Liposomes: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: From Olive Oil Emulsions to COVID-19 Vaccines: Liposomes Came First
1 Pliny the Elder, Benjamin Franklin, Singer, and Nicolson
2 The Penny Dropped: Membranes Came First
3 Multilamellar Smectic Mesophases
4 Liposomes as Immunological Adjuvants
5 Liposomes and Targeted Drug Delivery
6 Liposomal DNA Vaccines and Non-viral Transfection Vectors
7 COVID-19 Vaccines
8 Conclusion
References
Chapter 2: Preparation of DRV Liposomes
Abbreviations
1 Introduction
1.1 Drug Encapsulating DRVs
1.2 Antigen Encapsulating DRVs
2 Materials
2.1 Low MW Drug Encapsulating DRV
2.2 Protein (or Peptide) and/or Particulate Encapsulating DRV
2.3 Entrapment of Large Particles, Viruses, or Bacteria into Giant Liposomes
3 Methods
3.1 General Methodology for DRV Preparation: Preparation of Hydrophilic Compound or Small Molecule Encapsulating DRV Liposomes
3.1.1 Preparation of CF-Encapsulating DRV´s with High Entrapment Yield
Thin Film Formation (Step 1)
Hydration of Thin Film: MLV Formation (Step 2)
SUV Formation (Step 3)
Preparation of SUV-Solute Mixture (Step 4)
Freeze-Drying (Step 5) (See Note 11)
Rehydration or Reconstitution: DRV Formation (Step 6)
DRV Separation from Non-entrapped Solutes (Step 7)
Determination of Entrapped CF (or Calcein)
Determination of Lipid Concentration
3.1.2 Preparation of Amphiphilic/Lipophilic Drug Incorporating or Encapsulating DRVs (as Reported for PRE, Curcumin, Ibuprofen...
3.1.3 Preparation of Drug/CD Complex Encapsulating DRVs (as Reported in)
Formation of Drug-CD Inclusion Complexes
Preparation of DRVs
3.1.4 Preparation of Multicomponent DRVs for Photolabile Drugs
3.1.5 Preparation of DRV Liposomes with Controlled Entrapment Yield and Vesicle Size (as Reported in 2)
3.2 Preparation of Protein (or Peptide) and DNA Vaccines (as Reported in [1, 47, 63, 70 ])
3.2.1 Lipid Compositions for Protein and DNA Vaccines
3.2.2 Liposome Preparation Procedure: Vaccine Material Addition
3.2.3 Separation of DRVs from Non-entrapped (Vaccine) Material
3.2.4 Vaccine Entrapment Yield Estimation
3.2.5 Vaccine-Containing DRV Size Reduction
3.3 Preparation of Giant Liposome DRVs that Entrap Large Particles, Viruses, or Bacteria (as Described in)
3.3.1 Giant Liposome Preparation
3.3.2 Giant DRV Liposome Separation from Non-entrapped Particulates
4 Notes
References
Chapter 3: Preparation of Small Unilamellar Vesicle Liposomes Using Detergent Dialysis Method
1 Introduction
2 Materials and Equipment
3 Methods
3.1 Preparation of a Dry Lipid Film
3.2 Rehydration of the Dry Lipid Film
3.3 Detergent Removal by Dialysis
3.4 Particle Size Determination
3.5 Surface Charge Determination
4 Notes
References
Chapter 4: Thin-Film Hydration Followed by Extrusion Method for Liposome Preparation
1 Introduction
2 Materials and Equipment
3 Methods
3.1 Preparation of the Thin Film
3.2 Hydration of the Thin Film
3.3 Extrusion
3.4 Particle Size and Surface Charge Measurement
4 Notes
References
Chapter 5: Ethanol Injection Method for Liposome Preparation
1 Introduction
2 Materials
3 Methods
3.1 Preparation of Working Solutions
3.2 Fabrication of Liposomes by Ethanol Injection Method
3.3 Size and Surface Charge Characterization
4 Notes
References
Chapter 6: Preparation of Giant Vesicles with Mixed Single-Tailed and Double-Tailed Lipids
1 Introduction
2 Materials
2.1 General Materials
2.2 Pre-mixed GVs
2.2.1 Paper-Based Thin-Film Hydration
2.2.2 Emulsion Transfer Method
2.2.3 Fatty Acid Self-Assembly Method
2.3 Post-mixed GVs
2.3.1 Adding Lipid 2 Via Solvent
2.3.2 Adding Lipid 2 as Pre-formed Vesicles or Micelles
2.3.3 Phospholipase Incubation
3 Methods
3.1 Pre-mixed GVs
3.1.1 Paper-Based Thin-Film Hydration
3.1.2 Emulsion Transfer Method
3.1.3 Fatty Acid Self-Assembly Method
3.2 Post-mixed GVs
3.2.1 Adding Lipid 2 Via Solvent
3.2.2 Adding Lipid 2 as Pre-formed Vesicles or Micelles
3.2.3 Phospholipase Incubation
4 Notes
References
Chapter 7: Scalable Liposome Synthesis by High Aspect Ratio Microfluidic Flow Focusing
1 Introduction
2 Materials
2.1 Flow Focusing Device Fabrication
2.2 Lipid Preparation
2.3 Flow Focusing Device Operation
3 Methods
3.1 Flow Focusing Device Fabrication
3.2 Lipid Solution Preparation
3.3 Flow Focusing Device Operation
4 Notes
References
Chapter 8: Preparation of Doxorubicin Liposomes by Remote Loading Method
1 Introduction
2 Materials
2.1 Sucrose Solution (10% W/V)
2.2 (NH4)2SO4 Solution (250 mM, pH 4.0)
2.3 Lipid Solution
2.4 Doxorubicin Solution (10 mg/mL)
2.5 Triton X100 Solution (0.5%V/V)
3 Methods
3.1 Preparation of Blank Liposomes (Film Forming Method)
3.1.1 Lipid Film Forming
3.1.2 Blank Liposomes Forming
3.1.3 Transmembrane Ion Gradient Formation
3.2 Drug Loading
3.3 Characterize of Doxorubicin Liposomes
3.3.1 Size Detection
3.3.2 Encapsulation Efficiency
3.3.3 Liposome Morphology by Cryo-TEM
4 Notes
References
Chapter 9: Magnetic Thermosensitive Liposomes Loaded with Doxorubicin
1 Introduction
2 Materials
2.1 Liposomes Formulation
2.2 Dynamic Light Scattering (DLS)
2.3 Laser Doppler Velocimetry (LDV)
2.4 Encapsulation Efficiency
2.5 Atomic Force Microscopy (AFM)
2.6 Transmission Electronic Microscopy (TEM)
2.7 Differential Scanning Calorimetry (DSC)
2.8 Drug Release
2.9 Ultra-High-Field Magnetic Resonance Imaging (UHF-MRI)
2.10 Cell Viability
2.11 Activated Partial Thromboplastin Time (aPTT)
2.12 Ex Vivo Hemolysis Assay
3 Methods
3.1 Preparation of Magnetic Thermosensitive Liposomes
3.2 Size and Zeta Potential of the Liposomes
3.3 Loading Efficiency of DOX
3.4 Atomic Force Microscopy
3.5 Transmission Electronic Microscopy
3.6 Differential Scanning Calorimetry
3.7 Drug Release
3.8 Ultra-High-Field Magnetic Resonance Imaging
3.9 Thermal Therapy Under Magnetic Field
3.10 MTT Cell Viability Assay
3.11 Activated Partial Thromboplastin Time
3.12 Ex Vivo Hemolysis Assay
4 Notes
References
Chapter 10: Preparation and Physical Characterization of DNA Binding Cationic Liposomes
1 Introduction
2 Materials (Reagents and Equipments)
3 Methods-A
4 Notes-A
5 Methods-B
6 Notes-B
7 Methods-C
8 Notes-C
References
Chapter 11: Tunable pH Sensitive Lipoplexes
1 Introduction
2 Materials
2.1 Abbreviations of the Lipids Used
2.2 Chemicals and DNA Provided or Synthesized
2.3 Equipment
2.4 Buffer Preparation
3 Methods
3.1 Preparation of Cationic Liposomes
3.2 Preparation of Cationic Lipoplexes
3.3 Preparation of Anionic Pegylated Liposomes
3.4 Preparation of Anionic Pegylated Lipoplexes
3.5 Titration Experiments
3.6 DNA Complexation Checked by Fluorescence
3.7 In Vitro Experiments
4 Notes
References
Chapter 12: Solid Lipid Nanoparticles for Drug Delivery
1 Introduction
2 Materials
3 Methods
3.1 Preparation of Placebo SLNs without Drugs
3.2 Preparation of Dexamethasone Palmitate-Loaded SLNs
3.3 Purification of Dexamethasone Palmitate-Loaded SLNs
3.4 SLN Characterization
4 Notes
References
Chapter 13: Stable Discoidal Bicelles: Formulation, Characterization, and Functions
1 Introduction
2 Materials
2.1 Phospholipids
2.1.1 Long-Chain Phosphatidylcholines
2.1.2 Short-Chain Phosphatidylcholines or Detergents
2.1.3 Charged Long-Chain Phospholipid
2.1.4 Polyethylene Oxide Conjugated (PEGylated) Phospholipid
2.1.5 Lipophilic or Amphiphilic Molecules
2.2 Solvent
3 Methods
3.1 Calculation of the Required Lipid Quantities
3.2 Dissolving Lipids in Organic Solvent
3.3 Evaporation of the Solvent
3.4 Sample Hydration
4 Structural Characterization
4.1 Dynamic Light Scattering (DLS)
4.2 Negatively Stained (ns-) and Cryo-TEM
4.3 Small-Angle X-Ray Scattering (SAXS) and Small-Angle Neutron Scattering (SANS)
5 Stability and Cellular Uptake
6 Hydrophobic Confinement
References
Chapter 14: The Post-insertion Method for the Preparation of PEGylated Liposomes
1 Introduction
2 Materials
3 Methods
3.1 Preparation of Preformed Liposomes
3.2 Characterization of Preformed Liposomes
4 Preparation of PEGylated Lipid (PEG2000-DSPE)-Based Micelles
4.1 Transfer of PEG2000-DSPE from the Micelles to the Preformed Liposomes
4.2 Characterization of the Prepared PEGylated Liposomes
5 Notes
References
Chapter 15: Click Chemistry for Liposome Surface Modification
1 Introduction
2 Materials
2.1 Synthesis
2.2 Liposome Preparation
2.3 Azide-Alkyne Coupling Reaction by Click Chemistry
3 Methods
3.1 Copper-Catalyzed Click Reaction
3.1.1 Synthesis of the Terminal Alkyne-Functionalized Lipid Anchor (2) (Scheme 2)
3.1.2 Synthesis of the Azido-Functionalized Mannosyl Ligand (5) (Scheme 3)
3.1.3 Liposome Preparation
3.1.4 Liposome Characterization
3.1.5 Conjugation of the Azido-Functionalized Ligand to Liposomes by Click Reaction (Scheme 1)
3.1.6 Quantification of the Conjugation Reaction
3.1.7 Liposome Stability under Coupling Conditions
3.2 Copper-Free Reaction
3.2.1 Synthesis of Fluorocyclooctyne-Functionalized Lipid Anchor (9) (Scheme 4)
3.2.2 Synthesis of the Azido-Functionalized Biotinyl Ligand (11) (Scheme 5)
3.2.3 Conjugation of the Azido-Functionalized Ligand to Liposomes by Click Reaction (Scheme 6)
3.2.4 Biotin Quantification
4 Notes
References
Chapter 16: Surface Modification of Liposomes Using Folic Acid
1 Introduction
2 Materials
3 Methods
3.1 Preparation of the Thin Lipid Film
3.2 Hydration of the Dry Lipid Film
3.3 Preparation of FA-Modified Liposomes Loading with Lipophilic Drugs
3.4 Characterization of the FA-Modified Liposomes
4 Notes
References
Chapter 17: Preparation and Characterization of Trastuzumab Fab-Conjugated Liposomes (Immunoliposomes)
1 Introduction
2 Materials
2.1 Fab Conjugation and Purification
2.2 Liposome and Immunoliposome Preparation
2.3 Immunoliposome Characterizations
3 Methods
3.1 Fab Coupling and Purification
3.2 Immunoliposome Preparation
3.3 Characterization of Immunoliposomes
4 Notes
References
Chapter 18: Pyrophosphorylated-Cholesterol-Modified Bone-Targeting Liposome Formulation Procedure
1 Introduction
2 Materials
3 Methods
3.1 Synthesis of Pyrophosphorylated Cholesterol (Fig. 1)
3.1.1 Synthesis of Triethylene-Glycol-Conjugated Cholesterol Compound 2 (Fig. 2)
3.1.2 Synthesis of Compound 3 (Fig. 3)
3.1.3 Synthesis of Pyrophosphorylated Cholesterol Compound 4 (Cholesterol-PPi, Fig. 4)
3.2 Pyrophosphorylated-Cholesterol-Modified Bone-Targeting Liposome Formulation
3.2.1 Liposome Formulation
3.3 Liposome Purification
3.3.1 PD-10 Desalting Column Preparation
3.3.2 Column Equilibration
3.3.3 Sample Application
3.3.4 Elution
3.4 Characterization of Pyrophosphorylated-Cholesterol-Modified Bone-Targeting Liposomes
3.4.1 Liposome Characterization
3.5 Bone-Targeting Property Characterization of the Liposome
3.5.1 HA Binding Affinity Test
3.5.2 Bone-Targeting Test In Vivo
4 Prospects of Application
5 Notes
References
Chapter 19: Method of Simultaneous Analysis of Liposome Components Using HPTLC/FID
1 Introduction
2 Materials
2.1 Liposomal Sample Lyophilization
2.2 Sample Preparation
2.3 Sample Spotting
2.4 Sample Development
3 Methods
3.1 Liposomal Sample Lyophilization
3.2 Sample Preparation
3.3 Sample Spotting
3.4 Sample Development
3.5 Sample Scanning
3.6 Calibration Curve
3.7 Qualitative and Quantitative Analysis
4 Notes
References
Chapter 20: High-Performance Liquid Chromatography Coupled with Tandem Mass Spectrometry Method for the Identification and Qua...
1 Introduction
2 Materials
2.1 Lipid Extraction
2.2 Preparation of Stock and Working Solution
2.3 HPLC-MS/MS Analysis
2.4 Data Acquisition and Analysis
3 Methods
3.1 Lipid Extraction
3.2 Optimization of MS Conditions
3.2.1 Q1 Scan to Determine the Mass-to-Charge Ratio of Precursor Ions
3.2.2 Product Ion Scan (MS2) to Determine the Mass-to-Charge Ratio of Product Ions
3.3 FIA-Optimize Source Parameters
3.4 Optimization of Liquid Chromatographic Conditions
3.5 The Optimized HPLC Conditions for Lipid Analysis (See Note 15)
3.6 Analytical Method Validation
3.6.1 Selectivity
3.6.2 Linearity and Range
3.6.3 The Limit of Detection (LOD) and the Limit of Quantitation (LOQ)
3.6.4 Precision and Accuracy
3.6.5 Carryover
3.7 HPLC Analysis of Lipids
4 Notes
References
Chapter 21: DPH Probe Method for Liposome-Membrane Fluidity Determination
1 Introduction
2 Materials
3 Methods
3.1 Preparation of Liposomes
3.2 Incubation of DPH with Liposomes
3.3 Fluorescence Measure
4 Notes
References
Chapter 22: Imaging of Liposomes by Negative Staining Transmission Electron Microscopy and Cryogenic Transmission Electron Mic...
1 Introduction
2 Negative Staining Transmission Electron Microscopy
3 Cryogenic Transmission Electron Microscopy (Cryo-TEM)
4 Materials
4.1 Sample Preparation
4.2 TEM Sample Handling
5 Methods
5.1 Negative Staining Transmission Electron Microscopy (TEM)
5.2 Cryogenic Transmission Electron Microscopy (Cryo-TEM)
6 Notes
References
Chapter 23: Visualization and Characterization of Liposomes by Atomic Force Microscopy
1 Introduction
2 Materials
2.1 Atomic Force Microscope
2.2 Silicon Wafer
2.3 Liposomes
3 Methods
3.1 Preparation and Cleaning of Silicon Wafers
3.2 Preparation of Samples
3.3 AFM Settings
3.3.1 Imaging
3.4 Postprocessing of Images
4 Notes
References
Chapter 24: Determination of the Subcellular Distribution of Fluorescently Labeled Liposomes Using Confocal Microscopy
1 Introduction
2 Materials
2.1 Liposome Components
2.2 Cell Culture Components
2.3 Dyes
2.4 Microscopy Components
3 Methods
3.1 Preparation of Fluorescently Labeled Liposomes
3.2 Treatment of Cells for Microscopy
3.3 Image Acquisition by Confocal Microscopy
3.4 Analysis of Subcellular Distribution by Colocalization Analysis
4 Notes
References
Chapter 25: Liposome Biodistribution via Europium Complexes
1 Introduction
2 Materials
2.1 Abbreviations Used
2.2 Chemicals Provided or Synthesized
2.3 Equipment
3 Methods
3.1 Lipid Labeling
3.2 Preparation of the Conventional Liposomes Labeled with Europium
3.3 Preparation of the Conventional Liposomes Labeled with Rhodamine
3.4 Phosphorescence Spectra
3.5 Test in Buffers
3.6 Biodistribution In Vivo (See Note 13)
4 Notes
References
Chapter 26: Quantification of a Fluorescent Lipid DOPE-NBD by an HPLC Method in Biological Tissue: Application to Study Liposo...
1 Introduction
2 Materials
2.1 Lipids for Liposome Preparation
2.2 Reagents for Placenta Tissue Sampling and Culturing
2.3 Reagents for Extraction and HPLC Analysis of DOPE-NBD
2.4 Equipment
3 Methods
3.1 Preparation of Neutral and Cationic Liposomes with Fluorescent Labeling
3.1.1 Preparation of Neutral Liposomes Containing DOPE-NBD
3.1.2 Preparation of Cationic Liposomes Containing DOPE-NBD
3.2 Dissection of Placental Explants from Human Placenta
3.3 Incubation of Liposomes with Placental Explants
3.4 Liquid-Liquid Extraction of Fluorescent Probe DOPE-NBD from Human Placenta
3.5 Preparation of Calibration Curves of DOPE-NBD in Placental Tissues
3.6 HPLC Quantification of DOPE-NBD in Samples
3.7 Calculation of the Amount of DOPE-NBD Inside Placental Tissue Sample
4 Notes
References
Index