Photodynamic Therapy: Methods and Protocols (Methods in Molecular Biology, 2451)

✍ Scribed by Mans Broekgaarden (editor), Hong Zhang (editor), Mladen Korbelik (editor), Michael R. Hamblin (editor), Michal Heger (editor)

• Publisher: Humana
• Year: 2022
• Tongue: English
• Leaves: 741
• Category: Library

✦ Synopsis

This collection explores state-of-the-art methods and protocols for research on photodynamic therapy (PDT) and its use in a wide range of medical applications, from antiviral to anticancer. Beginning with an extensive section on in vitro and in vivo models, the volume continues with chapters on oxygen-independent photosensitizers, next-generation photosensitization strategies, contemporary insights into the immunomodulatory effects of PDT, antimicrobial effects of PDT, as well as a variety of general biochemical and molecular biological techniques. Written for the highly successful Methods in Molecular Biology series, chapters include the kind of detailed implementation advice that ensures successful results in the lab.
Thorough and authoritative, Photodynamic Therapy: Methods and Protocols serves as an ideal source of inspiration for both new and established PDT scientists and a guide for designing innovative research programs in this continuously advancing and multidisciplinary field.

✦ Table of Contents

Preface
Contents
Contributors
Part I: In Vitro and In Vivo Models to Study Photodynamic Therapy
Chapter 1: Application of Monolayer Cell Cultures for Investigating Basic Mechanisms of Photodynamic Therapy
1 Introduction
2 Methods
2.1 Photosensitization and Photosensitizer Localization
2.2 In Vitro Photodynamic Therapy
2.3 Combined Assessment of Viability and Metabolic Activity
2.4 Cell Cycle Profiling
2.5 Determination of Cell Death Modes
2.6 Assessment of Mitochondrial Membrane Potential
3 Data Analysis and Interpretation
4 Discussion
References
Chapter 2: The Negative Impact of Cancer Cell Nitric Oxide on Photodynamic Therapy
1 Introduction
2 Materials
2.1 Reagents and Antibodies
2.2 Cell Culture Analyses
2.3 Bystander Model Supplies
3 Methods
3.1 Cell Sensitization and Irradiation
3.2 Western Blot Analyses
3.3 Detection of Cellular NO
3.4 Evaluation of Cell Photokilling and NO-Mediated Resistance
3.5 iNOS/NO Effects on Surviving Cell Proliferation
3.6 Effects of iNOS/NO on Migration of Surviving Cells
3.7 Effects of iNOS/NO on Surviving Cell Invasiveness
3.8 Evaluation of PDT Bystander Effects and NO Involvement
4 Notes
References
Chapter 3: Microtumor Models as a Preclinical Investigational Platform for Photodynamic Therapy
1 Introduction
2 Materials
2.1 Cell Lines and Culture Medium
2.2 Generating Suspended Spheroids
2.3 Generating Adherent Microtumor Cultures
2.4 Materials for Performing PDT
2.5 Sample Preparation for Immunoblotting
2.6 Flow Cytometry Sample Preparation
3 Methods
3.1 Generating Suspended Spheroids
3.2 Treatment of Suspended Spheroid Cultures with PDT
3.3 Harvesting Suspended Spheroids for Immunoblotting
3.4 Harvesting Suspended Spheroids for Flow Cytometry
3.5 Generating Adherent Microtumor Cultures
3.6 Treatment of Adherent Microtumor Cultures with PDT
3.7 Harvesting Adherent Microtumors for Immunoblotting
3.8 Harvesting Adherent Microtumors for Flow Cytometry
4 Notes
References
Chapter 4: A Perfusion Model to Evaluate Response to Photodynamic Therapy in 3D Tumors
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Chip Fabrication
2.3 Cell Infusion
3 Methods
3.1 Preparation of Materials
3.2 Chip Assembly
3.3 Cell Preparation and Infusion
3.4 In Situ Imaging for Viability Assay
3.5 Protein Extraction for WB
3.6 Nucleic Acid Extraction for Transcriptomics/RT-qPCR
4 Notes
References
Chapter 5: Analysis of Treatment Effects on Structurally Complex Microtumor Cultures Using a Comprehensive Image Analysis Proc...
1 Introduction
2 Materials
3 Methods
3.1 Preparing the Total Killing Controls on Adherent Microtumor Cultures
3.2 Live/Dead Staining on Adherent Microtumor Cultures
3.3 Preparing the Total Killing Controls on Suspended Organoid Cultures
3.4 Live/Dead Staining on Suspended Organoid Cultures
3.5 Quantitative Live/Dead Imaging
3.6 CALYPSO Image Analysis: Data Organization
3.7 CALYPSO Image Analysis: Background Subtraction
3.8 CALYPSO Image Analysis: Thresholding the Fluorescence Intensities
3.9 CALYPSO Image Analysis: Data Extraction
3.10 Data Analysis and Interpretation
4 Notes
References
Chapter 6: High-Throughput Examination of Therapy-Induced Alterations in Redox Metabolism in Spheroid and Microtumor Models
1 Introduction
2 Materials
2.1 Determination of Spectral Overlap
2.2 Suspended Spheroid Cultures
2.3 Liquid-Overlay Adherent Microtumor Cultures
2.4 For the Redox State Controls and Treatment Groups
2.5 Imaging of NAD(P)H and FAD Autofluorescence Intensities and Image Analysis
3 Methods
3.1 Determination of Spectral Overlap
3.2 Control Experiment to Ensure Correct Image Acquisition and Processing
3.3 Treatment of the Organoid Cultures
3.4 Data Analysis and Representation
4 Notes
References
Chapter 7: Spatiotemporal Tracking of Different Cell Populations in Cancer Organoid Models for Investigations on Photodynamic ...
1 Introduction
2 Materials
3 Methods
3.1 Plate the Unlabeled Control (MIA PaCa-2 and MRC5) (see Note 2)
3.2 Label First Cell line (MIA PaCa-2 Cells) Using QTracker 655
3.3 Label Second Cell line (MRC5 Cells) with QTracker 525
3.4 Imaging the Spheroids Using Two-Photon Microscopy
3.5 Extract Quantitative Data from the Images
4 Notes
References
Chapter 8: Generating Large Numbers of Pancreatic Microtumors on Alginate-Gelatin Hydrogels for Quantitative Imaging of Tumor ...
1 Introduction
2 Materials
2.1 Plate Coating
2.2 Hydrogel Preparation
2.3 Microtumor Culture
2.4 Liposome Preparation
2.5 Quantification of Liposome Uptake
2.6 Quantification of Liposome Toxicity
3 Methods
3.1 Preparation of Stock Solutions
3.2 Plate Coating
3.3 Hydrogel Fabrication
3.4 Microtumor Culture
3.5 Liposome Uptake Assay
3.6 Toxicity Analysis
3.7 Photodynamic Therapy
3.8 Data Analysis and Visualization
4 Notes
References
Chapter 9: The Chicken Embryo Chorioallantoic Membrane as an In Vivo Model for Photodynamic Therapy
1 Introduction
2 Methods
2.1 CAM Incubation and Development
2.2 Tumors Grown on CAM
2.3 The CAM as a Model to Study and Optimize PDT
2.3.1 Photosensitizer Administration Protocols to the CAM
2.4 Light Dosimetry in the CAM Model
3 Data Analysis and Interpretation
3.1 CAM´s Vascular response to PDT
3.2 Quantification Methods of the PDT Effects on the CAM Vascular Network
3.2.1 Image Processing-Based Techniques
3.2.2 Biochemical and Histopathological Based Techniques
3.3 Effects Induced by Sub-thermal Irradiances of Visible or NIR Light on the CAM angiogenesis and Metabolic Activity
4 Discussion and Conclusion
References
Chapter 10: Subcutaneous Xenograft Models for Studying PDT In Vivo
1 Introduction
2 Methods
2.1 Implantation of Human Cell Line-Based Xenografts
2.2 Implantation of Syngeneic Cell Line-Based Tumors
2.3 Implanting Patient-Derived Xenografts (PDX)
2.4 PDT Treatment Regimens
2.4.1 Photosensitizer Administration Routes
2.4.2 Selection of PS-Light Interval
2.4.3 Light Application
3 Data Analysis and Interpretation
3.1 Longitudinal Monitoring of Treatment Efficacy
3.2 Intermediary Response Monitoring Using In Vivo Functional Imaging
4 Discussion
References
Chapter 11: In Vivo Models for Studying Interstitial Photodynamic Therapy of Locally Advanced Cancer
1 Introduction
2 Materials
2.1 Syngeneic SCCVII Squamous Cell Carcinoma Model
2.2 Rabbit VX2 Carcinoma
3 Methods
3.1 I-PDT in Murine Syngeneic SCCVII Squamous Cell Carcinoma
3.2 Post-procedure Follow-up and Treatment Assessment in the SCCVII/C3H Mice
3.3 Acquisition and Maintenance of the Rabbit VX2 Carcinoma Model
3.4 Implantation of the VX2 Model in Rabbits
3.5 Pretreatment Planning
3.6 Preparing the Rabbit and VX2 Tumor for I-PDT Treatment
3.7 Posttreatment Follow-up and Treatment Assessment in the Rabbit VX2 Model
4 Notes
References
Chapter 12: Orthotopic Models of Pancreatic Cancer to Study PDT
1 Introduction
2 Methods
2.1 Cell Line-Based Orthotopic PDAC Models
2.2 Genetically Engineered Mouse (GEM) Models
2.3 Prospective Orthotopic Models
3 Data Analysis and Interpretation
4 Discussion
References
Chapter 13: An Orthotopic Murine Model of Peritoneal Carcinomatosis of Ovarian Origin for Intraoperative PDT
1 Introduction
2 Materials
2.1 Cell line and Culture Conditions
2.2 Animal Preparation
2.3 Animal Imaging
3 Methods
3.1 Preparation of Cells for Injection
3.2 Anesthesia and Analgesia
3.3 Intratubal Injection Procedure
3.4 Tumor Growth and Metastasis Monitoring by Noninvasive Bioluminescence Imaging
4 Notes
References
Chapter 14: Photodynamic Treatments for Disseminated Cancer Metastases Using Fiber-Optic Technologies
1 Introduction
2 Methods
2.1 Mouse Model and Catheter Placement
2.2 Microendoscope Development
2.3 Photosensitizer Administration
2.4 Fiber-Optic Light Delivery for PDT
2.5 In Vivo Fluorescence Microendoscopy
2.6 Longitudinal Monitoring of Metastatic Burden
2.7 Ex Vivo Histopathology Co-registered with In Vivo Images
2.8 Ex Vivo Whole Peritoneal Cavity Measurement of Metastatic Burden
3 Discussion
3.1 Data Analysis and Interpretation
3.2 Concluding Remarks
4 Notes
References
Chapter 15: Stereotaxic Implantation of F98 Cells in Fischer Rats: A Syngeneic Model to Investigate Photodynamic Therapy Respo...
1 Introduction
2 Materials
3 Methods
4 Notes
References
Part II: Inorganic Photosenstizers and Light-Activatable Anti-Cancer Prodrugs
Chapter 16: New Generation of Photosensitizers Based on Inorganic Nanomaterials
1 Introduction
2 Inorganic Nanophotosensitizers
2.1 Titanium Dioxide
2.2 Zinc Dioxide
2.3 Fullerene
2.4 Carbon Nanotubes, Dots, and Graphene
2.5 Black Phosphorus Nanosheets
3 Inorganic Nanomaterials Used as Photosensitizer Carriers
3.1 Quantum Dots
3.2 Upconversion Nanomaterials
3.3 Silicon Nanomaterials
3.4 Metallic Nanomaterials
3.5 Magnetic Nanomaterials
3.6 Self-Illuminating Nanoparticles
4 Conclusions and Perspective
References
Chapter 17: Cytotoxicity of Metal-Based Photoactivated Chemotherapy (PACT) Compounds
1 Introduction
2 Materials
3 Methods
3.1 Cell Preparation and Seeding
3.2 Treatment
3.3 Illumination Under Normoxia
3.4 Illumination Under Hypoxia
3.5 The Sulforhodamine B (SRB) assay
3.6 Tris Base and Absorbance Reading
4 Notes
References
Part III: Third- and Fourth-Generation Photosensitizers and Targeting Strategies
Chapter 18: Photosensitized Oxidation of Intracellular Targets: Understanding the Mechanisms to Improve the Efficiency of Phot...
1 Introduction
2 Choosing Diseased Cells Over Healthy Ones
3 The Outcome of Attacking Specific Intracellular Targets
4 The Turn On/Off Button for Oxidative Species Production
5 Survival of the Fittest
6 Conclusions and Challenges
References
Chapter 19: Inhibition of the HIF-1 Survival Pathway as a Strategy to Augment Photodynamic Therapy Efficacy
Abbreviations
1 Introduction
2 HIF-1 in Cancer Biology
2.1 Activation of HIF-1
2.1.1 HIF-1 Activation by Hypoxia
2.1.2 HIF-1 Activation by ROS
2.1.3 HIF-1 Activation by NF-κB
2.1.4 HIF-1 Activation Through Loss/Gain-of-Function Mutations
2.2 HIF-1-Mediated Angiogenesis
2.3 Regulation of Cancer Cell Metabolism by HIF-1
2.3.1 Glucose Metabolism
2.3.2 HIF-1-Mediated Glucose Regulation
2.3.3 HIF-1 Modulation of Mitochondrial Activity
2.4 Cell Cycle and Proliferation Control by HIF-1
2.4.1 Proliferation and Its Regulation Through the Cell Cycle
2.4.2 Cell Cycle Modulation by HIF-1
2.5 Regulation of Cell Death and Survival by HIF-1
2.5.1 Modes of Cell Death
2.5.2 Cell Death and Survival Modulation by HIF-1α
2.6 Regulation of Cancer Metastasis by HIF-1
2.6.1 Metastasis
2.6.2 Metastasis Modulation by HIF-1
3 PDT and HIF-1 Signaling
4 Inhibition of the HIF-1 Pathway and Its Implications in PDT
4.1 17-AAG (Tanespimycin)
4.2 Acriflavine
4.3 Amphotericin B
4.4 Ascorbic Acid (Vitamin C)
4.5 Baicalein
4.6 Berberine
4.7 Bortezomib
4.8 Daunorubicin
4.9 Diphenylene Iodonium (DPI)
4.10 Doxorubicin
4.11 Epirubicin
4.12 Hypericin
4.13 LY294002
4.14 Microtubule-Targeting Drugs
4.14.1 2-Methoxyestradiol (2ME2)
4.14.2 Docetaxel
4.14.3 Vincristine
4.15 Minocycline
4.16 Rapamycin
4.17 Resveratrol
4.18 Silibinin
4.19 SN38
4.20 Sodium Butyrate
4.21 Sorafenib
4.22 Topotecan
4.23 Trichostatin A
4.24 Valproic Acid
4.25 Verteporfin
4.26 Vorinostat (SAHA)
4.27 Wortmannin
References
Chapter 20: Strategies for Improving Photodynamic Therapy Through Pharmacological Modulation of the Immediate Early Stress Res...
Abbreviations
1 Introduction
2 ASK-1 Pathway
2.1 JNK and p38
2.2 AP-1 Transcription Factor
2.2.1 JUN
2.2.2 FOS
2.2.3 ATF/CREB
3 ASK-1 Pathway in PDT
3.1 ASK-1 Signaling: Apoptosis
3.2 ASK-1 Signaling: Inflammation
4 Inhibition Strategies for the ASK-1 Pathway
4.1 ASK-1 Inhibitors
4.1.1 Gilead Science´s ASK-1 Inhibitors (GS-4997/Selonsertib, GS-444217, GS-459679)
4.1.2 ASK-1 Inhibitor 10
4.1.3 MSC2032964A
4.1.4 NQDI-1
4.2 p38 Inhibitors
4.2.1 CMPD1
4.2.2 PD 169316
4.2.3 Ralimetinib (LY2228820)
4.2.4 RWJ 67657
4.2.5 SB202190 (FHPI)
4.2.6 SB203580
4.2.7 SB239063
4.2.8 Thymoquinone
4.2.9 VX-702
4.3 Other Inhibitors of the ASK-1 Pathway
5 Conclusions
References
Chapter 21: Nanobody-Targeted Photodynamic Therapy: Nanobody Production and Purification
1 Introduction
2 Materials
2.1 General
2.2 Production with Fermentor (or Bioreactor)
2.3 Purification with Beads (Medium Scale)
2.3.1 Purification of Nanobodies Containing Histidine-Tag
2.3.2 Purification of Nanobodies Containing EPEA-Tag (C-Tag)
2.4 Purification with ÄKTAxpress (Large Scale)
2.4.1 Purification of Nanobodies Containing Histidine-Tag
2.4.2 Purification of Nanobodies Containing EPEA-Tag (C-Tag)
2.4.3 Purification of Nanobodies with Affinity for Protein A (see Note 1)
2.4.4 Purification of Nanobodies with Cation-Exchange Column
2.4.5 Purification of Nanobodies with Anion-Exchange Column
3 Methods
3.1 Medium-Scale Production of Nanobody (800 mL of Culture Media)
3.2 Large-Scale Production of Nanobody (5 L of Culture Media)
3.2.1 Preparation of Bacteria
3.2.2 Vessel Sterilization and O/N Bacterial Pre-culture
3.2.3 Production in Fermentor
3.2.4 Harvesting Bacterial Culture
3.3 Purification of Nanobodies with Affinity Chromatography Approach Using Gravity-Flow Column
3.4 Purification of Nanobodies Through Affinity chromatography with ÄKTAxpress System
3.4.1 Sample Preparation and Column Attachment
3.4.2 Nanobody Purification and Buffer Exchange
3.5 Purification of Nanobodies Using Ion-Exchange Chromatography
3.5.1 Sample Preparation and Column Attachment
3.5.2 Nanobody Purification Using the HiTrap SP HP Cation-Exchange Chromatography Column (STEP 1)
3.5.3 Nanobody Purification Using the HiPrep Q XL 16/10 Column Anion-Exchange Chromatography Column (STEP 2)
4 Notes
References
Chapter 22: Conjugation of IRDye Photosensitizers or Fluorophores to Nanobodies
1 Introduction
2 Materials
2.1 General
2.2 Site-Directed Conjugation
3 Methods
3.1 Random Conjugation
3.2 Site-Directed Conjugation
3.2.1 Protocol 1
3.2.2 Protocol 2
4 Notes
References
Chapter 23: In Vitro Assessment of Binding Affinity, Selectivity, Uptake, Intracellular Degradation, and Toxicity of Nanobody-...
1 Introduction
2 Materials
2.1 General Materials
2.2 Binding Assay on Cells for Affinity Determination
2.3 In Vitro Nanobody-Targeted PDT and Toxicity Assessment
2.4 Live/Dead Cell Assay with Mono- and Co-Cultures
2.5 Internalization Assay
2.6 Intracellular Degradation Assay
3 Methods
3.1 Binding Assay on Cells for Affinity Determination
3.2 In Vitro Nanobody-Targeted PDT and Toxicity Assessment
3.3 Live/Dead Cell Assays with Mono- and Co-Cultures
3.4 Internalization Assay
3.4.1 Determination of the Binding Equilibrium of the Nanobody Over Time
3.4.2 Optimization of the Acid Wash
3.4.3 Determining Internalized Nanobody-Photosensitizer Fraction Using Fluorescence
3.4.4 Determining Internalized Nanobody-Photosensitizer Fraction Using ELISA
3.5 Assessment of Intracellular Degradation of Nanobody-Photosensitizer Conjugates
4 Notes
References
Chapter 24: Investigation of the Therapeutic Potential of Nanobody-Targeted Photodynamic Therapy in an Orthotopic Head and Nec...
1 Introduction
2 Materials
2.1 Cell Lines
2.2 Mice
2.3 Equipment and Solutions for the Inoculation of Tumor Cells and for Monitoring Tumor Growth
2.4 Equipment and Solutions for Photodynamic Therapy
2.5 Nanobody-Photosensitizer Conjugates
2.6 Medication
2.7 Laser
3 Methods
3.1 Culturing Cell Line
3.1.1 Thawing Cryopreserved OSC-19-luc2-cGFP Cells
3.1.2 Culture OSC-19-luc2-cGFP Cells
3.1.3 Dilution of 40,000 OSC-19-luc2-cGFP cells in 20 μL PBS
3.2 Inoculation of Tumor Cells in the Tongue
3.3 Monitoring Mice and Tumor Growth
3.4 Photodynamic Therapy
3.4.1 Intravenous Injection of Nanobody-PS Conjugates
3.4.2 Illumination
3.5 Histological Assessment Post-PDT
4 Notes
References
Chapter 25: Assessment of the In Vivo Response to Nanobody-Targeted PDT Through Intravital Microscopy
1 Introduction
2 Materials
2.1 Mice
2.2 Tumor Cell Line
2.3 Analgesia and Anesthesia Procedure
2.4 Equipment and Solutions for the Skinfold Chamber Operation
2.5 Equipment and Solutions for Intravital Microscopy
3 Methods
3.1 Tumor Collection
3.2 Operation Procedure
3.3 IVM
3.3.1 Imaging Photosensitizer Distribution
3.3.2 Imaging PDT-Induced Vascular Damage
3.4 Image Analysis
3.4.1 Determining the Fluorescence Kinetics Over Time in Regions of Interest
3.4.2 Colocalization Analysis
3.4.3 Changes Vascular Architecture in the Chamber
3.4.4 Vascular Flow and Leakage in Tumor
4 Notes
References
Chapter 26: Orthotopic Breast Cancer Model to Investigate the Therapeutic Efficacy of Nanobody-Targeted Photodynamic Therapy
1 Introduction
2 Materials
2.1 Mice
2.2 Cell Lines
2.3 Equipment and Solutions for the Inoculation of Cells Under Direct Vision and for Monitoring Tumor Growth
2.4 Equipment for Photodynamic Therapy
2.5 Nanobody-Photosensitizer Conjugates
2.6 Medication
2.7 Laser
3 Methods
3.1 Culturing Cell Line
3.1.1 Thawing Cryopreserved HCC1954 and MCF7 cells
3.1.2 Culture HCC1954 or MCF7 cells
3.1.3 Preparing HCC1954 or MCF7 Cells for Inoculations in 30 μL PBS
3.2 Inoculation of Tumor Cells in the Mammary Fat Pad
3.3 Monitoring Mice and Tumor Growth
3.4 Photodynamic Therapy
3.4.1 Intravenous Injection of Nanobody-PS Conjugates
3.4.2 Illumination
3.5 Follow-Up and Tumor Measurements
3.6 Histological Assessment Post-PDT
4 Notes
References
Part IV: Photodynamic Therapy-Induced Immune Signaling
Chapter 27: Evaluation of the Antitumor Immune Response Following Photofrin-Based PDT in Combination with the Epigenetic Agent...
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Tumor Implantation and Treatment
2.3 Ex Vivo Studies
2.4 Staining and Flow Cytometry
3 Methods
3.1 Antitumor Photodynamic Therapy in Combination with 5-Aza-dC in EMT6 Tumor Model
3.1.1 Inoculation of Tumor Cells
3.1.2 Tumor Treatment and Monitoring
3.2 Analysis of Activation of Antitumor Immune Response
3.2.1 Isolation of Murine Cells
3.2.2 Ex Vivo Cell Stimulation
3.2.3 Lymphocyte Depletion
3.2.4 Adoptive Transfer
4 Notes
References
Chapter 28: Controlling Immunoregulatory Cell Activity for Effective Photodynamic Therapy of Cancer
1 Introduction
2 Materials
2.1 Treatment of SCCVII Tumors by Temoporfin-PDT Plus Adjuvant LCL521
2.2 Monitoring the Levels of MDSCs and Tregs
3 Methods
3.1 Tumor Implantation, PDT ± LCL521 Treatment and Tumor Response Assessment
3.2 Determination of MDSC and Treg Levels in Mice Following Tumor PDT Treatment with or Without Adjuvant LCL521
4 Notes
References
Chapter 29: Measuring the Antitumor T-Cell Response in the Context of Photodynamic Therapy
1 Introduction
2 Materials
2.1 T-Cell Depletion
2.2 Taking and Preparing Blood Samples for Ex Vivo Analysis
2.3 Taking and Preparing Organs for Ex Vivo Analysis
2.4 Phenotypic T-Cell Analysis
2.5 Functional T-Cell Analysis
3 Methods
3.1 T-Cell Depletion
3.2 Taking and Preparing Blood Samples for Ex Vivo Analysis
3.3 Taking and Preparing Organs for Ex Vivo Analysis
3.4 Phenotypic T-Cell Analysis
3.5 Functional T-Cell Analysis
3.6 Interpretation of Flow Cytometry Data
3.6.1 Phenotypic Analysis
3.6.2 Functional Analysis
4 Notes
References
Chapter 30: Combination of Photodynamic Therapy and Immune Checkpoint Blockade
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Tumor Inoculation and Measurement
2.3 Antibody Injection
2.4 Photodynamic Therapy
3 Methods
3.1 Cell Culture
3.2 Tumor Inoculation and Measurement
3.3 Antibody Injection
3.4 Photodynamic Therapy
4 Notes
References
Chapter 31: Combination of Photodynamic Therapy and Therapeutic Vaccination
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Tumor Inoculation and Measurement
2.3 Peptide Vaccination
2.4 Photodynamic Therapy
3 Methods
3.1 Cell Culture
3.2 Tumor Inoculation and Measurement
3.3 Peptide Vaccination
3.4 PDT
4 Notes
References
Part V: Antimicrobial Photodynamic Therapy
Chapter 32: In Vitro Potentiation of Antimicrobial Photodynamic Inactivation by Addition of Potassium Iodide
1 Introduction
2 Materials
2.1 Microorganisms
2.2 Equipment
2.3 Buffers, Reagents, Solutions
2.4 Light Source
2.5 Power Meter
2.6 Disposable Plasticware
3 Methods
3.1 Preparation of Suspension of Microbial Cells
3.2 Incubation with PS and KI Solution
3.3 Light Delivery
3.4 In vitro aPDI Experiments
3.5 Serial Dilutions
3.6 Data Interpretation of In Vitro aPDI
3.7 Concluding Remarks
4 Notes
References
Chapter 33: In Vivo Potentiation of Antimicrobial Photodynamic Therapy in a Mouse Model of Fungal Infection by Addition of Pot...
1 Introduction
2 Materials
2.1 Equipment
2.2 Buffers, Reagents, Solutions
2.3 Photosensitizers and Light Source
2.4 Power Meter
2.5 Microorganisms
2.6 Animal Model
3 Methods
3.1 Preparation of Suspension of Microbial Cells
3.2 In Vitro aPDI Studies on C. albicans
3.3 Experimental Candidiasis
3.4 In Vivo PDT of Oral Candida Infection in a Mouse Model
3.5 Bioluminescence Imaging
3.6 Pathological Examination
3.7 Interpretation of Results
3.8 Concluding Remarks
References
Chapter 34: Bioluminescent Models to Evaluate the Efficiency of Light-Based Antibacterial Approaches
1 Introduction
2 Bioluminescence
2.1 Bioluminescence systems in bacteria
2.2 Transformation of Bacteria with Bioluminescence Genes
2.3 Bioluminescence Applications
3 Bioluminescence to Monitor aPDT
3.1 Bioluminescent Models in aPDT Clinic Applications
3.1.1 Studies Using Poly-l-Lysine Chlorin e6 Conjugates, and Other Porphyrin Analogs as PS
3.1.2 Studies Using Non-Porphyrinic PS
3.2 Bioluminescent Models in aPDT Under Environmental Context
3.2.1 Studies Using Non-Immobilized PS
3.2.2 Studies Using Immobilized PS
3.3 Bioluminescent Models in the Photodynamic Efficiency Screening of Novel PS
3.4 Studies Performed Using Inorganic Salts as Potentiation Agents of Photosensitizers
4 Bioluminescence to Monitor Antimicrobial Blue-Light Therapy (aBL)
5 Conclusions and Perspectives
References
Chapter 35: Photochemical Internalization as a New Strategy to Enhance Efficacy of Antimicrobial Agents Against Intracellular ...
1 Introduction
2 Materials
2.1 Bacterial Strains and Inoculum Preparation
2.2 Quantitative Culture of Bacteria
2.3 Cell Maintenance and Culture
2.4 Antibiotics, Photosensitizers, and Light Source
2.5 Cytotoxicity Test
2.6 Phagocytosis Assay
2.7 Experimentation with Zebrafish Embryos
2.8 Imaging
3 Methods
3.1 Preparation of Bacterial Inoculum
3.2 Cell Culture and Maintenance
3.3 Cytotoxicity of Agents to RAW 264.7 Cells
3.4 Phagocytosis Assay (Cell Infection Model)
3.5 Intracellular Antimicrobial Activity Assay
3.6 Visualization of Intracellular Distribution of Agents In Vitro
3.7 Zebrafish Embryo Infection Model
3.8 In Vivo Visualization of Cell-Pathogen Interactions in Zebrafish Embryos
3.9 In Vivo Antimicrobial Activity Test
4 Notes
References
Chapter 36: Determination of the Efficiency of Photodynamic Decontamination of Food
Abbreviations
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Sample Preparation
2.2.1 Vegetables
2.2.2 Salads
2.2.3 Beans and Sprouts
2.3 PDc
3 Methods
3.1 Photodynamic Decontamination on Flat Surfaces
3.2 PDc on Round Objects
4 Notes
References
Part VI: Molecular Techniques and Tools in Photodynamic Therapy Research
Chapter 37: Super-Resolution Imaging of Intracellular Lipid Nanocarriers to Study Drug Delivery in Photodynamic Therapy
1 Introduction
2 Materials
2.1 UltraClean Coverslips
2.2 Fluorescently Labeled Liposomes
2.3 Paraformaldehyde Fixation
2.4 Immunolabeling
2.5 OxeA Imaging Buffer
2.6 Sample Mounting
3 Methods
3.1 Preparation of Ultraclean Coverslips
3.2 Plating Cells and Incubation with Liposomes
3.3 Paraformaldehyde Fixation
3.4 Immunostaining (Optional)
3.5 Preparation of OxeA Imaging Buffers
3.6 Imaging Setup
4 Notes
References
Chapter 38: Detection of Paraptosis After Photodynamic Therapy
1 Introduction
2 Materials
2.1 Cell Culture Procedures
2.2 Photosensitizing Agents and Irradiation
2.3 Microscopy
3 Methods
3.1 Cell Culture
3.2 Photosensitization and Irradiation
3.3 Microscopy
3.4 Fluorescent Probes
3.5 Inhibition of Paraptosis
4 Notes
References
Chapter 39: Optimal Use of 2′,7′-Dichlorofluorescein Diacetate in Cultured Hepatocytes
1 Introduction
2 Experimental Procedures
2.1 Reagents and Buffers
2.2 Preparation of DCFH2
2.3 Determination of Molar Extinction Coefficients
2.4 Spectral Properties of DCFH2-DA, DCFH2, and DCF
2.5 Stability of DCFH2-DA and DCFH2 in Solvent
2.6 Cell Culture
2.7 Cellular DCFH2-DA Uptake
2.8 Cellular DCF Uptake
2.9 Intracellular DCF Retention and Transmembrane Diffusion
2.10 Basal Oxidant Formation and Cellular Metabolic Rate
2.11 Real-Time Analysis of Oxidant Formation During In Vitro Anoxia/Reoxygenation in HepG2 Cells
2.12 Statistical Analysis
3 Results
3.1 The Spectral Properties of DCFH2-DA and Derivatives Are pH-Dependent
3.2 The Stability of DCFH2-DA and DCFH2 in Aqueous Solvent and Medium Is Dependent on the Composition of the Solution
3.3 DCFH2-DA Rapidly Accumulates in HepG2 and HepaRG Cells
3.4 DCF Accumulates in HepG2 and HepaRG Cells and Is Poorly Retained
3.5 DCF Crosses Membranes
3.6 Basal Oxidant Formation and Cellular Metabolic Rate Differ Between HepG2 and HepaRG Cells
3.7 Oxidative Stress During In Vitro Anoxia/Reoxygenation Can Be Visualized in Real Time Using DCFH2-DA
4 Discussion
5 Conclusions
References
Index