Cell penetrating peptides : methods and protocols

Preface
	References
Preface to the Second Edition [1]
	References
Contents
Contributors
Part I: Introduction, Selection, Mechanisms
	Chapter 1: Cell-Penetrating Peptides
		1 Introduction
			1.1 What Is a CPP?
			1.2 Short History of CPPs
			1.3 Classes of CPPs
				1.3.1 Protein Derived Versus Designed
				1.3.2 Physicochemical Properties Versus Structural Properties
				1.3.3 Predicted Versus Random
				1.3.4 Linear Versus Cyclic
				1.3.5 Protein-Mimicking Versus Cargo Delivery Vectors
				1.3.6 Nonspecific Versus Targeted
				1.3.7 Direct Translocators Versus Endocytosis Enhancers
				1.3.8 Nontoxic Versus Antimicrobial
		2 Penetration of the CPPs into the Cells
			2.1 Different Ways of Entering the Cell
			2.2 Mechanism of Penetration into the Cells
			2.3 Kinetics and Thermodynamics of the Penetration into the Cell
		3 Cargo Delivery
		References
	Chapter 2: Shuttling Homeoproteins and Their Biological Significance
		1 Homeoprotein Transduction, a Primitive and Widely Used Signaling Pathway
		2 Homeoprotein Transduction in Early Patterning
		3 Engrailed and VAX1 Homeoprotein Transduction Regulates Axon Navigation
		4 OTX2 Postnatal and Adult Non-cell Autonomous Activity Regulates Cerebral Cortex Plasticity
		5 Consequences of HP Transduction in Translational Research
		6 Conclusion
		References
	Chapter 3: Artificial Evolutionary Optimization Process to Improve the Functionality of Cell Penetrating Peptides
		1 Introduction
		2 Materials
			2.1 Genetic Algorithm for Processing Peptide Sequence Information
			2.2 Peptide Synthesis
			2.3 Adjustment of Peptide Concentrations
			2.4 Cell Culture
			2.5 CPP Uptake Experiments Using HeLa Cells
			2.6 Cytotoxicity Measurements (MTT Assay)
		3 Methods
			3.1 Evolutionary Peptide Optimization
			3.2 Peptide Synthesis
			3.3 Adjustment of CPP Concentrations
			3.4 Cell Culture
			3.5 CPP Uptake Experiments Using HeLa Cells
			3.6 Cytotoxicity Measurements Using the MTT Assay
		4 Notes
		References
	Chapter 4: In Silico Design of Chemically Modified Cell-Penetrating Peptides
		1 Introduction
		2 Materials
			2.1 Dataset
		3 Methods
			3.1 CellPPDMod: In Silico Tool for Prediction of Cell-Penetrating Peptides Containing Natural As Well As Modified Residues
			3.2 Prediction of Modified Cell-Penetrating Peptides
			3.3 Subsidiary Modules
				3.3.1 Sequence-Based Prediction
				3.3.2 Mutational Series
			3.4 Guidelines for Designing Natural As Well As Modified Therapeutic Peptides
		4 Notes
		5 Limitations and Future Prospects
		References
	Chapter 5: Synthetic Molecular Evolution of Cell Penetrating Peptides
		1 Introduction
		2 Materials
			2.1 Combinatorial Peptide Library Synthesis
		3 Methods
			3.1 Kaiser (Ninhydrin)  Test
			3.2 Preparation of the Resin
			3.3 Addition of a Common Amino  Acid
			3.4 Addition of Amino Acids to a Combinatorically Varied  Site
			3.5 Deprotection of the Sidechains
			3.6 Release of Library Members for Screening
			3.7 Synthesis Quality Control
			3.8 Determination of Sequence
		4 Notes
		References
	Chapter 6: A Native CPP from Rattlesnake with Therapeutic and Theranostic Properties
		1 Introduction
		2 Materials
			2.1 Antibacterial and Antifungal Susceptibility Tests
				2.1.1 Microorganisms
				2.1.2 Minimal Inhibitory Concentration (MIC) (See Note 2)
				2.1.3 Fungicidal Assay
			2.2 Tumor Engraftment and In Vivo Treatment with Crotamine
			2.3 Alterations in Metabolism
				2.3.1 Treatment of Animals
				2.3.2 Insulin Tolerance Test (ITT)
				2.3.3 Glucose Tolerance Test (GTT)
				2.3.4 Brown Preadipocyte Cell Culture and Confocal Microscopy
			2.4 Crotamine-Nucleic Acid Nanoparticle Formation and Characterization
			2.5 Delivery of Crotamine-Nucleic Acid Nanoparticles into Cells and Animals
				2.5.1 In Vitro Cell Transfection
				2.5.2 In Vivo Delivery
		3 Methods
			3.1 Minimal Inhibitory Concentration (MIC)
			3.2 Fungicidal Assay
			3.3 Tumor Engraftment and In Vivo Oral Treatment with Crotamine
			3.4 Alterations in Metabolism
				3.4.1 Treatment of Animals
				3.4.2 Insulin Tolerance Test (ITT)
				3.4.3 Glucose Tolerance Test (GTT)
				3.4.4 Brown Preadipocyte Cell Culture and Confocal Microscopy
			3.5 Crotamine-Nucleic Acid Nanoparticle Formation and Characterization
			3.6 Delivery of Crotamine-Nucleic Acid Nanoparticles into Cells and Animals
				3.6.1 In Vitro Cell Transfection
				3.6.2 In Vivo Delivery of Crotamine-Nucleic Acid Nanoparticles
		4 Notes
		5 Conclusion
		References
	Chapter 7: Synthesis of Cell-Penetrating Peptide Coated Silica Nanoparticles and Their Physicochemical and Biological Characte...
		1 Introduction
			1.1 Nanoparticle-CPP Conjugates
			1.2 Application of Nanoparticle-CPP Conjugates
			1.3 Synthesis of Nanoparticle-CPP Conjugates
			1.4 Characterization of Nanoparticle-CPP Conjugates
		2 Materials
			2.1 Silica Nanoparticle Synthesis
			2.2 Peptide Conjugation to Silica Nanoparticles
			2.3 Electron Microscopy
			2.4 Zeta Potential and Dynamic Light Scattering
			2.5 Bradford Assay
			2.6 CD Spectroscopy
			2.7 Cell Culture
			2.8 Reagents for Confocal Microscopy
			2.9 Reagents for Flow Cytometry
			2.10 Reagents for Cytotoxicity Experiments
		3 Methods
			3.1 Synthesis of Silica Nanoparticles
			3.2 Surface Modification of Silica Nanoparticles with Peptides
				3.2.1 Non-covalent Immobilization of Peptides
				3.2.2 Covalent Immobilization of Peptides
			3.3 Nanoparticle-Peptide Conjugate Characterization
				3.3.1 Physicochemical Characterization
					Electron Microscopy
					Zeta Potential and Dynamic Light Scattering (DLS)
					Surface Quantification of CPP Via Bradford Assay
					Determination of Secondary Structure Via CD Spectroscopy
				3.3.2 Bioactivity of Nanoparticle-Peptide Conjugates
					Cell Culturing and Seeding
					Qualitative Cellular Uptake Experiments Using Confocal Microscopy
					Quantitative Cellular Uptake Experiments Using Flow Cytometry
					Evaluation of Cytotoxic Profile Using a Resazurin-Based Assay
		4 Notes
		References
	Chapter 8: Enhancing the Therapeutic Potential of Extracellular Vesicles Using Peptide Technology
		1 Introduction
		2 Targeting Peptides
			2.1 Endogenous EV-Peptide Modification
			2.2 Exogenous EV-Peptide Modification
		3 Modification of EVs with Endosomal Escape Peptides
		4 Multifunctional EV-Peptide Modification
		5 Conclusions
		References
	Chapter 9: Challenges and Methods for the Study of CPP Translocation Mechanisms
		1 Introduction
		2 Materials
			2.1 Translocation with DIBs
			2.2 Translocation with Cells
		3 Methods
			3.1 Translocation with DIBs
			3.2 Translocation with Cells
		4 Notes
		References
	Chapter 10: Membrane Molecular Interactions and Induced Structures of CPPs
		1 Introduction
			1.1 Phospholipid Large Unilamellar Vesicles
			1.2 Leakage Experiment
			1.3 Categories of CPPs
			1.4 Structural Induction of CPPs
		2 Materials
			2.1 Preparation of the Large Unilamellar Vesicle
			2.2 Circular Dichroism Spectroscopy
			2.3 Calcein Leakage Experiment
		3 Methods
			3.1 Preparation of the Large Unilamellar Vesicle (Extrusion Method)
			3.2 Circular Dichroism Spectroscopy
			3.3 Calcein Leakage Experiment
		4 Notes
		References
	Chapter 11: A Single GUV Method for Revealing the Action of Cell-Penetrating Peptides in Biomembranes
		1 Introduction
		2 Materials
			2.1 Chemicals
			2.2 Synthesis and Purification of CF-TP10
		3 Methods
			3.1 Preparation and Purification of GUVs Containing Small GUVs
			3.2 Preparation and Purification of GUVs Containing LUVs
			3.3 Interaction of CF-TP10 with Single GUVs Containing Small GUVs (or LUVs)
			3.4 Data Analysis
		4 Notes
		References
	Chapter 12: Methods for Small-Angle Scattering Measurements on Peptiplexes of DNA with Cell-Penetrating Peptides
		1 Introduction
			1.1 The Structural Landscape of CPP-Nucleic Acid Complexes
			1.2 Structural Tools for Investigating  CPPs
			1.3 Small-Angle Scattering to Study the Structure of Peptiplexes
		2 Materials
			2.1 Reagents
			2.2 Instruments
			2.3 Software for Data Treatment
		3 Methods
			3.1 Sample Preparation
			3.2 Data Collection
			3.3 Data Analysis
		4 Notes
		References
	Chapter 13: Imaging of CPP Delivery Mechanisms of Oligonucleotides
		1 Introduction
		2 Materials
			2.1 Cell Culture and Transfection with Endosomal Markers
			2.2 Polyplex Formation
			2.3 Live Cell CLSM
			2.4 Image Analysis
		3 Methods
			3.1 Formation of Oligonucleotide Nanoparticles/Polyplexes
			3.2 Transfection of HeLa Cells with Marker Plasmids
			3.3 Incubation of Cells with Polyplexes
			3.4 Live Cell Confocal Laser Scanning Microscopy
				3.4.1 Colocalization Analysis (Fig. 2, Table 1)
				3.4.2 To Assess Nuclear Localization (Fig. 3)
		4 Notes
		References
	Chapter 14: Quantitative Subcellular Analysis of Cyclic Cell-Penetrating Peptide EJP18 in Nonadherent Cells
		1 Introduction
		2 Materials
			2.1 Cloning and Bacterial Expression
			2.2 Cell Culture and Imaging
			2.3 Quantification
		3 Methods
			3.1 Cloning of eGFP-EJP18 and eGFP-cEJP18
			3.2 Bacterial Transformation
			3.3 Bacterial Protein Expression
			3.4 Protein Purification
			3.5 SDS-PAGE Analysis
			3.6 Buffer Exchange
			3.7 Mass Spectrometry Analysis
			3.8 Quantification of eGFP Content
			3.9 Preparing Nonadherent Cells for Imaging
			3.10 Confocal Imaging
			3.11 Quantification of Subcellular CPP Fluorescence
				3.11.1 Generating Whole Cell Mask and Quantifying Total Cell Fluorescence
				3.11.2 Quantifying Intracellular Fluorescence
				3.11.3 Quantifying Nuclear Fluorescence
				3.11.4 Data Analysis
		4 Notes
		References
	Chapter 15: PepFect14 Signaling and Transfection
		1 Introduction
			1.1 Energy Independent and Dependent Uptake for  CPPs
			1.2 PepFect14
				1.2.1 What Is PepFect14?
				1.2.2 PepFect14/Oligonucleotide (PF14/ON) Complexes: Uptake and Transfection Mechanisms
			1.3 PepFect14 Uptake Involves Scavenger Receptors Class  A
				1.3.1 Scavenger Receptors Class A (SCARAs)
				1.3.2 Role of SCARA Receptors in PepFect14 Transfection and Signaling
				1.3.3 PepFect14 Intracellular Trafficking and Autophagy
		2 Materials
			2.1 Tryptophan Fluorescence Extinction
			2.2 Dynamic Light Scattering (DLS)
			2.3 Cell Culture
			2.4 Peptide Synthesis and Purification
			2.5 Epi-Fluorescence Microscopy
		3 Methods
			3.1 PepFect 14 Synthesis
			3.2 Tryptophan Extinction and Kd Calculation
			3.3 Hydrodynamic Diameter and zeta-Potential Measurement
			3.4 Hsp70 Uptake Mediated by Pepfect14 Detection
		4 Notes
		References
	Chapter 16: Utilization of Cell-Penetrating Peptides for In Vivo Delivery of Bioactive Cargo: The Effect of Nanoparticle Formu...
		1 Introduction
		2 Materials
			2.1 Solutions Needed
			2.2 Charge-Ratio Values of CPP-Cargo Complexes
		3 Methods
			3.1 Standard Formulation of CPP-Nucleic Acid Cargo Nanoparticles for In Vivo Delivery
			3.2 Formulation of Cryo-concentrated Complexes
			3.3 Transfection In Vivo
				3.3.1 In Case of Standard Complex Formation (See Subheading 3.1)
				3.3.2 In Case of Cryo-concentrated Complexes (See Subheading 3.2)
		4 Notes
		References
Part II: Delivery of Proteins and Protein Mimics
	Chapter 17: Protein Delivery by PTDs/CPPs
		1 Introduction
		2 Materials
			2.1 pTAT-HA Expression
			2.2 TAT-Fusion Protein Purification
			2.3 Equipment
		3 Methods
			3.1 Isolation of TAT-Fusion Protein Expression Vector
			3.2 First Step in TAT-Fusion Protein Purification: Ni-NTA Column
			3.3 Second Step in TAT-Fusion Protein Purification: FPLC Cationic-Exchange Column
			3.4 Alternative Second Step in TAT-Fusion Protein Purification: Gravity Flow IEX Column
		4 Notes
		References
	Chapter 18: Protein Delivery to Cytosol by Cell-Penetrating Peptide Bearing Tandem Repeat Penetration-Accelerating Sequence
		1 Introduction
		2 Materials
			2.1 Cell Culture, CPP, and Cargo Protein
			2.2 Endocytosis Assay Using Inhibitors
			2.3 Endocytosis Assay Using siRNA
		3 Methods
			3.1 Delivery of Cargo by Pas2r12 and Observation Using Confocal Microscopy
			3.2 Endocytosis Assay Using Inhibitors
			3.3 Endocytosis Assay Using siRNA
		4 Notes
		References
	Chapter 19: Recombinant Transcription Factors (TFs) Fused to ZEBRA Minimal Transduction Domain (MD) for Modulation of mRNA Tra...
		1 Introduction
		2 Materials
			2.1 Molecular Constructs
			2.2 Clonage and Transfection
			2.3 Culture and Selection of Bacteria
			2.4 Protein Expression, Cell Lysis, and Protein Purification
			2.5 Validations of the CPP-FT (Western Blotting)
			2.6 Fibroblasts
			2.7 HSC
			2.8 Treatment of Cells
			2.9 Reverse Transcriptase and Quantitative  PCR
		3 Methods
			3.1 Molecular Constructs and Transfer to Expression Plasmid
			3.2 Expression and Purification of Recombinant MD11 Proteins
			3.3 Treatment of Fibroblasts with MD11-Pluripotency Activators
			3.4 Treatment of HSC with MD11-Pluripotency Activators
			3.5 FT Targets Validations (qPCR)
		4 Notes
		References
	Chapter 20: Protein Mimicry and the Design of Bioactive Cell-Penetrating Peptides: The Genesis of STOPSPERM Bioportides
		1 Introduction
		2 Materials
			2.1 Isolation of Spermatozoa
				2.1.1 Bovine Sperm
				2.1.2 Highly Motile Human Sperm
			2.2 Confocal Analysis of CPP Import into Sperm Compartments
			2.3 Measurement of Sperm Viability
			2.4 Determination of Sperm Motility
		3 Methods
			3.1 Peptide Design
			3.2 Sperm Isolation
				3.2.1 Bovine Sperm
				3.2.2 Highly Motile Human Sperm
			3.3 Confocal Studies
			3.4 Measurement of Sperm Viability
			3.5 Determination of Sperm Motility
		4 Notes
		References
	Chapter 21: Lipopeptide Pepducins as Therapeutic Agents
		1 Introduction
		2 Pepducin Mechanism of Action: P1pal-19
		3 Pepducin-Based Therapeutics
			3.1 Protease-Activated Receptors
				3.1.1 PAR1
				3.1.2 PAR2
			3.2 Chemokine Receptors
				3.2.1 IL-8 Receptors (CXCR1/2)
				3.2.2 GPR35 (CXCR8)
			3.3 Formyl Peptide Receptors
				3.3.1 Formyl Peptide Receptor 3 (FPR3)
			3.4 Other Receptors
				3.4.1 GPR31
				3.4.2 Dopamine Receptor D1 (DRD1)
				3.4.3 Neurotensin Receptor Type 1 (NTS1)
				3.4.4 β2-Adrenergic Receptor (β2AR)
				3.4.5 PAC1
		References
	Chapter 22: Delivery of Antibiotics by Cell-Penetrating Peptides to Kill Intracellular Pathogens
		1 Introduction
		2 Materials
			2.1 Conjugation of CPP with Antibiotic (Tat-Gentamycin)
			2.2 Cell Line and Culture
			2.3 Culturing of Bacterial Strains
			2.4 Antimicrobial Activity Assays
				2.4.1 Minimal Inhibitory Concentration (MIC Assay)
				2.4.2 Growth Inhibition Assay
				2.4.3 Intracellular Killing Assay
			2.5 Cellular Toxicity Assay
		3 Methods
			3.1 Conjugation of CPP Conjugation Methods
			3.2 Cell Line and Culture
			3.3 Culturing of Bacterial Strains
			3.4 Antimicrobial Activity Assays
				3.4.1 Minimal Inhibitory Concentration (MIC Assay)
				3.4.2 Growth Inhibition Assay
				3.4.3 Intracellular Killing Assay
			3.5 Cellular Toxicity Assay
		4 Notes
		References
	Chapter 23: CPP Applications in Immune Modulation and Disease Therapy
		1 Introduction
		2 Allergic Diseases
		3 Autoimmune Diseases
		4 Cancer
		5 Neurological Diseases
		6 Vaccines
		7 Clinical Trials
		8 Perspectives
		References
Part III: Targeting
	Chapter 24: Cell-Penetrating Peptides as Carriers for Transepithelial Drug Delivery
		1 Introduction
		2 Materials
			2.1 Cell Culture
			2.2 Sample Preparation
			2.3 Permeation and Cell Viability Studies
			2.4 Visualization of TJ Protein and Actin Cytoskeleton
		3 Methods
			3.1 Test Sample Preparation
			3.2 Permeation and Cell Viability Studies
				3.2.1 Cargo Peptide Quantification
				3.2.2 Recovery Assessment After Permeation Study
			3.3 Immunostaining and Visualization of TJ and Actin Cytoskeleton
		4 Conclusions
		5 Notes
		References
	Chapter 25: New Tools for Streamlined In Vivo Homing Peptide Identification
		1 Introduction
		2 Materials
			2.1 In Vivo Phage Display
				2.1.1 General Reagents and Materials
				2.1.2 Basic Manipulation of T7 Phages: Cloning, Titering, and Sequencing
					Cloning T7 Peptide-Phage Libraries or Single Clones
					Titering T7 Phage
					Sequencing Peptide-Coding Region of Single T7 Phage Clones
				2.1.3 Phage Amplification and Purification
				2.1.4 In Vivo Phage Display
			2.2 Application of High-Throughput Sequencing and Bioinformatics Tools for Mapping of Vascular ZIP Codes
			2.3 In Vivo Auditioning of Homing Peptides Using Phage Playoff
			2.4 Identifying Homing Peptide Receptor Using Pull-Down Assay
		3 Methods
			3.1 In Vivo Phage Display
				3.1.1 Basic Manipulation of T7 Phages: Cloning, Titering, and Sequencing (Fig. 1)
					Cloning T7 Peptide-Phage Libraries or Single Clones
					Titering T7 Phage
					Sequencing Peptide-Coding Region of Single T7 Phage Clones
				3.1.2 Phage Amplification and Purification
				3.1.3 In Vivo Phage Display
					The Procedure of In Vivo Peptide-Phage Display
			3.2 Application of High-Throughput Sequencing and Bioinformatics Tools for Mapping of Vascular ZIP Codes
				3.2.1 Guidelines and Protocol for  HTS
				3.2.2 Mining of HTS Data for Differential Binding Analysis
				3.2.3 Mining of HTS Data for Enrichment Analysis
			3.3 In Vivo Auditioning of Homing Peptides Using Phage Playoff
				3.3.1 Guidelines and Protocol for In Vivo Playoff
			3.4 Identifying Homing Peptide Receptors Using Pull-Down Assay
				3.4.1 Preparing Tissue Lysate
				3.4.2 Peptide Conjugation to Magnetic Beads
				3.4.3 Peptide Receptor Pull-Down with Peptide Magnetic Beads
		4 Notes
		References
	Chapter 26: Isolation of Primary Hepatocytes for Testing Tumor Penetrating Peptides
		1 Introduction
			1.1 Liver Cancer
			1.2 Tumor Penetrating Peptides
			1.3 Tumor Penetrating and Interfering Peptides
			1.4 A Structural Perspective of  TPPs
		2 Materials
			2.1 FACS Analysis
			2.2 Microscopy
			2.3 Isolation of Primary Hepatocytes
		3 Methods
			3.1 Peptides
			3.2 Isolation and Culture of Primary Human Hepatocytes
			3.3 Quantification of Cellular Internalization
			3.4 Visualization of the Peptide Internalization by Fluorescence Microscopy
			3.5 Peptide Structure Prediction and PDB Mining
		4 Notes
		References
	Chapter 27: Mitochondrial Targeting Probes, Drug Conjugates, and Gene Therapeutics
		1 Introduction
			1.1 Mitochondria
			1.2 Mitochondria as a Target for Drug Discovery
			1.3 Cell-Penetrating Peptides
			1.4 Applications in Drug Delivery and Clinical Trials
			1.5 Targeting Intracellular Organelles
			1.6 Gene Therapy
		2 Materials
			2.1 Synthesis of mitFects
			2.2 Purification and Identification of mitFects
			2.3 Preparation of mitFects Nanoparticles
			2.4 In Vitro Studies Intracellular Delivery of Nanoparticles with mitFects
			2.5 Confocal Microscopy Studies
		3 Methods
			3.1 Overview of Procedure for the Synthesis of mitFects
			3.2 Synthesis and Cleavage of mitFects Library
			3.3 Purification and Characterization of mitFects
			3.4 Preparation of mitFect/SCO or mitFect/ASO (See Note 4)
			3.5 In Vitro Transfection of mitFect/SCO
			3.6 Physicochemical Characterization
				3.6.1 Size Distribution of mitFects Nanoparticles
				3.6.2 Zeta Potential and Efficiency of mitFects Complexes
			3.7 Internalization of peptide/oligonucleotide nanoparticles in cells by Confocal Microscopy
		4 Notes
		References
	Chapter 28: Novel Brain-Penetrating Single Chain Antibodies Directed Against 3RTau for the Treatment of Alzheimer´s Disease a...
		1 Introduction
		2 Materials (See Subheading 3)
		3 Methods
			3.1 Development of 3RTau Single Chain Antibody Clones
			3.2 Production and Purification of 3RT scFV for Protein Analysis
			3.3 Expression and Purification of 3RTau(352) for Protein Analysis
			3.4 Construction of Lentivirus Vectors Expressing scFV 3RTau with ApoB
			3.5 In Vitro Testing of the Lenti 3RTau scFV-ApoB in Neuronal Cells and Co-Cultures
			3.6 In Vivo Testing of LV-3RTau scFV-ApoB in Tau Transgenic Mice
		4 Notes
		5 Conclusions
		References
	Chapter 29: Strategies for Peptide-Mediated Cargo Delivery to Human Smooth Muscle Cells
		1 Introduction
		2 Materials
			2.1 Human Uterine Biopsies
			2.2 Human Uterine Smooth Muscle Cell Isolation and Primary Culture
			2.3 CPPs and Fluorescent Labeling
			2.4 Cell Microscopy
			2.5 Cell Microscopy Data Analysis
		3 Methods
			3.1 Human Uterine Biopsies
			3.2 Human Uterine Smooth Muscle Cell Isolation and Primary Culture
			3.3 Fluorescently Labeled  CPPs
			3.4 Treatment of Human Smooth Muscle Cells with CPP-Cargo Conjugates
			3.5 Treatment of Human Smooth Muscle-Enriched Tissues with CPP-Cargo Conjugates
			3.6 Imaging of Human Isolated Smooth Muscle Cell Uptake of CPP-Cargo Conjugates
			3.7 Imaging of Human Isolated Smooth Muscle Cell Uptake of CPP-Cargo Conjugates
		4 Notes
		References
Part IV: Oligonucleotide Delivery
	Chapter 30: Tips and Tools to Understand Direct Membrane Translocation of siRNA-Loaded WRAP-Based Nanoparticles
		1 Introduction
		2 Materials
			2.1 Synthesis of Peptides
			2.2 Formulation of the Nanoparticles
			2.3 Leakage Assays
			2.4 Cell Transfection with Endocytosis Inhibitors
			2.5 Cytotoxicity Assay (LDH)
			2.6 Luciferase Assay
			2.7 Co-localization Assays with Endocytosis or Vesicle Markers
		3 Methods
			3.1 Synthesis of Peptides
			3.2 Formulation of the Nanoparticles
			3.3 Leakage Assays
			3.4 Cell Transfection with Endocytosis Inhibitors
			3.5 Cytotoxicity Assay (LDH)
			3.6 Luciferase Assay
			3.7 Co-localization Assays with Endocytosis and Vesicle Markers
		4 Notes
		References
	Chapter 31: Evaluating Efficacy of Peptide-Delivered Oligonucleotides Using the Severe Taiwanese SMA Mouse Model
		1 Introduction
		2 Materials
			2.1 Synthesis and Preparation of Peptide-PMO Conjugates
			2.2 Tattooing Post-natal Day (PND) 0  Pups
			2.3 Intravenous Administration into  Pups
			2.4 Subcutaneous Administration into  Pups
			2.5 Measuring P-PMO Activity Through Survival
			2.6 Collection of Tissues
			2.7 Measuring PMO Tissue Concentrations Using ELISA
				2.7.1 Tissue Preparation
				2.7.2 ELISA
			2.8 Measuring PMO Activity (qPCR)
				2.8.1 Tissue RNA Extraction and Single-Stranded cDNA Synthesis
				2.8.2 Quantitative PCR of Tissue cDNA Using TaqMan Based Assays
			2.9 Measuring PMO Tissue Activity (Western Blot)
		3 Methods
			3.1 Preparation of the P-PMO Conjugates
				3.1.1 Synthesis of the Peptides
				3.1.2 Conjugation of the Peptide to the PMO (2μmol Scale)
			3.2 Tattooing PND0  Pups
			3.3 Intravenous Administration into  Pups
			3.4 Subcutaneous Administration into  Pups
			3.5 Measuring PMO Activity Through Survival
			3.6 Collection of Tissues in  Pups
			3.7 Measuring P-PMO Concentration Using ELISA
				3.7.1 Tissue Lysate Preparation
				3.7.2 ELISA
				3.7.3 ELISA Analysis: Determining P-PMO Concentration
			3.8 Measuring P-PMO Activity (qPCR)
				3.8.1 Tissue RNA Extraction and Single-Stranded cDNA Synthesis
				3.8.2 Quantitative PCR from Tissues Using TaqMan Based Assays
			3.9 Measuring P-PMO Tissue Activity (Western Blot)
				3.9.1 Protein Extraction and Preparation from Tissue
				3.9.2 Western  Blot
		4 Notes
		References
	Chapter 32: Development of CPP-Based Methods for Delivery of miRNAs into the Skin and Airways: Lessons from Cell Culture and M...
		1 Introduction
		2 Materials
			2.1 Cell-Penetrating Peptides (CPPs)
			2.2 MicroRNA Mimics
			2.3 Epithelial Cell Cultures
			2.4 Isolation of CD14+ Monocytes from Peripheral Blood ``Buffy Coat´´ Fraction and Culturing of moDCs
			2.5 Transfection Experiments in Cell Culture
			2.6 Mouse Model of  ICD
			2.7 Mouse Model of  AAI
		3 Methods
			3.1 Peptide Synthesis and Preparation of Stock Solutions
			3.2 Primary Human Epithelial Cell Cultures
			3.3 Preparation of CPP-miRNA Nanocomplexes and Transfection into Cultured Epithelial  Cell
			3.4 Isolation of CD14+ Monocytes and Culture of moDCs
			3.5 Preparation of CPP-miRNA Nanocomplexes and Transfection of moDCs
			3.6 Preparation of CPP-miRNA Nanocomplexes and Application in a Mouse Model  ICD
			3.7 Preparation of CPP-miRNA Nanocomplexes and Application in a Mouse Model of  AAI
		4 Notes
		References
	Chapter 33: Endpoint and Kinetic Approaches for Assessing Transfection Efficacy in Mammalian Cell Culture
		1 Introduction
		2 Materials
			2.1 Cell Culturing and Seeding
			2.2 Complex Formation
			2.3 Transfection of Cells
			2.4 Kinetic Measurement: SEAP Measurement from Media
			2.5 Endpoint Measurement: GFP Measurement by Flow Cytometry
			2.6 Lysis of the Cells
			2.7 Endpoint Measurement: Luciferase from Cell Lysate
			2.8 Protein Measurement
		3 Methods
			3.1 Cell Culturing and Seeding
				3.1.1 Reviving Mammalian Cell Cryo-Stocks
				3.1.2 Cell Culturing
				3.1.3 Seeding
			3.2 Complex Formation
			3.3 Transfection of Cells
			3.4 SEAP Analysis
			3.5 Flow Cytometry Analysis
			3.6 Lysis of Cells
			3.7 Luminescence Measurement
			3.8 Protein Measurement from Cell Lysate
		4 Notes
		References
	Chapter 34: Tissue Analysis of Lung-Targeted Delivery of siRNA and Plasmid DNA
		1 Introduction
		2 Materials
			2.1 List of Materials and Reagents: Animal Injection
			2.2 List of Reagents: Lung Inflation with Cryomatrix
			2.3 List of Reagents: Histology for the Pathological Analysis
		3 Methods
			3.1 In Vivo Transfection: Injection Procedure
			3.2 Lung Inflation with Cryomatrix: Preparation
			3.3 Lung Inflation with Cryomatrix: The Procedure
			3.4 Cryosectioning and Tissue Analysis
		4 Notes
		Reference
	Chapter 35: Improvement of Transfection with PepFects Using Organic and Inorganic Materials
		Abbreviations
		1 Introduction
		2 Materials
		3 Methods
		4 Notes
		Refernces
	Chapter 36: DNA Transfer into Animal Cells Using Stearylated CPP Based Transfection Reagent
		1 Introduction
		2 Equipment and Materials
			2.1 Cell Culture and Transfection
			2.2 SDS Polyacrylamide Gel Electrophoresis and Staining
		3 Methods
			3.1 Cell Culture and Transfection
			3.2 Preparation of Cells for Transfection in 6-Well Plate
			3.3 Reagent 007 Transfection
			3.4 PEI MAX Transfection
			3.5 Production
			3.6 10% SDS Polyacrylamide Gel Electrophoresis
			3.7 Staining
		4 Notes
		References
	Chapter 37: Use of PepFect14 and siRNA for Targeted Gene Silencing in Human Embryonic Stem Cells
		1 Introduction
		2 Materials
			2.1 Cell Culture
			2.2 Transfection Complexes
			2.3 Equipment
		3 Methods
			3.1 Preparation
			3.2 Formation of Transfection Complexes
			3.3 Plating Cells for Transfection
			3.4 Transfection
		4 Notes
		References
	Chapter 38: CRISPR/Cas9 Plasmid Delivery Through the CPP: PepFect14
		1 Introduction
		2 Materials
			2.1 Cell Culture
			2.2 CRISPR/Cas9 Plasmid Delivery and Luciferase Activity Assay
			2.3 CRISPR/Cas9 Plasmid Epi-Fluorescent Microscopy Detection
		3 Methods
			3.1 CRISPR/Cas Plasmid Delivery and Luciferase Activity Assay
			3.2 CRISPR/Cas Plasmid Labeled with Cy5 Delivery. Microscopy Detection
		4 Note
		References
	Chapter 39: Applications of CPPs in Genome Editing of Plants
		1 Introduction
		2 Cell Penetrating Peptides (CPPs)
		3 CPPs and Delivery of Biomolecules into Plant Cells
			3.1 DNA Delivery into Plant Cells Using  CPPs
			3.2 Delivery of Proteins into Plant Cells Using  CPPs
			3.3 Nucleo-protein Complexes as a Novel Cargo for  CPPs
			3.4 Gene Silencing Through CPP-Delivered  RNA
		4 Plant Genome Editing via Transgenic and Non-transgenic Means
		5 Genetic Engineering of Organelle Genomes
			5.1 Chloroplast Transformation
			5.2 Mitochondrial Transformation
		6 Conclusions
		References
	Chapter 40: Live Cell Genomics: Cell-Specific Transcriptome Capture in Live Single Cells from Complex Tissues
		1 Introduction
			1.1 TIVA Captures mRNA Cell-Specific Transcriptomes in Their Natural Environment
			1.2 How Does TIVA Technology Work?
			1.3 Photolysis to Cell-Specific mRNA Capture
			1.4 Specific Target mRNA Restoration from Heterogeneous mRNA Distribution
			1.5 TIVA Technology Summary
		2 Materials
			2.1 Buffers
			2.2 Other Reagents
			2.3 Brain Slices and Cell Culture
		3 Methods
			3.1 TIVA Tag Loading in Brain Slice
			3.2 TIVA Tag Loading Confirmation and Photolysis in Brain Slices
			3.3 TIVA Tag Loading in Cell Cultures
			3.4 TIVA Tag Loading Confirmation and Photolysis in Cell Cultures
			3.5 Harvesting Samples from the Collection Pipette to the Collection Tube
			3.6 Magnetic Bead Preparation
			3.7 TIVA Tag Affinity Purification and mRNA Isolation
			3.8 Eluting the TIVA-Captured mRNAs from Magnetic Beads
		4 Amplification, Library Construction, and RNA-seq
		5 Note
		6 Future of In Vivo Genomics Analysis
		References
Index