Liposome-Based Drug Delivery Systems

Series Preface
Volume Preface
Contents
About the Series Editor
About the Volume Editors
Contributors
Part I: General Protocols for Fabrication of Drug Liposomes
	1 Liposomes in Drug Delivery: Status and Advances
		1 Introduction
		2 Features of Liposomes
		3 Classification of Liposomes
			3.1 Passive Targeting Liposomes
				3.1.1 Conventional Liposomes
				3.1.2 Long Circulating Liposomes
			3.2 Active Targeting Liposomes
				3.2.1 Ligand-Mediated Liposomes
				3.2.2 Antibody-Mediated Liposomes
			3.3 Physicochemical Targeting Liposomes
				3.3.1 pH-Sensitive Liposomes
				3.3.2 Temperature-Sensitive Liposomes
				3.3.3 Enzyme-Sensitive Liposomes
				3.3.4 Physical Adsorption-Mediated Liposomes
			3.4 Multifunctional Liposomes
		4 Preparation Materials and Methods of Liposomes
			4.1 Preparation Materials
				4.1.1 Phospholipids
				4.1.2 Cholesterol
				4.1.3 Other Materials
			4.2 Preparation Methods
				4.2.1 Film Dispersion Method
				4.2.2 Reverse-Phase Evaporation Method
				4.2.3 Chemical Gradient Methods
					pH Gradient Method
					Ammonium Sulfate Gradient Method
					Calcium Acetate Gradient Method
				4.2.4 Additional Methods
		5 Application of Liposomes
		6 Concluding Remarks
		References
	2 Preparation of Drug Liposomes by Thin-Film Hydration and Homogenization
		1 Introduction
			1.1 Thin-Film Hydration
			1.2 Homogenization
				1.2.1 Sonication
				1.2.2 Extrusion
		2 Materials
			2.1 Preparation of DTX-Encapsulating Liposomes
			2.2 Preparation of siRNA-Encapsulating Liposomes
		3 Methods
			3.1 Preparation of DTX-Encapsulating Liposomes
				3.1.1 Thin Film Hydration-MLVs Formation
				3.1.2 Homogenization (Sonication)-SUVs Formation
			3.2 Preparation of siRNA-Incorporating Liposomes
				3.2.1 Thin Film Hydration- MLVs Formation
				3.2.2 Homogenization (Extrusion)-SUVs Formation
		4 Notes
		5 Conclusion
		References
	3 Preparation of Drug Liposomes by Reverse-Phase Evaporation
		1 Introduction
		2 Materials
			2.1 Chemicals
		3 Methods
			3.1 Preparation of Liposomes by Reverse-Phase Evaporation
			3.2 The Determination of Encapsulation Efficiency (%)
			3.3 Observation of Particle Size and Electron Micrograph
			3.4 Measurement of Surface Charge
			3.5 Diffusional Exchange Measurements
		4 Notes
		5 Conclusion
		References
4 Preparation and Characterization of Drug Liposomes by pH-Gradient Method
	1 Introduction
	2 Materials
	3 Methods
		3.1 Preparation of Liposomes Loaded by Fluorescent Probes or Adriamycin
		3.2 Fluorescence and Release Measurements
		3.3 Data Analysis
	4 Notes
	References
5 Preparation and Characterization of Drug Liposomes by Ammonium Sulfate Gradient
	1 Introduction
	2 Materials and Apparatuses
		2.1 Synthesis of Targeting Molecule (DSPE-PEG2000-PTDHIV-1 Conjugate)
		2.2 Preparation of the Liposomes
		2.3 Determination of Epirubicin and Celecoxib
		2.4 Characterization of the Targeting Epirubicin Plus Celecoxib Liposomes
		2.5 Cell Cultures
		2.6 Evaluations on Breast Cancer Cells
		2.7 Evaluations on Breast Cancer Spheroids
		2.8 Evaluations on Breast Cancer Bearing Nude Mice
		2.9 Statistics
	3 Procedures and Methods
		3.1 Synthesis of DSPE-PEG2000-PTDHIV-1 Conjugate
		3.2 Preparation of the Liposomes
			3.2.1 Preparation of Targeting Celecoxib Liposomes
			3.2.2 Preparation of Targeting Epirubicin Plus Celecoxib Liposomes
			3.2.3 Preparation of Epirubicin Liposomes
			3.2.4 Preparation of Targeting Epirubicin Liposomes
			3.2.5 Preparation of Fluorescence Labeling Liposomes
		3.3 Determination of Epirubicin and Celecoxib by HPLC
			3.3.1 Methodology of the HPLC Determination
			3.3.2 EE Determination of Epirubicin and Celecoxib in the Liposomes
		3.4 Characterization with DLS
		3.5 Morphology Characterization with AFM and TEM
		3.6 In Vitro Release of Epirubicin and Celecoxib
		3.7 Cell Cultures
		3.8 Evaluations on Breast Cancer Cells
			3.8.1 Cytotoxic Effects on Breast Cancer Cells
			3.8.2 Cellular Uptakes in Breast Cancer Cells
			3.8.3 Targeting Effects on Breast Cancer Cells
		3.9 Evaluations on Breast Cancer Cell Spheroids
			3.9.1 Establishing Multicellular Cancer Cell Spheroids
			3.9.2 Penetrating Effects in Breast Cancer Cell Spheroids
			3.9.3 Destructing Effects on the Breast Cancer Spheroids
		3.10 Evaluations on Breast Cancer-Bearing Nude Mice
			3.10.1 Establishing Breast Cancer-Bearing Nude Mice Models
			3.10.2 Anticancer Evaluations of the Targeting Liposomes on Nude Mice
			3.10.3 In Vivo Imaging in Mice
	4 Notes
	References
6 Preparation of Drug Liposomes by EDTA Gradient Methods
	1 Introduction
	2 Materials
		2.1 Materials for Preparation of Ammonium EDTA Solution
		2.2 Materials for Preparation of Empty Liposomes
		2.3 Materials for Preparation of EDTA Gradient Empty Liposomes
		2.4 Materials for Preparation of Drug-Loaded EDTA Gradient Liposomes
		2.5 Materials for Determination of Lipid Concentration Using a Phosphate Assay
	3 Methods
		3.1 Doxorubicin-Loaded EDTA Gradient Liposomes (Song et al. 2014)
			3.1.1 Preparation of Ammonium EDTA Solution (See Note 1)
			3.1.2 Preparation of Empty Liposomes (Modified Ethanol Injection Method) (See Note 3)
			3.1.3 Preparation of EDTA Gradient Empty Liposomes (Dialyzed Method) (See Note 10)
			3.1.4 Doxorubicin-Loaded EDTA Gradient Liposomes
		3.2 Preparation of Topotecan-Loaded EDTA Gradient Liposomes (Yang et al. 2012)
			3.2.1 Preparation of Ammonium EDTA Solution
			3.2.2 Preparation of Empty Liposomes (Modified Ethanol Injection Method)
			3.2.3 Preparation of EDTA Gradient Empty Liposomes (Anion and Cation Mixed Ion-Exchange Resin Mini-Column Method)
			3.2.4 Preparation of Topotecan-Loaded EDTA Gradient Liposomes
		3.3 Epirubicin-Loaded EDTA Gradient Liposomes (Yang et al. 2014)
			3.3.1 Preparation of Ammonium EDTA Solution
			3.3.2 Preparation of Empty Liposomes (Modified Ethanol Injection Method)
			3.3.3 Preparation of EDTA Gradient Empty Liposomes (Sephadex G-50 Mini-Column Method)
			3.3.4 Preparation of Epirubicin-Loaded EDTA Gradient Liposomes
		3.4 Idarubicin-Loaded EDTA Gradient Liposomes (Gubernator et al. 2010)
			3.4.1 Preparation of Diammonium EDTA Solution
			3.4.2 Preparation of Empty Liposomes (Thin Lipid Film Method)
			3.4.3 Preparation of EDTA Gradient Empty Liposomes (Sephadex G-50 Mini-Column Method)
			3.4.4 Preparation of Idarubicin-Loaded EDTA Gradient Liposomes
		3.5 Determination of Lipid Concentration Using a Phosphate Assay (Torchilin and Weissig 2003)
			3.5.1 Preparation of Solutions
			3.5.2 Determination of Lipid Concentration
	4 Notes
	5 Conclusion
	References
7 Lipid-Coated Cisplatin Nanoparticles for Insoluble Drug Loading
	1 Introduction
	2 Materials
		2.1 Materials
		2.2 Synthesis of cis-[Pt(NH3)2(H2O)2](NO3)2 Precursor
		2.3 Preparation of LPC NPs
		2.4 Characterization of Particle Size and Zeta Potential
		2.5 Transmission Electron Microscopy (TEM)
		2.6 Characterization of Drug-Loading Capacity
		2.7 In Vitro Release of Drug from NPs in Medium
		2.8 In Vitro Release of Drug from NPs in Cells
	3 Methods
		3.1 Synthesis of Cis-[Pt(NH3)2(H2O)2](NO3)2 Precursor
		3.2 Preparation of LPC NPs
		3.3 Characterization of Particle Size and Zeta Potential
		3.4 Transmission Electron Microscopy (TEM)
		3.5 Characterization of Drug-Loading Capacity
		3.6 In Vitro Release of Drug from NPs in Medium
		3.7 In Vitro Release of Drug from NPs in Cells
	4 Notes
	5 Conclusion
	References
8 Purification Method of Drug-Loaded Liposome
	1 Introduction
	2 Dialysis
	3 Column Chromatographic Separation Method
	4 Centrifugation
	5 Microcentrifugation
	6 Ion-Exchange Resin
	7 Ultrafiltration
	8 Protamine Aggregation Method
	9 Others
	10 Conclusion
	References
9 Quality Evaluation of Drug-Loaded Liposomes
	1 Introduction
	2 Particle Size
	3 Size Distribution
	4 Morphology
	5 Zeta Potential
	6 Entrapment Efficiency
		6.1 Gel Column Chromatography
		6.2 Dialysis
		6.3 Ultracentrifugation
		6.4 Ultrafiltration
		6.5 Protamine Aggregation Method
	7 In Vitro Release
	8 Osmotic Pressure
	9 Sterilization
	10 Oxidation Index
	11 Lysophospholipid
	12 Drug Contents
	13 Leakage Rate
	14 Stability
	15 Pharmacokinetics and Pharmacology
	16 Assessment of Efficacy
	17 Safety and Toxicity
	18 Conclusion
	References
Part II: Functionalized Liposome-Based Drug Delivery Systems and Potential Application
	10 Coupling Methods of Antibodies and Ligands for Liposomes
		1 Introduction
			1.1 Coupling Methods
			1.2 Targeted Antibody-Drug Conjugates
			1.3 Targeted Drug Liposomes
		2 Materials
			2.1 ADC Conjugates
				2.1.1 Preparation of Conjugate P/PEG(5%)GH-DOX (Rameshwar and Jose 2012)
				2.1.2 Preparation of mAb-taxoid Conjugates (Ojima and Geng 2002)
				2.1.3 Preparation of cAC10-Valine-Citrulline-MMAE (Russell and Sanderson 2004)
				2.1.4 Preparation of β-Galactosidase-Sensitive Antibody Drug Conjugates (Sergii and Chloe 2017)
			2.2 Preparation of Mitochondrial Targeting Topotecan-Loaded Liposomes (Yu et al. 2012)
		3 Methods
			3.1 ADC Conjugates
				3.1.1 Preparation of Conjugate P/PEG(5%)GH-DOX (Fig. 9, Notes 1, 2, 3)
					Preparation of Conjugate P/PEG(5%)GH
					Preparation of Conjugate P/PEG(5%)GH-DOX(5%)
				3.1.2 Preparation of mAb-taxoid Conjugates (Fig. 10, Note 4)
					Preparation of 3′-Dephenyl-3′-(2-methylprop-1-enyl)-10-(3-methyldisulfanylopropa- noyl-docetaxel)
					Preparation of Antibody-Taxoid Conjugates
				3.1.3 Preparation of cAC10-Valine-Citrulline-MMAE (Fig. 11, Notes 5, 6)
					Preparation of Valine-Citrulline-MMAE
					Preparation of cAC10-Valine-Citrulline-MMAE
				3.1.4 Preparation of β-Galactosidase-Sensitive Antibody Drug Conjugates (Figs. 12 and 13, Notes 7, 8)
					Preparation of Payloads
					Preparation of ADCs
			3.2 Preparation of Mitochondrial Targeting Topotecan-Loaded Liposomes (Yu et al. 2012)
		4 Notes
		5 Summary
		References
	11 Preparation and Evaluation of Folate Receptor Mediated Targeting Liposomes
		1 Overview
		2 Protocol
			2.1 Materials
				2.1.1 Synthesis of DPPE-PEG2000- Folate
				2.1.2 Characterization of DPPE-PEG2000-Folate
				2.1.3 Preparation of Doxorubicin Liposomes
				2.1.4 Characterization of Folate-Targeted Doxorubicin Liposomes
				2.1.5 Cellular Uptake Evaluation of Folate-Targeted Doxorubicin Liposomes
			2.2 Rationale and Procedures
				2.2.1 Synthesis of DPPE-PEG2000-Folate
				2.2.2 Characterization of DPPE-PEG2000-Folate
				2.2.3 Preparation of Folate-Targeted Doxorubicin Liposomes
					Liposome Formation
					Micellization of DPPE-PEG2000-Folate and Insert the Micelle into Preformed Liposomes
					Folate-Targeted Doxorubicin Liposomes
				2.2.4 Characterization of Folate-Targeted Doxorubicin Liposomes
				2.2.5 Cellular Uptake Evaluation of Folate-Targeted Doxorubicin Liposomes
		3 Notes
		References
	12 Preparation of Cell Penetrating Peptides-Mediated Targeting Drug Liposomes
		1 Introduction
		2 Materials
			2.1 Materials
			2.2 Synthesis of DSPE-PEG2000-R8
			2.3 Preparation of Liposomes
			2.4 Cell Culture, Cellular Uptake, and Cytotoxicity
		3 Rationale and Procedures
			3.1 Synthesis of DSPE-PEG2000-R8
			3.2 Preparation of Liposomes
			3.3 Determination of Particle Size and Zeta Potential
			3.4 Morphological Examination of Liposomes
			3.5 Stability of Liposomes in Serum
			3.6 PTX Release Study
			3.7 Cellular Uptake Study
			3.8 Cytotoxicity Study In Vitro
		4 Notes
		5 Conclusion
		References
13 Preparation of Multifunctional Paclitaxel Liposomes for Treatment of Brain Glioma
	1 Introduction
	2 Materials
		2.1 Synthesis of Targeting Molecule Conjugates
			2.1.1 MAN-TPGS1000 (See Note 1)
			2.1.2 DQA-PEG2000-DSPE (See Note 2)
		2.2 Preparation of Multifunctional Paclitaxel Liposomes
		2.3 Measurement of Encapsulation Efficiency of Paclitaxel and Artemether
		2.4 Measurement of Particle Size, Zeta Potential, and Polydispersity Index (PDI)
		2.5 Morphology Characterization with Atomic Force Microscope (AFM)
		2.6 In Vitro Release of Paclitaxel or Artemether
		2.7 Cell Culture
		2.8 Cytotoxic Effects on Brain Cancer Cells and Brain CSCs
		2.9 Transport Across the BBB and Targeting of Brain Cancer Cells
		2.10 In Vivo Imaging in Mice
		2.11 Anticancer Efficacy in Rats
	3 Methods
		3.1 Synthesis of Targeting Molecule Conjugates
			3.1.1 Synthesis of MAN-TPGS1000 Conjugate
			3.1.2 Synthesis of DQA-PEG2000-DSPE Conjugate
		3.2 Method for Determination of Paclitaxel and Artemether
			3.2.1 Linearity of Paclitaxel
			3.2.2 Linearity of Artemether
			3.2.3 HPLC Method for Measurement of Paclitaxel or Artemether
		3.3 Preparation of Multifunctional Paclitaxel Liposomes by Thin-Film Hydration
		3.4 Measurement of Encapsulation Efficiency of Paclitaxel or Artemether
		3.5 Measurement of Particle Size, Zeta Potential, and Polydispersity Index (PDI)
		3.6 Morphology Characterization with AFM
		3.7 In Vitro Release of Paclitaxel or Artemether
		3.8 Cell Culture
		3.9 Cytotoxic Effects on Brain Cancer Cells or Brain CSCs
			3.9.1 Cytotoxic Effects on Brain Cancer Cells
			3.9.2 Cytotoxic Effects on Brain CSCs
		3.10 Transport Across the BBB and Targeting of Brain Cancer Cells
		3.11 In Vivo Imaging in Mice
		3.12 Anticancer Efficacy in Rats
	4 Notes
	5 Conclusion
	References
14 Preparation and Evaluation of Integrin Receptor-Mediated Targeting Drug Liposomes
	1 Introduction
	2 Materials
		2.1 Synthesis of RGD Peptide
		2.2 Preparation of RGD Conjugated Polyethylene Glycol-Lipid (RGD-PEG-DSPE)
		2.3 Characterization of RGD-PEG-DSPE
		2.4 Preparation of RGD-Modified Liposome
			2.4.1 Preparation of RGD-Modified Liposome Loaded with Doxorubicin (RGD-LS/DOX)
			2.4.2 Preparation of RGD-Modified Liposome Loaded with pDP (RGD-LS/pDP)
		2.5 Characterization of RGD-Modified Liposomes
		2.6 Tumor Targeting Ability of RGD-Modified Liposomes
			2.6.1 Cellular Uptake
				Preparation of RGD-Modified Liposome Loaded with 5-Carboxyfluorescein (RGD-LS/FAM)
				Uptake by Glioblastoma Cells
			2.6.2 In Vivo Tumor Targeting Ability
				Preparation of RGD-Modified Liposome Loaded with Near-Infrared Fluorescent Dyes (RGD-LS/Dir)
				In Vivo Distribution
		2.7 Antitumor Activity of RGD-Modified Liposomes
			2.7.1 In Vitro Antitumor Activity
			2.7.2 In Vivo Antiglioblastoma Efficacy
	3 Methods
		3.1 Synthesis of RGD Peptide
		3.2 Preparation of RGD-PEG-DSPE
		3.3 Characterization of RGD-PEG-DSPE
		3.4 Preparation of RGD-Modified Liposomes
			3.4.1 Preparation of RGD-LS/DOX
			3.4.2 Preparation of RGD-LS/pDP
		3.5 Characterization of RGD-Modified Liposomes
			3.5.1 Physicochemical Characterization of RGD-Modified Liposomes
			3.5.2 Characterization of Conjugated RGD on the Surface of Liposomes
			3.5.3 DOX Encapsulation Efficacy
			3.5.4 pDP Encapsulation Efficacy
		3.6 Tumor Targeting Ability of RGD-Modified Liposomes
			3.6.1 Cellular Uptake
				Preparation of RGD-Modified Liposome Loaded with 5-Carboxyfluorescein (RGD-LS/FAM)
				Uptake by Glioblastoma Cells
					Cell Culture
					Observation Using Confocal Laser Microscope
			3.6.2 In Vivo Tumor Targeting Ability
				Preparation of RGD-Modified Liposome Loaded with Near-Infrared Fluorescent Dyes (RGD-LS/Dir)
				In Vivo Distribution
					Establishment of Intracranial Glioma Model
		3.7 Antitumor Activity of RGD-Modified Liposomes
			3.7.1 In Vitro Antitumor Activity
			3.7.2 In Vivo Antiglioblastoma Efficacy
	4 Notes
	5 Conclusion
	References
15 Preparation of Functional Vincristine Liposomes for Treatment of Invasive Breast Cancer
	1 Introduction
	2 Materials
		2.1 Synthesis of Targeting Molecule
		2.2 Preparation of the Liposomes
		2.3 Determination of Vincristine and Dasatinib
		2.4 Characterization of the Liposomes
		2.5 Cell Cultures
		2.6 Evaluations on Invasive Breast Cancer Cells
		2.7 Evaluations on Invasive Breast Cancer Spheroids
		2.8 Evaluations on Breast Cancer-Bearing Nude Mice
	3 Methods
		3.1 Synthesis of Targeting Molecule
		3.2 Preparation of the Liposomes
			3.2.1 Preparation of Functional Vincristine Plus Dasatinib Liposomes
			3.2.2 Preparation of Vincristine Plus Dasatinib Liposomes
			3.2.3 Preparation of Vincristine Liposomes
			3.2.4 Preparation of Dasatinib Liposomes
			3.2.5 Preparation of Functional Liposomes
			3.2.6 Preparation of Fluorescence Labeling Liposomes
		3.3 Determination of Vincristine and Dasatinib
			3.3.1 Linearity of Vincristine
			3.3.2 Linearity of Dasatinib
			3.3.3 Precision of Vincristine or Dasatinib
			3.3.4 Detection Limits of Vincristine or Dasatinib
		3.4 Characterization of the Liposomes
			3.4.1 Encapsulation Efficiency Determination of Drug-Loaded Liposomes
			3.4.2 Characterization with Dynamic Light Scatter
			3.4.3 Morphology Characterization with AFM
			3.4.4 In Vitro Release of Vincristine or Dasatinib
		3.5 Cell Cultures
		3.6 Evaluations on Invasive Breast Cancer Cells
			3.6.1 Cellular Uptakes in Breast Cancer Cells
		3.7 Evaluations on Invasive Breast Cancer Spheroids
			3.7.1 Construction of Multicellular Cancer Spheroids
			3.7.2 Penetrating Effect in Breast Cancer Spheroids
			3.7.3 Destructing Effect on the Breast Cancer Spheroids
		3.8 Evaluations on Breast Cancer-Bearing Nude Mice
			3.8.1 Establishing Breast Cancer-Bearing Nude Mice Models
			3.8.2 Anticancer Evaluations of the Functional Liposomes on Nude Mice
			3.8.3 In Vivo Imaging in Mice
		3.9 Statistics
	4 Notes
	5 Conclusion
	References
16 Preparation and Characterization of DNA Liposomes Vaccine
	1 Introduction
	2 Materials
		2.1 Preparation of Liposomes
		2.2 Preparation of Plasmid DNA Liposome Vaccine
		2.3 Encapsulation Efficiency of DNA Liposome Vaccine
		2.4 Size Reduction of DNA Liposome Vaccine
	3 Methods (Gregoriadis et al. 1999)
		3.1 Preparation of MLV and SUV
			3.1.1 Preparation of Phospholipid Membranes
			3.1.2 Preparation of MLV
			3.1.3 Preparation of SUV
		3.2 Encapsulation of Plasmid DNA by Liposomes
			3.2.1 Dehydration of SUV After Encapsulating Plasmid DNA
			3.2.2 Rehydration of the Freeze-Dried Material
			3.2.3 Depletion of Free DNA
		3.3 Encapsulation Efficiency of DNA in the Liposomes
		3.4 Reduction of DRV Liposomes Size
			3.4.1 Reduction of DRV Liposomes Size by a Microfluidizer
			3.4.2 Preparation of Small DNA Liposomes
			3.4.3 Separation of Free DNA
			3.4.4 Characterization of DNA Content and Properties of DRVs After Microfluidization
			3.4.5 An Example of DNA Liposomes Vaccine
	4 Notes
	5 Conclusions
	References
17 Preparation and Evaluation of Biomineral-Binding Antibiotic Liposomes
	1 Introduction
	2 Materials
		2.1 Preparation of Biomineral-Binding Lipid ALN-TEG-Chol
		2.2 Preparation and Characterization of Biomineral-Binding Liposomes (BBL)
		2.3 Binding Kinetics of Biomineral-Binding Liposomes (BBL) on HA
		2.4 In Vitro Oxacillin Release from Biomineral-Binding Liposomes (BBL)
		2.5 In Vitro Inhibition of S. aureus Biofilm Growth Using Oxacillin-Loaded Biomineral-Binding Liposomes (BBL) Staphylococcus a...
	3 Methods
		3.1 Preparation of Biomineral-Binding Lipid ALN-TEG-Chol (Fig. 2, Note 1)
			3.1.1 Preparation of Azido-Terminated Cholesterol (Azido-TEG-Chol, Compound 5) (Note 2)
			3.1.2 Preparation of Acetylene-Terminated Alendronate (Acetylene ALN, Compound 7) (Note 3)
			3.1.3 Preparation of Biomineral-Binding Alendronate-Cholesterol Lipid (ALN-TEG-Chol, Compound 8) Using Click Reaction (Note 4)
		3.2 Preparation and Characterization of Biomineral-Binding Liposomes (BBL) and Non-Binding Liposome (NBL)
			3.2.1 Preparation of Oxacillin-Loaded Liposomes by Extrusion Method (Gabizon et al. 2003; Note 5-8)
			3.2.2 Preparation of Oxacillin-Loaded Liposomes by Sonication Method (Note 9, 10)
		3.3 Binding Kinetics of Biomineral-Binding Liposomes (BBL) on HA
		3.4 In Vitro Oxacillin Release from Biomineral-Binding Liposomes (BBL)
		3.5 In Vitro Inhibition of S. aureus Biofilm Growth Using Oxacillin-Loaded Biomineral Binding Liposomes (BBL) (Note 11)
		3.6 Statistical Analysis
	4 Notes
	5 Conclusion
	References
18 Dual-Modified siRNA-Loaded Liposomes for Prostate Cancer Therapy
	1 Introduction
	2 Materials
		2.1 Synthesis of DSPE-PEG2000-PRP
		2.2 Synthesis of DSPE-PEG5000-Folate
		2.3 Preparation and Characterization of siRNA-Loaded Liposomes
		2.4 Gel Electrophoresis
		2.5 Cell Culture
		2.6 Cellular Uptake and Flow Cytometric Analysis
		2.7 Cell Apoptosis Assay
		2.8 Analytical Instruments
	3 Methods
		3.1 Synthesis of DSPE-PEG2000-PRP (See Note 2)
		3.2 Synthesis of DSPE-PEG5000-Folate (See Note 5)
		3.3 Preparation and Characterization of siRNA-Loaded Liposomes (See Note 8)
		3.4 Gel Electrophoresis
		3.5 Cell Culture
		3.6 Cellular Uptake and Flow Cytometric Analysis
		3.7 Cell Apoptosis Assay
	4 Notes
	5 Conclusion
	References
19 Fabrication and Evaluation of Dual Peptides-Modified Liposomes Coencapsulating siRNA and Docetaxel
	1 Introduction
	2 Materials
		2.1 Synthesis of DSPE-PEG2000-Angiopep
		2.2 Synthesis of DSPE-PEG2000- tLyP-1
		2.3 Preparation of Dual Peptides-Modified Liposomes Coencapsulating siRNA and Docetaxel
		2.4 Characterization of Dual Peptides-Modified Liposomes Coencapsulating siRNA and Docetaxel
		2.5 Gel Electrophoresis
		2.6 Determination of Entrapment Efficiency of DTX
		2.7 Antiproliferation Study
		2.8 Analytical Instruments
	3 Methods
		3.1 Synthesis of DSPE-PEG2000-Angiopep (see Note 1)
		3.2 Synthesis of DSPE-PEG2000-tLyP-1(see Note 1)
		3.3 Preparation of Liposomes Coencapsulating siRNA and Docetaxel
		3.4 Characterization of Liposomes
		3.5 Gel Electrophoresis
		3.6 Determination of Entrapment Efficiency of DTX
		3.7 In Vitro Antiglioblastoma Efficacy
			3.7.1 Cell Culture
			3.7.2 Antiproliferation Study
	4 Notes
	5 Conclusion
	References
20 Preparation and Evaluation of Rivastigmine Liposomes for Intranasal Delivery
	1 Overview
	2 Protocol
		2.1 Materials
			2.1.1 Preparation of Rivastigmine Liposomes
			2.1.2 Synthesis of DSPE-PEG2000-CPP
			2.1.3 Preparation of CPP-Modified Rivastigmine Liposomes
			2.1.4 Characterization of Liposomes
			2.1.5 Determination of Entrapment Efficiency of Rivastigmine
			2.1.6 In Vitro Release of Liposomes
			2.1.7 The BBB Model and Transport Across the BBB
			2.1.8 Distribution of Rivastigmine Liposomes
			2.1.9 Pharmacodynamic
			2.1.10 Analytical Instruments
		2.2 Methods
			2.2.1 Preparation of Rivastigmine Liposomes
			2.2.2 Synthesis of DSPE-PEG2000-CPP
			2.2.3 Preparation of CPP-Modified Rivastigmine Liposomes
			2.2.4 Characterization of Liposomes
			2.2.5 Determination of Entrapment Efficiency of Rivastigmine
			2.2.6 In Vitro Release of Liposomes
			2.2.7 The BBB Model and Transport Across the BBB
			2.2.8 Distribution of Rivastigmine Liposomes in the Brain
			2.2.9 Pharmacodynamic Study of Liposomes
	3 Discussion
	References
21 Dequalinium-Mediated Mitochondria-Targeting Drug Liposomes for the Treatment of Drug-Resistant Lung Cancer
	1 Introduction
	2 Materials
		2.1 Synthesis of DQA-PEG2000-DSPE Conjugate
		2.2 Preparation of Targeting Liposomes
		2.3 Characterization of Liposomes
		2.4 Cell Culture
		2.5 Cytotoxicity
		2.6 Targeting Mechanism and Effect
			2.6.1 Mitochondrial Co-localization
			2.6.2 Drug Content in Mitochondria
			2.6.3 Mitochondrial Depolarization
			2.6.4 Release of Cytochrome C from Mitochondria
			2.6.5 Caspase Activation
			2.6.6 Effects on Pro-apoptotic Bax and Anti-apoptotic Mcl-1
			2.6.7 ROS Assay
			2.6.8 ATP Assay
		2.7 Efficacy in Drug-Resistant Lung Cancer Xenografts
		2.8 In Vivo Imaging Observation
	3 Methods
		3.1 Synthesis of DQA-PEG2000-DSPE Conjugate
		3.2 Preparation of Liposomes
			3.2.1 Preparation of Targeting Lonidamine Liposomes
			3.2.2 Preparation of Lonidamine Liposomes
			3.2.3 Preparation of Targeting Coumarin Liposomes (See Note 3)
			3.2.4 Preparation of Coumarin Liposomes
			3.2.5 Preparation of Targeting Epirubicin Liposomes
			3.2.6 Preparation of Epirubicin Liposomes
		3.3 Characterization of Liposomes
		3.4 Cell Culture
		3.5 Cytotoxicity
		3.6 Targeting Mechanism and Effect
			3.6.1 Mitochondrial Co-localization
			3.6.2 Drug Content in Mitochondria
			3.6.3 Mitochondrial Depolarization
				Measure the Mitochondria Membrane Potential (DeltaPsim)
				Confirm the Specificity of Induced Mitochondrial Depolarization by Targeting Lonidamine Liposomes or Targeting Epirubicin Lipo...
			3.6.4 Release of Cytochrome C from the Mitochondria
			3.6.5 Caspase Activation
			3.6.6 Effects on Pro-apoptotic Bax and Anti-apoptotic Mcl-1
			3.6.7 ROS Assay
			3.6.8 ATP Assay
		3.7 Efficacy in Drug-Resistant Lung Cancer Xenografts
		3.8 In Vivo Imaging Observation
		3.9 Statistical Analysis
	4 Notes
	5 Conclusion
	References
22 Preparation of Anthracyclines Liposomes for Tumor-Targeting Drug Delivery
	1 Introduction
	2 Materials
		2.1 Preparation of Liposomes by pH Gradient Method
		2.2 Preparation of Liposomes Modified with MAN
		2.3 Daunorubicin Liposomes Modified with MAN and TF
		2.4 Determination of Encapsulation Efficiency, Drug Release, and Particle Size
		2.5 Determination of Morphology
		2.6 Cell Culture
		2.7 BBB Model In Vitro
		2.8 Transport Across the BBB and Competition Assay of MAN
		2.9 C6 Glioma Cellular Uptake and Competition Assay of TF
		2.10 Antiproliferative Activity Against C6 Glioma Cells
		2.11 Dual-Targeting Effects In Vitro
		2.12 Effect on the Avascular C6 Glioma Spheroids
		2.13 Effects on the Survival of Brain Tumor-Bearing Animals
		2.14 Measurement of Daunorubicin in Plasma
		2.15 Measurement of Daunorubicin in Tissues
		2.16 Pharmacokinetics and Biodistribution
	3 Methods
		3.1 Preparation and Characterization of the Liposomes
			3.1.1 Blank Liposomes
			3.1.2 Daunorubicin Liposomes (Note 1)
			3.1.3 Daunorubicin Liposomes Modified with MAN (Note 2)
			3.1.4 Daunorubicin Liposomes Modified with MAN and TF (Note 3)
			3.1.5 Daunorubicin Liposomes Modified with TF (Note 3)
			3.1.6 Encapsulation Efficiency (Note 4)
			3.1.7 Drug Release (Note 5)
			3.1.8 Particle Size (Note 6)
			3.1.9 Determination of the Morphology Under TEM (Note 6)
			3.1.10 Determination of the Morphology Under AFM (Note 6)
		3.2 Cell Culture
		3.3 BBB Model In Vitro (Note 7)
		3.4 Transport Across the BBB and Competition Assay of MAN
		3.5 C6 Glioma Cellular Uptake Assay of TF (Note 8)
		3.6 Competition Assay of TF
		3.7 Antiproliferative Activity Against C6 Glioma Cells (Note 9)
		3.8 Dual-Targeting Effects In Vitro (Note 10)
		3.9 Effect on the Avascular C6 Glioma Spheroids (Note 11)
		3.10 Effects on the Survival of Brain Tumor-Bearing Animals (Note 12)
		3.11 Measurement of Daunorubicin in Plasma (Note 13)
		3.12 Measurement of Daunorubicin in Tissues (Note 14)
		3.13 Pharmacokinetics and Biodistribution (Note 15)
		3.14 Statistical Analysis
	4 Notes
	5 Conclusion
	References
23 Preparation and Characterization of pH Sensitive Drug Liposomes
	1 Introduction
	2 Materials
		2.1 Synthesis of Zwitterionic Oligopeptide Lipid (e.g., 1,5-Dioctadecyl-L-Glutamyl 2-Histidyl-Hexahydrobenzoic Acid, HHG2C18)
		2.2 Preparation of pH-Sensitive Drug Liposomes by Thin-Film Dispersion Methods (HHG2C18-L)
		2.3 Preparation of Conventional Drug Liposomes by Thin-Film Dispersion Methods (SPC-L)
		2.4 Determination of Particle Size
		2.5 Determination of Zeta Potential
		2.6 Determination of the Buffering Capacity
		2.7 Determination of the Degradation of Hexahydrobenzoic Amide from the HHG2C18-L
		2.8 Determination of Zeta Potential Variation of the HHG2C18-L with the Degradation of the Amide
		2.9 Cell Culture
		2.10 Cellular Uptake of the HHG2C18-L Under Different pH Values
		2.11 Qualitatively Evaluation of Endolysosomal Escape and Mitochondrial Targeting of the HHG2C18-L by Confocal Laser Scanning ...
	3 Methods
		3.1 Synthesis of Zwitterionic Oligopeptide Lipid (e.g., HHG2C18)
		3.2 Preparation of pH-Sensitive Drug Liposomes by Thin-Film Dispersion Methods (HHG2C18-L)
		3.3 Preparation of Conventional Drug Liposomes by Thin-Film Dispersion Methods (SPC-L)
		3.4 Determination of Particle Size
		3.5 Determination of Zeta Potential
		3.6 Determination of the Buffering Capacity
		3.7 Determination of the Degradation of Hexahydrobenzoic Amide from the HHG2C18-L
		3.8 Determination of Zeta Potential Variation of the HHG2C18-L with the Degradation of the Amide
		3.9 Cell Culture
		3.10 Cellular Uptake of the HHG2C18-L Under Different pH Values
		3.11 Qualitatively Evaluation of Endolysosomal Escape and Mitochondrial Targeting of the HHG2C18-L by CLSM
	4 Notes
	5 Conclusion
	References
24 HER2-Specific PEGylated Immunoliposomes Prepared by Lyophilization/Rehydration Method
	1 Introduction
	2 Materials
		2.1 Reagents
		2.2 Preparation of Lyophilized PEGylated Immunoliposomes (LPIL)
		2.3 Determination of Particle Size and Zeta Potential
		2.4 Flow Cytometry
		2.5 SiRNA Serum Stability
	3 Methods
		3.1 Preparation of LPIL
		3.2 Encapsulation of siRNA into the Liposomes
		3.3 Determination of Particle Size and Zeta Potential
		3.4 Measurement of Transfection Efficiency
		3.5 Measurement of Gene Silencing Efficiency
		3.6 SiRNA Serum Stability
	4 Notes
	5 Conclusion
	References
25 Preparation and Characterization of Drug Liposomes by Nigericin Ionophore
	1 Introduction
	2 Materials
		2.1 Preparation of Ion Liposomes by Thin-Film Hydration
		2.2 Formation of Ion Gradient
		2.3 Loading of Vincristine or Topotecan and Measurement of Encapsulation Efficiency
		2.4 Measurement of Particle Size, Zeta Potential, and Polydispersity Index (PDI)
		2.5 Transmission Electron Microscopy (TEM)
		2.6 In Vitro Release of Vincristine or Topotecan
		2.7 Cell Culture
		2.8 Cytotoxicity to MDA-MB231or MCF-7 Cells
		2.9 In Vivo Inhibition of the Tumor Growth
		2.10 Pharmacokinetics and Tissue Distribution
	3 Methods
		3.1 Preparation of Standard Curve of Vincristine
		3.2 Preparation of Standard Curve of Topotecan
		3.3 Linearity
		3.4 HPLC Method for Measurement of Vincristine
		3.5 HPLC Method for Measurement of Topotecan
		3.6 Stability Testing of Vincristine or Topotecan
		3.7 Precision of Vincristine or Topotecan
		3.8 Experiment Design
		3.9 Preparation of Vincristine Liposomes and Measurement of Encapsulation Efficiency
			3.9.1 Preparation of K+ Liposomes by Thin-Film Hydration (Fig. 1, Note 1, 2, 3, 4, 5, and 6)
			3.9.2 Formation of K+ Gradient (Note 7, 8, 9, and 10)
			3.9.3 Loading of Vincristine and Measurement of Encapsulation Efficiency (Note 11 and 12)
		3.10 Preparation of Topotecan Liposomes and Measurement of Encapsulation Efficiency (Note 1, 2, 3, 4, 5, and 6)
			3.10.1 Preparation of Na+ Liposomes by Thin-Film Hydration (Fig. 2)
			3.10.2 Formation of Na+ Gradient (Note 7, 8, and 10)
			3.10.3 Loading of Topotecan and Measurement of Encapsulation Efficiency (Note 11 and 12)
		3.11 Measurement of Particle Size, Zeta Potential, and Polydispersity Index (PDI, Note 6)
		3.12 Transmission Electron Microscopy (TEM)
		3.13 In Vitro Release of Vincristine or Topotecan (Note 20)
		3.14 Cell Culture
		3.15 Cytotoxicity to MDA-MB231 or MCF-7 Cells
		3.16 Animal
		3.17 In Vivo Inhibition of the Tumor Growth
		3.18 Pharmacokinetics (Note 21, 22, and 23)
		3.19 Tissue Distribution (Note 24)
		3.20 Statistical Analysis
	4 Notes
	5 Conclusion
	References
26 Application of Labeled Liposomes in Imaging and Biodistribution Observation
	1 Introduction
	2 Materials
		2.1 Preparation of Liposomes Loaded with PET, MRI, and Optical Imaging Contrast
		2.2 Loading of PET, MRI, and Optical Imaging Contrast and Encapsulation Efficiency
		2.3 Measurement of Particle Size, Zeta Potential, and Polydispersity Index (PDI)
		2.4 Transmission Electron Microscopy (TEM)
		2.5 In Vitro Release
		2.6 Cell Culture
		2.7 Cytotoxicity to MDA-MB231, MDA-MB231-BR, or U87-Luc Cells
		2.8 MRI Relaxivity
		2.9 Establishment of the MDA-MB231 Tumor Model
		2.10 Establishment of the Orthotopic Glioma Model
		2.11 Establishment of the Breast Cancer Brain Metastasis Model
		2.12 Bioluminescence Imaging
		2.13 Optical Imaging
		2.14 MRI
		2.15 Pet/Ct
		2.16 Imaging of Biodistribution
	3 Methods
		3.1 Preparation of Liposomes Loaded with PET, MRI, and Optical Imaging Contrast (Note1)
		3.2 Loading of PET, MRI, and Optical Imaging Contrast and Encapsulation Efficiency (Note 2 and 3)
		3.3 Measurement of Particle Size, Zeta Potential, and Polydispersity Index (PDI)
		3.4 Transmission Electron Microscopy (TEM)
		3.5 In Vitro Release
		3.6 Cell Culture
		3.7 Cytotoxicity to MDA-MB231, MDA-MB231-BR, or U87-Luc Cells
		3.8 MRI Relaxivity (Note 4 and 5)
		3.9 Animal
		3.10 Establishment of the MDA-MB231 Tumor Model
		3.11 Establishment of the Orthotopic Glioma Model
		3.12 Establishment of the Breast Cancer Brain Metastasis Model (Note 6)
		3.13 Bioluminescence Imaging (Note 7 and 8)
		3.14 Optical Imaging (Fig. 1)
		3.15 MRI (Note 9 and 10)
		3.16 PET/CT Scan
		3.17 Imaging of Biodistribution
		3.18 Statistical Analysis
	4 Notes
	5 Conclusion
	References
27 Application of Drug Liposomes in Gene Transfection
	1 Introduction
	2 Materials
		2.1 Synthesis of MAL-PEG-DOPE
		2.2 Preparation of the Liposomes
		2.3 Characterization of the Liposomes
		2.4 In Vitro Cell Assays
		2.5 In Vivo Assays
	3 Methods
		3.1 Synthesis of MAL-PEG-DOPE (Fig. 2)
		3.2 Preparation of the Liposomes
			3.2.1 Preparation of DC-Chol/DOPE Liposomes
			3.2.2 Preparation of siRNA/Liposome Complexes
			3.2.3 Conjugation of AS1411 to the Surface of siRNA/Liposome Complexes
		3.3 Characterization of the Liposomes
			3.3.1 Confirm AS1411 Conjugation to Liposomes
			3.3.2 Morphology of Liposomes
			3.3.3 Particle Size and Zeta Potential
			3.3.4 Evaluate the siRNA Loading Ability of Liposomes
		3.4 In Vitro Cell Assays
			3.4.1 Targeting Effects on Melanoma Cells
			3.4.2 Cellular Uptakes in Melanoma Cells
			3.4.3 Verify the Silencing Efficiency by Real-Time PCR
			3.4.4 Assess the Downregulation of Protein by Western Blot Analysis
			3.4.5 Cytotoxicity Assays
			3.4.6 Antiproliferation Assays
		3.5 In Vivo Assays
			3.5.1 Establishing Melanoma Cancer-Bearing Nude Mice
			3.5.2 Tissue Distribution Study
			3.5.3 Tumor Uptake Study
			3.5.4 Gene Silencing In Vivo
			3.5.5 H&E Staining of Tumor Tissues
		3.6 Statistical Analysis
	4 Notes
	References
28 Application of Drug Liposomes in the Hormone Therapy
	1 Introduction
	2 Materials and Apparatuses
		2.1 Preparation of Dex-Loaded Liposomes (Dex-Lips)
		2.2 Characterization of Dex-Loaded Liposomes (Dex-Lips)
		2.3 In Vitro Release of Encapsulated Dex
		2.4 Pharmacokinetic Studies of Dex-Lips in Healthy Rats
		2.5 Establishment of the Adjuvant-Induced Arthritis (AIA) Rat Model
		2.6 Biodistribution of DiD Loaded Liposomes (DiD-Lips) in Arthritic Rats
		2.7 Therapeutic Efficacy of Dex-Lips In Vivo
		2.8 Safety Evaluation in Arthritic Rats
		2.9 Statistical Analysis
	3 Methods
		3.1 Preparation of Liposomes
			3.1.1 Preparation of Dex-Loaded Liposomes (Dex-Lips)
			3.1.2 Preparation of Fluorescence Labeling Liposomes
		3.2 Characterization of Dex-Loaded Liposomes (Dex-Lips)
			3.2.1 Size and Zeta Potential
			3.2.2 Morphology Characterization with TEM
		3.3 Encapsulation and Drug Loading Efficiencies
			3.3.1 Methodology of the HPLC Determination
			3.3.2 Determination of Encapsulation and Drug Loading Efficiencies
		3.4 Stability of Dex-Lips
		3.5 In Vitro Release of Encapsulated Dex
		3.6 Pharmacokinetic Studies of Dex-Lips in Healthy Rats
		3.7 Establishment of the Adjuvant-Induced Arthritis (AIA) Rat Model
		3.8 Biodistribution of DiD Loaded Liposomes (DiD-Lips) in Arthritic Rats
		3.9 Therapeutic Efficacy of Dex-Lips In Vivo
			3.9.1 Paw Thickness Measurement in Dex-Treated Arthritic Rats
			3.9.2 Cytokine Levels in Serum After Therapy
			3.9.3 Histopathology of Ankle Joints from Arthritic Rats After Therapy
		3.10 Safety Evaluation in Arthritic Rats
	4 Notes
	References
Index