Supramolecules in Drug Discovery and Drug Delivery: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: Application of Neutralization and Freeze-Drying Technique for the Preparation of the Beneficial in Drug Delivery 2-Hydroxypropyl-β-Cyclodextrin Complexes with Bioactive Molecules
	1 Introduction
	2 Materials
	3 Methods
		3.1 Steps to Follow for the Preparation of Solid-State Irbesartan–2-HP-β-CD Inclusion Complex [25, 26]
		3.2 Steps to Follow for the Preparation of Solid-State LOS–2-HP-β-CD Inclusion Complex
		3.3 Steps to Follow for the Preparation of Solid-State CAN–2-HP-β-CD and CC–2-HP-β-CD Inclusion Complexes [27]
		3.4 Steps to Follow for the Preparation of Solid-State RA–2-HP-β-CD and CA–2-HP-β-CD Inclusion Complexes
			3.4.1 CA–2-HP-b-CD Inclusion Complex
			3.4.2 RA–2-HP-b-CD Inclusion Complex
		3.5 Steps to Follow for the Preparation of Solid State SLB–2-HP-β-CD Inclusion Complex [28]
		3.6 Steps to Follow for the Preparation of Solid-State CRM–2-HP-β-CD Inclusion Complex
		3.7 Steps to Follow for the Preparation of Solid-State QUE–2-HP-β-CD and QUE–Me-β-CD Inclusion Complexes [29–31]
			3.7.1 QUE–2-HP-β-CD Inclusion Complex [29, 30]
			3.7.2 QUE–Me-β-CD Inclusion Complex [31]
	4 Notes
	5 Conclusions
	References
Chapter 2: Functionalized Carbon Nanohorns as Drug Delivery Platforms
	1 Introduction
	2 Materials
		2.1 Preparation of CNHs
		2.2 Drying Solvents
		2.3 Synthesis of BOC-Aniline Derivative
		2.4 Synthesis of BOC-CNHs and the Corresponding NH2-CNHs
		2.5 Oxidation of CNHs
		2.6 Conjugation of Drugs/Biomolecules
		2.7 Nanomesh CNHs
		2.8 Encapsulation of Drugs/Biomolecules
	3 Methods
		3.1 Preparation of CNHs
		3.2 Procedures for Drying Solvents
		3.3 Covalent Incorporation of Amine Moieties on CNHs
		3.4 Incorporation of Carboxylic Acid Moieties on CNHs
		3.5 Conjugation of Drugs/Biomolecules on Pre-modified CNHs
		3.6 Encapsulation of Drugs/Biomolecules Within CNHs
	4 Notes
	5 Conclusions
	References
Chapter 3: Ultrasonics-Assisted Effective Isolation and Characterization of Exosomes from Whole Organs
	1 Introduction
	2 Materials
		2.1 Reagents Used for Exosome Isolation
		2.2 Reagents Used for the Characterization of Isolated Exosomes
			2.2.1 Exosomal Protein Quantification
			2.2.2 Western Blot Analysis
			2.2.3 Transmission Electron Microscopy Imaging
			2.2.4 RNA Isolation
	3 Methods
		3.1 Isolation of Exosomes from Brain, Heart, and Liver
		3.2 Protein Quantification of Exosomes Using BCA Test
		3.3 Exosome Particle Counting with CD63 ELISA Kit
		3.4 Western Blot Analysis
		3.5 Preparation of TEM Samples
		3.6 RNA Isolation and Quantification
	4 Notes
	References
Chapter 4: Aggregate Determination by Permeation Technique
	1 Introduction
	2 Materials
		2.1 Tested Solutions or Donor Solutions: Saturated Guest/Host Aggregate Solution
		2.2 Self-Aggregate or Cluster Solutions
		2.3 Receptor Solutions
		2.4 Semipermeable Membrane
	3 Methods
		3.1 Permeation Studies
			3.1.1 Franz Diffusion Cells
			3.1.2 Micro-Equilibrium Dialysis Device
			3.1.3 Cuplike MINI Dialysis Device
			3.1.4 Determination of Permeation Profiles
		3.2 Evaluation of Guest/Host Aggregate Profiles
		3.3 Evaluation of Critical Aggregation Concentration (cac)
	4 Notes
	References
Chapter 5: Study of Candesartan Cilexetil: 2-Hydroxypropyl-β-Cyclodextrin Interactions: A Computational Approach Using Steered Molecular Dynamics Simulations
	1 Introduction
	2 Software
	3 Methods
		3.1 System Preparation
			3.1.1 Manually Create the Drug: CD Complex
			3.1.2 Minimize Structure
			3.1.3 Create the Topology Files for Both Drug and Host
			3.1.4 Create the Gromacs Input Files
			3.1.5 Manually Create the Topology File for the Run
			3.1.6 Create the Simulation Box
			3.1.7 Solvate the System
		3.2 Energy Minimization
			3.2.1 Create Input
			3.2.2 Run the Energy Minimization Simulation
		3.3 Equilibration
			3.3.1 NVT Ensemble Equilibration
				Create Input
				Create Input
				Run the Equilibration Simulation
			3.3.2 NPT Ensemble Equilibration
				Create Input
				Run the Equilibration Simulation
		3.4 Steered Molecular Dynamics Simulation
			3.4.1 Create Input for the Pulling Simulation
			3.4.2 Run the Pulling Simulation
			3.4.3 Collect Frames
			3.4.4 Choose the Starting Configurations
		3.5 Umbrella Sampling
			3.5.1 Equilibration
			3.5.2 Umbrella Sampling Simulations
		3.6 Analysis
			3.6.1 Create Input
			3.6.2 Run the WHAM Analysis
	4 Notes
	References
Chapter 6: Drug Delivery: Hydrophobic Drug Encapsulation into Amphiphilic Block Copolymer Micelles
	1 Introduction
	2 Materials
	3 Methods
		3.1 Solution of Pluronic F-127 with 50% Curcumin Encapsulated (Organic Cosolvent Protocol)
		3.2 Solution of  Pluronic F-127 with 50% Curcumin Encapsulated (Thin-Film Protocol)
		3.3 Solution of PEO-b-PCL with 20% IND Encapsulated (Organic Solvent Protocol)
		3.4 Solutions of PEO-b-PCL with 20% IND Encapsulated (Thin-Film Protocol)
		3.5 UV-Vis Spectroscopy
		3.6 Fourier Transform Infrared Spectroscopy (ATR-FTIR)
		3.7 Dynamic Light Scattering (DLS)
	4 Notes
	References
Chapter 7: Multisensitive Polymeric Nanocontainers as Drug Delivery Systems: Biological Evaluation
	1 Introduction
	2 Materials
		2.1 Evaluation of Drug Loading and Release
			2.1.1 Buffer Solutions [19–21]
			2.1.2 Drug-Loading Suspensions
			2.1.3 Drug-Release Suspensions
		2.2 Biological Evaluation
			2.2.1 Culture Media
			2.2.2 MTT Solution Preparation
			2.2.3 Other Reagents and Materials
	3 Methods
		3.1 Drug Loading and Release
			3.1.1 Drug Loading in Nanocontainers
			3.1.2 Drug Release from Nanocontainers
		3.2 Hyperthermia Measurements
		3.3 Cytotoxicity and Biocompatibility Evaluation
			3.3.1 Cell Culture
			3.3.2 MTT Cell Proliferation Assay
			3.3.3 In Vitro Cytotoxicity Studies Under Hyperthermia
		3.4 Scratch-Wound Healing
		3.5 Imaging via Fluorescent Confocal Microscopy
	4 Notes
	References
Chapter 8: Drug Incorporation in the Drug Delivery System of Micelles
	1 Introduction
	2 Materials
	3 Methods
	4 Results and Discussion
	5 Notes
	References
Chapter 9: Molecular Dynamics Protocols for the Study of Cyclodextrin Drug Delivery Systems
	1 Introduction
	2 Materials
	3 Methods
		3.1 Molecular Modeling
			3.1.1 Structure Retrieval
			3.1.2 Molecular Docking Calculations
		3.2 Structure Preparation for Molecular Dynamics
		3.3 Molecular Dynamics Simulation of the Complex
			3.3.1 Energy Minimization
			3.3.2 Heating
			3.3.3 Density Equilibration
			3.3.4 MD Production Simulation
		3.4 Energetic Analysis with the  MM–PBSA Method
		3.5 Conformational Analysis
	4 Notes
	References
Chapter 10: Drug Delivery Through Multifunctional Polypeptidic Hydrogels
	1 Introduction
	2 Materials
	3 Methods
		3.1 Instrumentation
		3.2 Synthesis of Monomers
			3.2.1 Synthesis of ε-tert-Butoxycarbonyl-l-Lysine N-Carboxy Anhydride (Νε BOC-l-LYS NCA)
			3.2.2 Synthesis of Nim-trityl-l-Histidine-N-Carboxy Anhydride (Trt-HIS-NCA) [4]
			3.2.3 Synthesis of γ-Benzyl-l-Glutamate N-Carboxy Anhydride (BLG-NCA)
			3.2.4 Synthesis of l-Leucine N-Carboxy Anhydride (LEU-NCA)
		3.3 Synthetic Approach of Polypeptides PLys-b-(PHIS-co-PBLG (or PLEU))-b-PLys-b-(PHIS-co-PBLG (or PLEU))-b-PLys (See Fig. 2)
		3.4 Formation of Hydrogels
		3.5 Formation of Hydrogels Loaded with Gemcitabine [5, 6]
	4 Notes
	5 Results
	References
Chapter 11: Polymersomes from Hybrids-Polypeptides for Drug Delivery Applications
	1 Introduction
	2 Materials
	3 Methods
		3.1 Instrumentation
		3.2 Synthesis of Monomers
			3.2.1 Synthesis of ε-tert-Butoxycarbonyl-l-Lysine N-Carboxy Anhydride, (Νε BOC-l-LYS NCA)
			3.2.2 Synthesis of γ-Benzyl-l-Glutamate N-Carboxy Anhydride (BLG-NCA)
		3.3 Synthesis of the Triblock Copolypeptide PLL-b-PBLG-d7-b-PLL
		3.4 Synthesis of the Novel Triblock Copolypeptide PEO-b-PBLG-b- PLL
		3.5 Drug Loading (See Fig. 3) [4, 5]
			3.5.1 Doxorubicin Loading
			3.5.2 Pacl Loading
	4 Notes
	5 Results
		5.1 Synthesis and Characterization of the Polymers and Determination of the Concentration of Pacl in Polymersomes [6]
		5.2 Loading and Stability
		5.3 Polymersome Characteristics
		5.4 In Vitro Release and Activity
		5.5 In Vivo Toxicity Study
	6 Conclusions
	References
Chapter 12: Drug Delivery Systems Based on Modified Polysaccharides: Synthesis and Characterization
	1 Introduction
	2 Materials
	3 Methods
		3.1 Synthesis of PMMA@HPC@CS@CH Nanospheres (NCs)
		3.2 Drug Encapsulation
		3.3 Characterization of Vehicles
			3.3.1 SEM Preparation
			3.3.2 TEM Preparation
			3.3.3 AT-IR Preparation
	4 Notes
	References
Chapter 13: Differential Scanning Calorimetry (DSC) on Sartan/Cyclodextrin Delivery Formulations
	1 Introduction
	2 Materials
		2.1 Lyophilization of Raw Materials
		2.2 Complex of Sartan with 2-HP-β-CD
		2.3 Differential Scanning Calorimetry (DSC)
	3 Methods
		3.1 Lyophilization of Raw Materials
		3.2 Complexation of the Sartans with 2-HP-β-CD
		3.3 DSC Sample Preparation
		3.4 DSC Analysis
		3.5 DSC Diagram and Thermodynamic Parameter Extraction
		3.6 DSC Profile Analysis of the Cyclodextrin:Sartan Systems
	4 Notes
	References
Chapter 14: Encapsulation of Small Drugs in a Supramolecule Enhances Solubility, Stability, and Therapeutic Efficacy Against Glioblastoma Multiforme
	1 Introduction
	2 Materials
		2.1 Synthesis and Characterization of Encapsulated TMZ
		2.2 UV-Vis Spectroscopy
		2.3 Liquid Chromatography and Mass Spectrometry
		2.4 In Vitro Assays of TMZ@PSC4 Activity
	3 Methods
		3.1 Synthesis, Characterization, and Quantification of the Encapsulated TMZ
			3.1.1 Synthesis and Purification of Nanocapsule
			3.1.2 Characterization of the Nanocapsule
			3.1.3 Quantification of Encapsulated TMZ Using UV-Vis Spectroscopy
				Preparation of the Calibration Curve Using UV-Vis Spectroscopy
				Quantification of Encapsulated Drug Using UV-Vis Spectroscopy
		3.2 Determination of Chemical Stability of TMZ and ΤΜΖ@PSC4 in Buffer Solutions Using UV-Vis, 1H-NMR, and LC-MS/MS
			3.2.1 Determination of Chemical Stability of ΤΜΖ@PSC4 by UV-Vis
			3.2.2 Determination of Chemical Stability of ΤΜΖ@PSC4 Using 1H-NMR
			3.2.3 Determination of Chemical Stability of ΤΜΖ@PSC4 Using Liquid Chromatography and Mass Spectrometry
				Method Development and Optimization
				Buffer Chemical Stability Assay
		3.3 Cell Culture
		3.4 In Vitro Cytotoxicity: Sulforhodamine B (SRB) and Cell Counting Kit 8 (CCK8) Assays
		3.5 In Vivo Pharmacokinetic Analysis
	4 Notes
	References
Chapter 15: Unveiling the Thermodynamic Aspects of Drug-Cyclodextrin Interactions Through Isothermal Titration Calorimetry
	1 Introduction
	2 Materials
		2.1 Samples
		2.2 Instrumentation
	3 Methods
		3.1 Experimental Design
		3.2 Sample Preparation and Loading
		3.3 Run Parameters
		3.4 Data Analysis
	4 Notes
	References
Chapter 16: Antitumor Efficacy of Ceranib-2 with Nano-Formulation of PEG and Rosin Esters
	1 Introduction
		1.1 Sphingolipid Metabolism and Cancer
		1.2 Ceranib-2 as Potent Ceramidase Inhibitor
		1.3 Therapeutic Limitation of Ceranib-2 as Anticancer Drug
		1.4 Nanostructure Material in Anticancer Drug Delivery System
		1.5 Polymeric Nanoparticle Drug Delivery Systems
		1.6 Rosin Ester Nanoparticles in Drug Formulation
		1.7 Improving the Structure
		1.8 PEGylation
		1.9 Preparation Methods for Nanoparticle Drug Delivery System
		1.10 Solvent Evaporation
		1.11 Ultrasonic Cavitation
	2 Materials and Methods
		2.1 Synthesis of PREC-2 NPs
		2.2 Cell Culture
		2.3 In Vitro Release Assessment
		2.4 Preparation MTT Assay Stock Solution
		2.5 Equipment and Instruments
		2.6 Statistical Analysis
	3 Methods
		3.1 Preparation of Dispersion Phase
		3.2 Preparation of Continuous Phase
		3.3 Synthesis of PREC-2 NPs
		3.4 Preparation of Physiological Reception Solution for In Vitro Release Experiment
		3.5 In Vitro Drug-Release Profile of PREC-2 NPs
		3.6 Preparation of DMEM Culture Media for Cell Line
		3.7 Determination of the Efficacy and Therapeutic Effect of PREC-2 NPs
	4 Notes
	References
Chapter 17: Association of the Thermodynamics with the Functionality of Thermoresponsive Chimeric Nanosystems
	1 Introduction
	2 Materials
		2.1 Preparation of Thermoresponsive Chimeric Bilayers
		2.2 Differential Scanning Calorimetry (DSC)
		2.3 Preparation of Thermoresponsive Chimeric Liposomes
		2.4 Dynamic Light Scattering (DLS) and Heating Protocol
	3 Methods
		3.1 Preparation of Thermoresponsive Chimeric Bilayers
		3.2 DSC Sample Preparation
		3.3 DSC Analysis
		3.4 DSC Profile and Thermodynamic Parameter Extraction
		3.5 DSC Profile Analysis of the Thermoresponsive Chimeric Systems
		3.6 Preparation of Thermoresponsive Chimeric Liposomes Through the Thin-Film Hydration Method
		3.7 Dynamic Light Scattering (DLS)
		3.8 Heating of the Thermoresponsive Chimeric Liposomes
		3.9 Physicochemical Properties of  the Thermoresponsive Chimeric Liposomes
	4 Notes
	References
Chapter 18: 2D DOSY NMR: A Valuable Tool to Confirm the Complexation in Drug Delivery Systems
	1 Introduction
	2 Materials
	3 Methods
		3.1 Preparation of the Complex
			3.1.1 Preparation of the Que-HP-β-CD Complex
			3.1.2 Preparation of the TMZ-Calixarene Complex
		3.2 NMR Spectroscopy
			3.2.1 1H NMR Spectrum
			3.2.2 2D DOSY NMR Spectrum
	4 Notes
	References
Chapter 19: Drug-Encapsulated Cyclodextrin Nanosponges
	1 Introduction
	2 Encapsulated Cyclodextrin Nanosponges
		2.1 Cyclodextrin Nanosponges for Anticancer Drugs
		2.2 Cyclodextrin Nanosponges for Anti-inflammatory Drugs
		2.3 Cyclodextrin Nanosponges for Antiviral Drugs
		2.4 Cyclodextrin Nanosponges for Antifungal Drugs
		2.5 Cyclodextrin Nanosponges for Antioxidants
		2.6 Cyclodextrin Nanosponges for Polyphenols
		2.7 Cyclodextrin Nanosponges for Antibacterial Drug
		2.8 Cyclodextrin Nanosponges for Anti-Zika
		2.9 Molecularly Imprinted Cyclodextrin Nanosponges for Parkinson’s Disease
		2.10 Cyclodextrin Nanosponges for Hypertension Medication
		2.11 Cyclodextrin Nanosponges for Anti-seizure
		2.12 Cyclodextrin Nanosponges for Sleep Disorder
		2.13 Cyclodextrin Nanosponges for Gastric Reflux
		2.14 Cyclodextrin Nanosponges for Gas Delivery
		2.15 Cyclodextrin Nanosponges for Antihyperglycemic Agent
		2.16 Cyclodextrin Nanosponges for Calcium Delivery
		2.17 Cyclodextrin Nanosponges for Peptides and Steroids
		2.18 Cyclodextrin Nanosponges with Photosensitizing/Photooxidation Agents
		2.19 Cyclodextrin Nanosponges for Miscellaneous Uses
	3 Conclusion
	References
Chapter 20: Electrochemistry Investigation of Drugs Encapsulated in Cyclodextrins
	1 Introduction
		1.1 The CD-Drug Complex Is Oxidized at the Surface of Electrode and the Formation of a Complex Between CD and Oxidated Drug Is Observed
		1.2 The CD-Drug Complex Dissociates and Afterwards the Free Drug Molecule Is Oxidized at the Surface of Electrode
	2 Materials
		2.1 Reagents
		2.2 Cyclic Voltammetry
		2.3 Electrolysis
		2.4 UV–Vis Spectro-electrochemistry
		2.5 Gas Chromatography
		2.6 HPLC-DAD Chromatography
		2.7 HPLC-MS/MS Chromatography
	3 Methods
		3.1 Cyclic Voltammetry
		3.2 The Generation of Oxidation Products Electrochemically
			3.2.1 In Situ UV–Vis Spectroelectrochemistry
			3.2.2 The Generation of Oxidation Products by Potential Controlled Coulometry (Exhaustive Electrolysis)
		3.3 Identification of Products
			3.3.1 GC-MS Chromatography
			3.3.2 HPLC-DAD Chromatography
			3.3.3 HPLC-MS/MS Chromatography
	4 Notes
	References
Chapter 21: A Differential Scanning Calorimetry (DSC) Experimental Protocol for Evaluating the Modified Thermotropic Behavior of Liposomes with Incorporated Guest Molecules
	1 Introduction
	2 Materials
		2.1 Preparation of  Liposomes with Incorporated Guest Molecules
	3 Methods
		3.1 Differential Scanning Calorimetry (DSC) Measurements
	4 Notes
	References
Chapter 22: Applications of NMR in Drug:Cyclodextrin Complexes
	1 Introduction
	2 Materials
		2.1 General Practical Aspects
		2.2 Materials for Liquid NMR Experiments
		2.3 Materials for ssNMR Experiments
	3 Methods
		3.1 Investigation of the Structural Properties of Irbesartan and Irbesartan–2-Hydroxypropyl-β-Cyclodextrin Complex in Micelles
		3.2 The Application of ssNMR Spectroscopy to Study Drug:Membrane Interactions
	4 Notes
		4.1 Notes Related to Subheading 3.1
		4.2 Notes Related to Subheading 3.2 (Figs. 4 and 5)
	References
Chapter 23: Construction of Peptide-Drug Conjugates for Selective Targeting of Malignant Tumor Cells
	1 Introduction
	2 Materials and Equipment
		2.1 Synthesis of PDCs
			2.1.1 Synthesis of PDCs Using Succinic Acid as Linker (Ester and Amide Bonds)
			2.1.2 Synthesis of PDCs Using Carbamate Bond in the Linker
			2.1.3 Synthesis of PDCs Using Aminooxy-PEG4-CH2CO2H as Linker (Amide and Bond)
		2.2 Purification and Characterization of Intermediates and Final Conjugates
			2.2.1 Column Purification
			2.2.2 RP-HPLC Purification
			2.2.3 Characterization with Mass Spectrometry and/or NMR Spectroscopy
	3 Methods
		3.1 Synthesis of PDCs
			3.1.1 Synthesis of PDCs Using Succinic Acid as Linker (Ester and Amide Bonds) (Scheme 1)
				Preparation of Gemcitabine-Linker (diBoc-Gemcitabine-Hemisuccinate)
				Preparation of the Final Conjugate (Gemcitabine-Hemisuccinate-D-Lys6- GnRH)
			3.1.2 Synthesis of PDCs Using Carbamate Bond in the Linker (Scheme 2)
				Preparation of Gemcitabine-Linker (diBoc-Gemcitabine-Bis (4-Nitrophenyl)Carbonate)
				Preparation of the Final Conjugate (Gemcitabine-carbamate-D-Lys6-GnRH)
			3.1.3 Synthesis of PDC Using Aminooxy-PEG4-CH2CO2H as Linker (Amide and Oxime Bond) (Scheme 3)
				Preparation of Gemcitabine-Linker (diBoc-Gemcitabine-Boc-Aminooxy-PEG4-CH2CO2H)
				Attachment of Aldehyde Group on D-Lys6-GnRH, as previously reported [14], and described below (D-Lys6-GnRH aldehyde)
				Preparation of the Final Conjugate (Gemcitabine-PEG4CH2CO2H-D-Lys6-GnRH)
	4 Notes
	References
Index