SUVs......Page 81
Removal of Nonentrapped DNA......Page 82
Preparation of DNA-Containing Small Liposomes by Sucrose Method......Page 83
DRVs and Small Sucrose Liposomes......Page 84
Measurement of DNA Retention by Microfluidized DRVs and Vesicle Characteristics......Page 86
Interdigitation-Fusion Process......Page 87
Preparation of Ethanol-Induced Interdigitation-Fusion Liposomes......Page 94
Preparation of Pressure-Induced Interdigitation-Fusion Liposomes......Page 99
Interdigitation-Fusion Liposome Properties and Applications......Page 100
Acknowledgment......Page 105
Introduction......Page 106
Freezing Phase......Page 107
Primary Drying......Page 111
Secondary Drying......Page 113
Selection of Liposome Type......Page 114
Lyoprotectants and Other Excipients......Page 115
Freeze-Drying of Liposomes Loaded with Non-Water-Soluble Compounds......Page 116
Freeze-Drying Protocol......Page 117
Introduction......Page 118
Preparation of Proliposome Mixture and Its Conversion into Liposomes......Page 120
Assay of Encapsulation Efficiency for 5,6-Carboxyfluorescein......Page 123
Effect of Proliposome Mixture Composition on Entrapment Efficiency......Page 124
Preparation of Frozen-and-Thawed Multilamellar Vesicles......Page 126
Determination of Captured Volume......Page 127
Effect of Transformation Temperature on Entrapment Efficiency......Page 128
Statistical Analyses......Page 129
Concluding Remarks......Page 130
Acknowledgments......Page 132
Introduction......Page 133
Lipid Purity......Page 135
Lipid Dispersions......Page 136
Unilamellar Lipid Vesicles......Page 137
Differential Scanning Calorimetry......Page 138
X-Ray Diffraction: Methods and Examples......Page 143
Instrumentation......Page 145
Salt Effects......Page 148
Phase Diagram Mapping......Page 149
Concluding Remarks......Page 151
Introduction......Page 152
Equipment: Rank Brothers Apparatus......Page 153
Cell and Electrodes......Page 154
Optics......Page 156
Temperature Control......Page 157
Stationary Layers......Page 158
Experimental Determination......Page 159
Theoretical Determination......Page 160
Cell Constant......Page 162
Solutions......Page 163
Liposome Preparation......Page 165
Blackening Electrodes......Page 168
Cleaning Cell, Glassware, and Electrodes......Page 169
Measurements: Electrophoretic Mobility......Page 170
Other Devices......Page 171
Relation between Electrophoretic Mobility and Liposome Charge......Page 173
Calculation of Zeta Potential......Page 174
Calculation of Surface Potential......Page 175
Spherical Case, Small Potentials......Page 176
Planar Case, Small Potentials......Page 177
Spherical Case, Small Potentials......Page 178
Ion Binding......Page 179
Acknowledgments......Page 180
Viscosity of Small Unilamellar Vesicle Suspension......Page 181
Determination of Average Volume Fraction for Small Unilamellar Vesicles......Page 183
Viscometry of Multilamellar Vesicle Suspension......Page 184
Viscometry of DNA-Lipid Complex and DNA-Polymer Complex......Page 188
Minicapillary Viscometer......Page 189
Problems with Minicapillary Viscometers......Page 190
Automation of Viscosity Measurement......Page 191
Materials......Page 192
Lipoplex Preparation by Flow Impinger......Page 193
Viscosity Measurements......Page 194
Results......Page 195
Discussion......Page 196
Concluding Remarks......Page 202
Acknowledgment......Page 203
Introduction......Page 204
Substrates......Page 206
Liposomes......Page 207
Preparing Samples for Imaging in Air......Page 208
Preparing Samples for Imaging in Liquid......Page 210
Mode of AFM Imaging......Page 212
Image Presentation and Interpretation......Page 213
Example of Imaging Liposomes......Page 214
Conclusion......Page 218
Introduction: What Is the Lipid Interface?......Page 219
Properties of Lipid Interface......Page 220
Factors Affecting Surface Properties of Liposomes......Page 222
Merocyanine Partition between Lipid and Bulk Water Phase......Page 223
Surface Properties at Phase Transition as Determined by Merocyanine 540 Absorbance and Fluorescence......Page 226
Interfacial Properties Derived from Equilibrium Processes at Interface......Page 228
Determination of Dimerization Constant of MC540 at Membrane Interface......Page 230
Changes in Dimerization Constant due to Packing......Page 232
Packing and Polarity Changes Measured with MC540 and Fluorophores Anchored to Lipid Phase......Page 233
Steady-State Fluorescence Anisotropy Determinations Performed with Dansyldihexadecylamine......Page 235
Dipolar Relaxation Determined by Laurdan and Prodan Probes......Page 236
Packing and Dipole Potential......Page 237
Final Remarks......Page 239
Introduction......Page 240
Direct Visualization of Lipid Domain Coexistence in Bilayers......Page 241
Some Considerations about Giant Unilamellar Vesicles......Page 242
General Considerations for Preparation of Giant Unilamellar Vesicles......Page 244
Comparison of Methods Used to Generate GUVs......Page 245
Why Two-Photon Excitation Microscopy?......Page 246
Fluorescent Probes......Page 248
Extracting Lipid Domain Phase State Information Directly from Fluorescence Intensity Images......Page 250
Photoselection Effect and LAURDAN Generalized Polarization Function......Page 251
Direct Visualization of Lipid Domains......Page 254
Lipid Domain Shape Information......Page 256
LAURDAN GP Can Be Related to Compositional Differences between Coexisting Lipid Domains......Page 258
Acknowledgments......Page 260
Introduction......Page 261
Fluorescent Probe Solutions......Page 263
Small Lipid Vesicles Bearing Fluorophore......Page 264
Calibration of Dielectric Constants of Environment of DPE in Various Solvents......Page 265
Emission Spectra......Page 266
Surface Dielectric Constants of Liposomes Determined from Emission Spectra......Page 267
Determination of Surface Hydrohobicity of Liposome Membranes......Page 270
Surface Tension Measurements of Monolayers Formed at Air-Water Interface......Page 271
Formation of Lipid Monolayer at the Oil-Water Interface and Measurements of Interfacial Tension......Page 272
Determination of Surface Hydrophobicity of Vesicle Membranes......Page 273
Theory......Page 274
Some Experimental Results......Page 277
Some Comments and Possible Applications......Page 278
Introduction......Page 281
General Principles......Page 282
General Limitations of the Use of the Enzymatic Assays......Page 283
Cholesterol Reagent......Page 286
Comparison of Enzymatic and HPLC Cholesterol Determinations......Page 287
Background......Page 289
Preparation of Standard Curve (See Table II)......Page 290
Reagents......Page 292
Assay Procedure......Page 293
Specific Limitations of Enzymatic Assay......Page 294
Background......Page 295
Advantages and Limitations......Page 296
Reagents......Page 297
Assay Procedure......Page 298
Conclusion......Page 299
Acknowledgments......Page 301