دانلود کتاب Gene Delivery (Biomaterial Engineering)

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Series Preface
Volume Preface
Contents
About the Series Editor
About the Volume Editors
Contributors
1 Molecular Strings Modified Gene Delivery System
1 Overview
2 Protocol and Discussion
3 Materials
3.1 Synthesis of Lys(Z)-NCA
3.2 Synthesis of PLL
3.3 Synthesis of PLL-RT4
3.4 Synthesis of PLL-MS
3.5 Synthesis of PLL-Too
3.6 Synthesis of PLL-Tos
3.7 Synthesis of PLL-Orn
3.8 Synthesis of PLL-Arg
3.9 Synthesis of PLL-Orn(Tos)
3.10 Synthesis of PLL-Arg(NO2)
3.11 Synthesis of PEI25k-RT2
3.12 Synthesis of G4-RT2
3.13 Preparation of Carrier/DNA Nanoparticles
3.14 Measurements of Zeta Potential and Particle Size
3.15 Determination of Molecular Weight and Molecular Weight Distribution
3.16 In Situ Surface-Enhanced Infrared Absorption Spectroscopy (SEIRAS)
3.17 Isothermal Titration Calorimetry (ITC) Measurement
3.18 Measurement of Circular Dichroism (CD) Spectra
3.19 Cell Culture
3.20 In Vitro DNA Transfection
3.21 Flow Cytometry Assay
3.22 Confocal Laser Scanning Microscopy (CLSM) to Observe the Cellular Uptake
3.23 CLSM to Observe Endosomal Escape
3.24 Cytotoxicity Assay
3.25 Antiserum Transfection
3.26 In Vitro Gene Silencing
3.27 Construction of Tumor Model
3.28 Antitumor Treatment
3.29 Hematoxylin-Eosin (H&E) Staining
3.30 Immunofluorescent Staining for Tumor Vessels
3.31 qRT-PCR Assay
3.32 Enzyme Linked Immunosorbent Assay (ELISA)
4 Methods
4.1 Synthesis of Lys(Z)-NCA
4.2 Synthesis of PLL
4.3 Synthesis of PLL-RT4
4.4 Synthesis of PLL-MS
4.5 Synthesis of PLL-Too
4.6 Synthesis of PLL-Tos
4.7 Synthesis of PLL-Orn
4.8 Synthesis of PLL-Arg
4.9 Synthesis of PLL-Orn(Tos)
4.10 Synthesis of PLL-Arg(NO2)
4.11 Synthesis of PEI25k-RT2
4.12 Synthesis of G4-RT2
4.13 Preparation of Polymer/DNA Nanoparticles
4.14 Measurements of Zeta Potential and Particle Size
4.15 Determination of Molecular Weight and Molecular Weight Distribution
4.16 In Situ Surface-Enhanced Infrared Absorption Spectroscopy (SEIRAS)
4.17 Isothermal Titration Calorimetry (ITC) Measurement
4.18 Measurement of Circular Dichroism (CD) Spectra
4.19 Cell Culture
4.20 In Vitro DNA Transfection
4.21 Flow Cytometry Assay
4.22 CLSM to Observe the Cellular Uptake
4.23 CLSM to Observe Endosomal Escape
4.24 Cytotoxicity Assay
4.25 Antiserum Transfection
4.26 In Vitro Gene Silencing
4.27 Construction of Tumor Model
4.28 Antitumor Treatment
4.29 Histological Analyses
4.30 Immunofluorescent Staining for Tumor Vessels
4.31 qRT-PCR Assay
4.32 Elisa
4.33 Statistical Analysis
5 Conclusion
References
2 Charge/Size Dual-Rebound Gene Delivery System
1 Overview
2 Materials
2.1 Synthesis and Characterization of Poly-l-Glutamate (PLG)
2.2 Synthesis of Aldehyde Group Modified PEG (OHC-PEG-CHO) and Characterization
2.3 Preparation of NPs
2.4 Zeta Potential, Particle Size, and Morphology
2.5 In Vitro DNA Transfection
2.6 Cytotoxicity Assay
2.7 Cell Uptake
2.8 Confocal Laser Scanning Microscopy (CLSM)
2.9 Tumor Accumulation
2.10 In Vivo Antitumor Therapeutic Efficacy
2.11 Histology and Immunofluorescence Analyses
2.12 Photoacoustic Imaging
2.13 VEGF Gene Expression
2.14 Enzyme-Linked Immunosorbent Assay (ELISA)
2.15 Western Blot
3 Methods
3.1 Synthesis and Characterization of PLG (Note 1)
3.2 Synthesis of OHC-PEG-CHO and Characterization (Note 2)
3.3 Preparation of NPs (Note 3)
3.4 Zeta Potential, Particle Size, and Morphology (Note 4)
3.5 In Vitro DNA Transfection (Note 5)
3.6 Cytotoxicity Assay
3.7 Cell Uptake
3.8 CLSM
3.9 Tumor Accumulation (Note 6)
3.10 In Vivo Antitumor Therapeutic Efficacy (Note 7)
3.11 Histology and Immunofluorescence Analyses
3.12 Photoacoustic Imaging (Note 8)
3.13 VEGF Gene Expression
3.14 Elisa
3.15 Western Blot
3.16 Statistical Analysis
4 Notes
5 Conclusion
References
3 Pulmonary Co-delivery of DOX and siRNA
1 Overview
2 Protocol
2.1 Materials
2.1.1 Synthesis of PEI-CA-DOX
2.1.2 Characterization
2.1.3 Preparation of PEI/siRNA and PEI-CA-DOX/siRNA
2.1.4 Drug Release Experiment
2.1.5 Particle Sizes and Zeta Potential Analysis
2.1.6 Cell Uptake Study
2.1.7 Quantification of Bcl2 Gene Expression by qRT-PCR
2.1.8 Cytotoxicity Assay
2.1.9 In Vivo Antitumor Therapy
2.1.10 Biodistribution
2.1.11 Intracellular Uptake of DOX and siRNA In Vivo
2.2 Methods
2.2.1 Synthesis of PEI-CA-DOX and Characterization (Note 1)
2.2.2 Characterization of PEI-CA-DOX (Note 2)
2.2.3 Preparation of PEI/siRNA and PEI-CA-DOX/siRNA (Note 3)
2.2.4 Drug Release Experiment (Note 4)
2.2.5 Zeta Potential and Particle Size Analysis (Note 5)
2.2.6 Cell Uptake Study
2.2.7 Quantification of Bcl2 Gene Expression by qRT-PCR (Note 6)
2.2.8 Cytotoxicity Assay
2.2.9 In Vivo Antitumor Therapy (Note 7)
2.2.10 Biodistribution
2.2.11 Intracellular Uptake of DOX and siRNA In Vivo (Note 8)
3 Discussion
4 Conclusion
References
4 Fluorinated α-Helical Polypeptides Toward Pulmonary siRNA Delivery
1 Overview
2 Protocol
2.1 Materials
2.1.1 Synthesis of 3F-Cl and 5F-Cl
2.1.2 Synthesis of 7F-Cl
2.1.3 Synthesis of nF-N3 (n = 3, 5, and 7)
2.1.4 Synthesis of Polypeptide PPOBLG
2.1.5 Synthesis of PmFx and PG1
2.1.6 Preparation of Polypeptide/siRNA Polyplexes
2.1.7 Gel Electrophoresis
2.1.8 Cell Culture
2.1.9 Cell Uptake
2.1.10 In Vitro TNF-α Knockdown Efficiency
2.1.11 In Vitro Permeation Across Calu-3 Cell Monolayers (Huang et al. 2017) (See Sect. 3 ``6´´)
2.1.12 Multiple Particle Tracking (Huang et al. 2017)
2.1.13 In Vivo Gene Knockdown Efficiency
2.1.14 Lung Function Assessment
2.2 Methods
2.2.1 Synthesis of 3F-Cl and 5F-Cl
2.2.2 Synthesis of 7F-Cl
2.2.3 Synthesis of nF-N3 (n = 3, 5, and 7)
2.2.4 Synthesis of Polypeptide PPOBLG
2.2.5 Synthesis of PmFx and PG1
2.2.6 Preparation of Polypeptide/siRNA Polyplexes
2.2.7 Gel Electrophoresis
2.2.8 Cell Culture
2.2.9 Cell Uptake
2.2.10 ELISA Assay
2.2.11 PCR Assay
2.2.12 In Vitro Permeation Across Calu-3 Cell Monolayers
2.2.13 Multiple Particle Tracking
2.2.14 In Vivo Gene Knockdown Efficiency
2.2.15 Lung Function Assessment
3 Discussion
4 Conclusion
References
5 Preparation and Evaluation of Polymeric Hybrid Micelles to Co-deliver Small Molecule Drug and siRNA for Rheumatoid Arthritis...
1 Overview
2 Protocol
2.1 Materials
2.1.1 Synthesis of PCL-PEG and PCL-PEI Polymers
2.1.2 Preparation and Characterization of Polymeric Hybrid Micelles
2.1.3 Optimization of N/P Ratio Based on Cellular Internalization
2.1.4 RNase Protection Assay of siRNA-Loaded Hybrid Micelles
2.1.5 Measurement of Cell Viability
2.1.6 Endosome Escape
2.1.7 Gene Silencing Study In Vitro
2.1.8 Ability of Dual Drugs-Loaded Micelles to Inhibit the Production of Inflammatory Cytokines
2.1.9 Immunofluorescent Staining of Nuclear Translocation of p65 Subunit
2.1.10 Western Blotting
2.1.11 Establishment of Collagen-Induced Arthritis Model
2.1.12 Accumulation of Hybrid Micelles in Arthritic Joints
2.1.13 Therapeutic Efficacy of Micelles Co-Loaded with Dex and siRNA In Vivo
3 Methods
3.1 Synthesis of PCL-PEG and PCL-PEI Polymers (Fig. 2)
3.2 Preparation of Polymeric Hybrid Micelles Co-Loading with Dex and siRNA (Fig. 3)
3.3 Characterization of Polymeric Hybrid Micelles Co-Loading Dex and p65 siRNA
3.4 Optimization of N/P Ratio Based on Cellular Internalization
3.5 RNase Protection Assay of siRNA Loaded into Hybrid Micelles
3.6 Cell Viability
3.7 Endosome Escape
3.8 In Vitro Gene Silencing Study
3.9 Ability of Drugs-Loaded Hybrid Micelles to Inhibit the Production of Inflammatory Cytokines
3.10 Immunofluorescent Staining of Nuclear Translocation of p65 Subunit
3.11 Western Blotting
3.12 Establishment of the Collagen-Induced Arthritis Model (CIA)
3.13 Biodistribution of the Hybrid Micelles in CIA Mice
3.14 Therapeutic Efficacy of Hybrid Micelles Co-Loading with Dex and p65 siRNA In Vivo
3.15 Statistical Analysis
4 Discussion (Table 2)
5 Notes
6 Conclusion
References
6 Preparation and Application of MPEG-PCL-g-PEI Cationic Micelles in Cancer Therapy
1 Overview
2 Materials
2.1 Preparation of MPEG-PCL-g-PEI Micelles
2.2 Preparation of Doxorubicin and Msurvivin T34A Loaded MPEG-PCL-g-PEI Micelles
2.3 Evaluation of MPEG-PCL-g-PEI Micelles Morphology
2.4 Determination of Doxorubicin and Msurvivin T34A Loaded Micelles In Vitro and In Vivo
3 Protocol
3.1 Preparation and Characterization of MPEG-PCL-g-PEI Copolymer
3.1.1 Preparation and Characterization of MPEG-PCL Copolymer
3.1.2 Preparation and Characterization of MPEG-PCL-GMA Copolymer
3.1.3 Preparation and Characterization of MPEG-PCL-g-PEI Copolymer
3.2 Preparation and Characterization of Blank MPEG-PCL-g-PEI Micelles
3.2.1 Preparation of Blank MPEG-PCL-g-PEI Micelles
3.2.2 Morphology of Blank MPEG-PCL-g-PEI Micelles
3.2.3 Cytotoxicity of Blank MPEG-PCL-g-PEI Micelles In Vitro
3.2.4 Preparation of pDNA/MPEG-PCL-g-PEI Complexes
3.2.5 Gel Retardation Assay of Blank MPEG-PCL-g-PEI Micelles
3.2.6 Transfection Efficiency of Blank MPEG-PCL-g-PEI Micelles In Vitro
3.3 Preparation and Characterization of Doxorubicin and Msurvivin T34A Loaded MPEG-PCL-g-PEI Micelles
3.3.1 Preparation of Doxorubicin and Msurvivin T34A Loaded Micelles
3.3.2 Morphology of Doxorubicin and Msurvivin T34A Loaded Micelles
3.3.3 Uptake of Doxorubicin Loaded Micelles In Vitro
3.3.4 Drug Release of Doxorubicin Loaded Micelles In Vitro
3.3.5 Transfection Efficiency of Doxorubicin and Msurvivin T34A Loaded Micelles In Vitro
3.4 Bio-distribution of Doxorubicin Loaded Micelles In Vivo
3.5 Effect of Doxorubicin and Msurvivin T34A Loaded Micelles in Lung Metastases Tumor Model
3.6 Effect of Doxorubicin and Msurvivin T34A Loaded Micelles in an Abdominal Cavity Metastases Tumor Model
3.7 Effect of Doxorubicin and Msurvivin T34A Loaded Micelles in a Subcutaneous Tumor Model
3.8 Statistical Analysis
4 Notes
5 Discussion
References
7 Preparation and Evaluation of Lipopeptides with Arginine-Rich Periphery for Gene Delivery
1 Introduction
2 Protocol
2.1 Materials
2.1.1 Preparation of Dendritic Arginine-Containing Cationic Peptides
2.1.2 Preparation of Dendritic Arginine and Disulfide Bond-Containing Lipopeptides (RLS)
2.1.3 Synthesis of Low Molecular Drug Camptothecin and Candesartan as Hydrophobic Segment of Lipopeptides
2.1.4 Synthesis of the Dendritic Arginine-Containing Cationic Peptides-Prodrug Conjugation
2.1.5 Synthesis of Disulfide Bond-Modified Cyanine Dyes with Two Long Carbon Chains
2.1.6 Preparation of Assemblies and Gene Complexes and Characterization of Gene Complexes
2.1.7 Drug Release from Cationic Assemblies
2.1.8 Cell Culture and Gene Transfection
2.1.9 Intracellular Tracking of Gene Complexes
2.2 Methods
2.2.1 Preparation of Dendritic Arginine-Containing Cationic Peptides (Fig. 1(I))
2.2.2 Preparation of Dendritic Arginine and Disulfide Bond-Containing Lipopeptides (RLS)
2.2.3 Synthesis of Disulfide Bond-Modified Camptothecin and Candesartan Prodrug (Fig. 1(II), (III))
Synthesis of Disulfide Bond-Modified Camptothecin
Synthesis of Disulfide Bond-Modified Candesartan Prodrug
2.2.4 Synthesis of the Dendritic Arginine-Containing Cationic Peptides-Prodrug Conjugation
2.2.5 Synthesis of Disulfide Bond-Modified Cyanine Dyes with Two Long Carbon Chains (Fig. 1(IV))
2.2.6 Preparation of Assemblies and Gene Complexes and Characterization
2.2.7 Release of Drug from Cationic Assemblies (Fig. 2)
2.2.8 Cell Culture and Gene Transfection (Fig. 3a)
Cell Culture
Gene Transfection
2.2.9 Intracellular Tracking of Gene Complexes (Fig. 3b, c)
3 Discussion
4 Notes
5 Conclusion
References
8 Preparation and Evaluation of Multistage Delivery Nanoparticle for Efficient CRISPR Activation In Vivo
1 Overview
2 Materials
2.1 Synthesis of PEI-PBA, mPEG113-b-PLys100, mPEG113-b-PLys100/DMMA, and mPEG113-b-PLys100/SA
2.2 Investigation on pH-Responsiveness of mPEG113-b-PLys100/DMMA
2.3 Preparation of SDNP and MDNP
2.4 Characterization of MDNP and SDNP
2.5 Fluorescence Resonance Energy Transfer (FRET) Assay
2.6 Nonspecific Protein Adsorption Assay
2.7 Cell Culture
2.8 Cellular Internalization and Endosome Escape
2.9 Transfection Efficiency of MDNP in Cancer Cells
2.10 CRISPR Activation of miR-524 Expression with MDNP in Cancer Cells
2.11 In Vitro Antitumor Study
2.12 Tumor-Targeting Capability of MDNP in Mice
2.13 In Vivo Tumor Growth Inhibition
2.14 Safety Evaluation
3 Methods
3.1 Synthesis of PEI-PBA
3.2 Synthesis of mPEG113-b-PLys100/DMMA and mPEG113-b-PLys100/SA
3.2.1 mPEG113-b-PLys100
3.2.2 mPEG113-b-PLys100/DMMA and mPEG113-b-PLys100/SA
3.3 pH-Responsiveness of mPEG113-b-PLys100/DMMA
3.4 Preparation and Characterization of MDNP and SDNP
3.4.1 Preparation of SDNP and MDNP
3.4.2 Determination of Particle Size and Zeta Potential
3.4.3 Zeta Potential Variation of MDNP and SDNP with pH Adjustment
3.4.4 FRET Assay on SDNP and MDNP at Different pHs
3.5 Quantification of Nonspecific Protein Adsorption
3.6 Cellular Internalization and Endosomal Escape
3.6.1 Cell Culture
3.6.2 Cellular Internalization
3.6.3 Endosomal Escape
3.7 In Vitro Gene Transfection
3.8 In Vitro Cytotoxicity Analysis
3.8.1 In Vitro Cytotoxicity of Polymer
3.8.2 Activation of miR-524 Expression in Vitro
3.8.3 In Vitro Antitumor Effect of MDNP
3.9 In Vivo Distribution of MDNP
3.10 Tumor Growth Inhibition
3.11 Analysis of the Activation of miR-524 Expression in Mice
3.12 Safety Evaluation of MDNP
3.13 Statistical Analysis
4 Notes
5 Conclusion
References
9 Preparation and Evaluation of Rationally Designed Polymers for Efficient Endosomal Escape of siRNA
1 Overview
2 Protocol
2.1 The Synthesis of CTAm, mPEG2k-CTAm, and TDMAEMA
2.2 The Synthesis of mPEG2k-P(DPAx-co-DMAEMAy)-PT (PDDT) Polymers
2.3 The pH-Sensitivity and the pH-Dependence of PDDT-Ms
2.4 siRNA Transfection In Vitro
3 Method
3.1 The Synthesis of mPEG2k-P(DPAx-co-DMAEMAy)-PTn (PDDT) Polymers
3.1.1 The Synthesis of PEG2k-CTAm
3.1.2 The Synthesis of TDMAEMA
3.1.3 The Synthesis of PDDT
3.2 The pH-Sensitivity and Characterization of PDDT-Ms/siRNA Nanomicelles
3.2.1 The pH-Sensitivity of PDDT-Ms Assessed with Nile Red
3.2.2 pH-Responsive Dissociation of PDDT-Ms Examined with DLS
3.2.3 The Size and Morphology of Polyplexes
3.2.4 Gel Retardation Assay
3.3 The Evaluation of PDDT-Ms/siRNA Polyplexes In Vitro
3.3.1 Cell Transfection
3.3.2 Cellular Uptake of Complexes
3.3.3 Cytotoxicity Assessment
3.3.4 Quantitative Real-Time PCR
3.4 The Endosomal Escape of PDDT-Ms/siRNA Polyplexes In Vitro
3.4.1 Cell Transfection in HepG2-Luc Cell
3.4.2 The Influence of Chloroquine and Bafilomycin A1 on Transfection
3.5 Antitumor Activity of PDDT-Ms/siPLK1 Complexes
4 Notes
5 Discussion
References
10 Molecular and Supramolecular Construction of Arginine-Rich Nanohybrids for Visible Gene Delivery
1 Overview
2 Materials
2.1 Synthesis of Arginine-Terminal PDs
2.2 Preparation of ARNHs
2.3 Preparation and Characterizations of ARNHS/DNA Complex
2.4 Investigation of In Vitro Gene Transfection
2.5 Cytotoxicity and Intracellular Tracking
2.6 In Vivo Gene Transfection
2.7 In Vivo Imaging
3 Protocol
3.1 Synthesis of Arginine-Terminal PDs
3.1.1 Synthesis of Arginine-Terminal Dendrons
3.1.2 Decoration of Lipoic Acid
3.1.3 Synthesis of PDs
3.1.4 Characterization of Compounds
3.2 Self-Assembly of PDs into ARNHs
3.3 Preparation of the ARNHS/DNA Complex and DNA Condensation Assay
3.4 Characterizations of ARNHs
3.4.1 Thermal Gravimetric Analysis
3.4.2 Size and Zeta Potential of ARNHs and ARNHS/DNA Complex
3.4.3 Morphology of ARNHS/DNA Complex
3.5 Investigation of In Vitro Gene Transfection Effect
3.5.1 In Vitro Luciferase Activity Assay
3.5.2 GFP Expression
3.6 Cytotoxicity and Intracellular Tracking in HepG2 Cells
3.6.1 Cytotoxicity of ARNHS/DNA Complex
3.6.2 Intracellular Tracking
3.6.3 Live Cell Imaging System (LCIS) Observation
3.6.4 Transmission Electron Microscope Imaging
3.7 In Vivo Gene Transfection
3.7.1 Tumor Model
3.7.2 pCMV-β-gal Transfection in Mouse Muscles
3.7.3 Luciferase Activity in Mouse Muscles
3.7.4 p-53 Gene Expression in Tumor Tissues
3.8 In Vivo Imaging
4 Discussion
References
11 Bioinspired Fabrication of Peptide-Based Capsid-Like Nanoparticles for Gene Delivery
1 Overview
2 Materials
2.1 Synthesis of Poly(L-lysine) Dendrimers
2.2 Synthesis of Poly(L-leucine)
2.3 Preparation of Capsid-Like Nanoparticles (CLNs)
2.4 pH Responsive Properties of CLNs
2.5 In Vitro Gene Condensation of CLNs
2.6 In Vitro Gene Transfection Investigation of CLNs
3 Protocol
3.1 Synthesis of Poly(L-lysine) Dendrimers
3.1.1 Synthesis of Octa(3-Aminopropyl)Silsesquioxane (OAS) Hydrochloride
3.1.2 Synthesis of Generation 2 OAS-poly(L-lysine) (G2-Lys) Dendrimers
3.2 Synthesis of Poly(L-leucine)
3.2.1 Synthesis of L-leucine N-carboxyanhydride (NCA-Leu)
3.2.2 Synthesis of Poly(L-leucine) with Terminal Group of Double Carboxyl
3.3 Preparation of Capsid-Like Nanoparticles (CLNs)
3.4 pH Responsive Properties of CLNs
3.4.1 Fluorescence Analysis
3.4.2 NMR Analysis of CLNs at Different pH Conditions
3.4.3 Nanostructure Changes of CLNs at Different pH Conditions
3.5 In Vitro Gene Condensation of CLNs
3.5.1 Gel Retardation Assay
3.6 In Vitro Gene Transfection Investigation of CLNs
4 Discussion
References
12 Peptide-Modified Polycations with Acid-Triggered Lytic Activity for Efficient Gene Delivery
1 Overview
2 Protocol
2.1 Materials
2.2 Methods
2.2.1 Synthesis of P(OEGMA-DMAEMA)-b-P(DIPAMA-(PDSEMA-Melittin))
Synthesis of P(OEGMA-DMAEMA)
Synthesis of P(OEGMA-DMAEMA)-b-P(DIPAMA-PDSEMA)
Conjugation of Cys-Melittin to P(OEGMA-DMAEMA)-b-P(DIPAMA-PDSEMA)
2.2.2 Synthesis of PDMAEMA-C6M3, PLL-C6M3, and PEI-C6M3
Synthesis of PDMAEMA-Co-PDSEMA
Synthesis of PLL-SPDP
Synthesis of PEI-SPDP
Synthesis of C6M3-Modified Polycations
2.2.3 Hemolysis of Polymers
2.2.4 Preparation and Characterization of DNA Polyplexes
2.2.5 Endosomal Escape of Polyplexes by Confocal Microscope
2.2.6 Polyplex Uptake Assay
2.2.7 In Vitro Transfection
3 Discussion
3.1 Optimization of P(OEGMA-DMAEMA)-b-P(DIPAMA-(PDSEMA-Melittin)) Synthesis
3.2 Preparation of P(OEGMA-DMAEMA)-b-P(DIPAMA-(PDSEMA-Melittin)) Micelles
3.3 Endo/Lysosome Release of the Polyplexes
3.4 In Vitro Transfection by P(OEGMA-DMAEMA)-b-P(DIPAMA-(PDSEMA-Melittin))
3.5 Optimization of the Synthesis of PDMAEMA-C6M3, PEI-C6M3, and PLL-C6M3
3.6 Polyplexes Formation by PDMAEMA-C6M3, PEI-C6M3, and PLL-C6M3 and Plasmid DNA
3.7 In Vitro Transfection by PDMAEMA-C6M3, PEI-C6M3, and PLL-C6M3
4 Conclusion
References
13 Preparation and Evaluation of Supramolecular Hydrogels for Localized Sustained Gene Delivery
1 Overview
2 Protocol
2.1 Materials
2.1.1 Synthesis and Characterization of Positively Charged Amphiphilic PPP Copolymer
2.1.2 Preparation of PPP/Nur77 Polyplex
2.1.3 Gel Retardation Assay of PPP/Nur77 Polyplex
2.1.4 Particle Size and Potential Measurement of PPP/Nur77 Polyplex
2.1.5 Preparation of PPP/α-CD/Nur77 Supramolecular Hydrogels
2.1.6 Sustained Gene Release Assay of Supramolecular Hydrogels
2.1.7 Cell Culture
2.1.8 In Vitro Gene Delivery Efficiency Assessment of PPP/Nur77 Polyplex
2.1.9 Construction of HepG2/Bcl-2 Hepatocellular Carcinoma Cell Lines Overexpressing Bcl-2
2.1.10 Cytotoxicity Verification of PPP/Nur77 Polyplex for Tumor-Resistant Cells
2.2 Methods
2.2.1 Preparation of Positively Charged Amphiphilic PPP Copolymer (Note 1, 2)
Synthesis Method of MPEG-PCL-OH
Synthesis Method for MPEG-PCL-Imidazole
Synthesis Method for PPP
2.2.2 Characterization of PPP Cationic Polymer
2.2.3 Preparation of PPP/Nur77 Polyplex (Note 3)
2.2.4 Gene Binding Capacity Characterization of PPP Polymer (Note 4)
Gel Retardation Assay
Particle Size and Potential Detection
2.2.5 Preparation of Supramolecular Hydrogels (Note 5)
2.2.6 Evaluation of Sustained Release of Supramolecular Hydrogels in Vitro
2.2.7 In Vitro Gene Transfection Evaluation of PPP Polymer (Note 6, 7)
2.2.8 Construction Method of Bcl-2 Overexpressed Tumor Cells (Note 8)
2.2.9 Therapeutic Effect Evaluation of PPP/Nur77 Polyplex on Bcl-2 Drug-Resistant Tumor Cells
3 Discussion
4 Conclusion
References
14 MRI-Visible Nanocarrier for Synergistic MicroRNA Therapy in Liver Fibrotic Rat
1 Overview
2 Protocol
3 Discussion
4 Conclusion
References
15 High DNA-Binding Affinity and Gene-Transfection Efficacy of Bioreducible Cationic Nanomicelles
1 Overview
2 Protocol
2.1 Materials
2.2 Methods
2.2.1 Synthesis of 2-(2-pyridyldithio)ethylamine Hydrochloride (Fig. 1)
2.2.2 Synthesis of N-(2-(2-pyridyldithio)ethyl)perfluorooctanamide (Fig. 2)
2.2.3 Synthesis of Fluorinated PEI (Fig. 3)
2.2.4 Preparation of Nanomicelles (Fig. 4)
2.2.5 Critical Micelle Concentration of PEI25k-SS-5C7F15 (Fig. 5)
2.2.6 Isothermal Titration Microcalorimetry (ITC) Measurement (Fig. 6)
2.2.7 Gene Transfection (Figs. 7 and 8)
2.2.8 Evaluation of Cytotoxicity of Fluorinated PEI Nanomicelles
2.2.9 Erythrocyte Aggregation Test
3 Discussion
4 Conclusion
References
16 Preparation of Chimeric Polymersomes for Gene Delivery
1 Overview
1.1 Introduction
2 Protocol
2.1 Materials
2.1.1 Preparation and Characterization of PEG-P(TMC-co-DTC)
2.1.2 Preparation and Characterization of PEG-P(TMC-co-DTC)-PEI/Spermine
2.1.3 Preparation and Characterization of Peptide-Decorated Polymers
2.1.4 Preparation of Polymersomes
2.1.5 Determination of siRNA Loading Content, Stability, and Reduction Responsivity of Chimeric Polymersomes (CP)
2.1.6 Characterization of CP by Transmission Electron Microscopy (TEM)
2.1.7 MTT Assays of Blank CP
2.1.8 Flow Cytometry Assays and Confocal Microscopy of siRNA-Loaded CP
2.1.9 In Vitro Gene Silencing Efficacy of CP-siRNA
2.1.10 Pharmacokinetic of CP-siRNA
2.1.11 In Vivo Gene Silencing Efficacy of CP-siRNA
2.2 Methods
2.2.1 Preparation and Characterization of PEG-P(TMC-co-DTC) (Fig. 2, Notes 1, 2, 3, 4, 5, and 6)
2.2.2 Preparation of PEG-P(TMC-co-DTC)-PEI/Spermine (Fig. 3, Notes, 7, 8, and 9)
2.2.3 Preparation of Peptide or N3-Functionalized PEG-P(TMC-co-DTC)
Synthesis of cRGD-Functionalized PEG-P(TMC-co-DTC) (Fig. 4, Notes 10, 11)
Synthesis of ANG-Functionalized PEG-P(TMC-co-DTC) (Fig. 5, Notes 12, 13)
Synthesis of N3-PEG-P(TMC-co-DTC) (Fig. 6)
2.2.4 Preparation of CP
Preparation Method 1 (DMSO as a Solvent) (Notes 14,15)
Preparation Method 2 (DMF as a Solvent) (Notes 16)
Preparation of Polypeptide Functionalized CP (Notes 17)
Preparation of Monoclonal Antibody Functionalized CP (Fig. 7, Notes 18)
2.2.5 Characterization of CP
siRNA Loading Efficiency of CP Was Characterized by Gel Electrophoresis (Fig. 8, Notes 19)
Evaluate Stability and Reduction Responsivity of CP-siRNA (Notes 20)
Characterization of CP by TEM (Fig. 9, Notes 21)
2.2.6 MTT Assay of CP (Fig. 10)
2.2.7 Flow Cytometry and Confocal Laser Scanning Microscopy Assays of CP-siRNA
Flow Cytometry Assays of CP-siRNA-Cy3 (Fig. 11)
Confocal Laser Scanning Microscopy of CP-siRNA (Fig. 12)
2.3 In Vitro Gene Silencing Determined by RT-PCR (Fig. 13)
2.4 Pharmacokinetics of CP-siRNA (Fig. 14)
2.5 The Gene Silencing Efficiency In Vivo (Fig. 15)
2.6 Statistical Analysis
3 Discussion
4 Notes
5 Conclusion
References
17 Preparation of Ultrasmall Gold Nanoparticles for Nuclear-Based Gene Delivery
1 Overview
2 Protocol
2.1 Materials
2.1.1 Preparation of 2 nm Au-TIOP NPs and Au-POY2T NPs
2.1.2 Characterization of 2 nm Au-TIOP NPs and Au-POY2T NPs
2.1.3 Cell Viability Study
2.1.4 Determination of the Gene Conjugation Efficiency
2.1.5 Determination of the mRNA Level and Protein Expression
2.2 Methods
2.2.1 Preparation Before the Experiment
Preparation of Aqua Regia
Clean the Glassware, Etc.
2.2.2 Preparation of 2 nm Au-TIOP NPs (Fig. 1)
2.2.3 Preparation of Au-POY2T NPs (Fig. 2)
2.2.4 Determination of the Gene Conjugation Efficiency
2.2.5 Cell Viability Study
2.2.6 Determination of the mRNA Level and Protein Expression
3 Discussion
4 Conclusion
References
18 Polypeptide Cationic Micelles–Mediated Co-delivery of Docetaxel and siRNA for Synergistic Tumor Therapy
1 Overview
2 Protocol
2.1 Materials
2.1.1 Preparation of Polypeptide PEG1-PLL10-PLLeu40
2.1.2 Characterization of Polypeptide PEG1-PLL10-PLLeu40
2.1.3 Preparation of Drug Loaded Micelle Nanoparticles (NPs)
2.1.4 Characterization of Drug Loaded Micelle NPs
2.1.5 Preparation of Micelleplex
2.1.6 Gel Retardation Assay
2.1.7 Stability Analysis of DTX-NPs and Characterization of Micelleplex
2.1.8 Cell Culture
2.1.9 Animals and Antitumor Model
2.1.10 In Vitro Study on Co-delivery of Drug and siRNA
2.1.11 In Vitro siRNA-Bcl-2 Transfection
2.1.12 Analysis of Bcl-2 Expression by PCR
2.1.13 Western Blot Analysis of Bcl-2 Expression
2.1.14 Analysis of Cell Proliferation
2.1.15 Micelle NPs Distribution in Nude Mice
2.1.16 Tumor Suppression Study
2.1.17 Detection of Bcl-2 Gene Expression in Tumor
2.2 Methods
2.2.1 Preparation of PEG1-PLL10-PLLeu40 Copolymer (Note 1, 2)
2.2.2 Characterization of PEG1-PLL10-PLLeu40 Copolymer (Note 3)
2.2.3 Preparation of DTX-Loaded Micelle NPs
2.2.4 Characterization of DTX-Loaded Micelle NPs (Note 4, 5, 6)
2.2.5 Preparation of Micelleplex (Note 7, 8, 9)
2.2.6 Gel Retardation Assay of siRNA-Loading Micelleplex
2.2.7 Stability of DTX-NPs and Characterization of Micelleplex (Note 10, 11)
2.2.8 Cell Culture
2.2.9 Animals and Tumor Model Study
2.2.10 Co-delivery Analysis of Drug and siRNA into Tumor Cells
2.2.11 siRNA-Bcl-2 Transfection in Cells
2.2.12 Analysis of Bcl-2 mRNA Expression by PCR Test
2.2.13 Analysis of Bcl-2 Protein Expression by Western Blot
2.2.14 Analysis of Cell Proliferation
2.2.15 Micelle NPs Distribution In Vivo
2.2.16 Tumor Suppression Assay (Note 12)
2.2.17 Analysis of Tumorous Bcl-2 Expression In Vivo
2.2.18 Statistical Analysis
3 Notes
4 Discussion
References
19 Preparation and Evaluation of Reduction-Controlled Hierarchical Unpacking Terplexes for Gene Delivery
1 Introduction
2 Materials
2.1 Preparation of 3, 3′-Diselanediyldipropanoic Acid
2.2 Preparation of Diselenide-Crosslinked Oligoethylenimine (OEI-SeSex)
2.3 Preparation of Disulfide Bonds Functionalized HA (HA-SS-COOH)
2.4 Preparation of Reduction-Controlled Hierarchical Unpacking Terplexes
2.5 Determination of Particle Size and Zeta Potential
2.6 Reduction-Responsive Degradability of OEI-SeSex and OEI-SSx
2.7 Reduction-Controlled Hierarchical Unpacking Behavior of Terplexes
2.8 Cell Culture and In Vitro Viability Assay
2.9 In Vitro Gene Transfection of Reduction-Controlled Hierarchical Unpacking Terplexes
2.10 In Vivo Gene Transfection of Reduction-Controlled Hierarchical Unpacking Terplexes
2.11 Analytical Instruments
3 Methods
3.1 Preparation of 3, 3′-Diselanediyldipropanoic Acid (Koch et al. 1990)
3.2 Preparation of Diselenide or Disulfide-Crosslinked Oligoethylenimine
3.3 Preparation of HA-SS-COOH (Note 1)
3.4 Preparation of Reduction-Controlled Hierarchical Unpacking Terplexes (Note 2)
3.5 Determination of Particle Size and Zeta Potential
3.6 Reduction-Responsive Degradability of OEI-SeSex and OEI-SSx (Note 3 and 4)
3.7 Reduction-Controlled Hierarchical Unpacking Behavior of Terplexes (Note 5 and 6)
3.8 Cell Culture and In Vitro Viability Assay
3.9 In Vitro Gene Transfection of Reduction-Controlled Hierarchical Unpacking Terplexes (Note 7)
3.10 In Vivo Gene Transfection of Reduction-Controlled Hierarchical Unpacking Terplexes
3.11 Statistical Analysis
4 Notes
5 Summary
References
20 Bioreducible Zinc (II)-Coordinative Polyethylenimine with Low Molecular Weight for Robust Gene Delivery of Primary and Stem...
1 Introduction
2 Protocol
2.1 Materials
2.1.1 Synthesis of Ligand DDAC
2.1.2 Synthesis of Zn-Coordinative Cationic Polymers
2.1.3 Gel Retardation Assays
2.1.4 Polyplex Size and Zeta Potential Measurements
2.1.5 In Vitro Gene Transfection
2.1.6 Cytotoxicity Assays
2.1.7 Cellular Uptake of Polyplexes
2.2 Methods
2.2.1 Synthesis of Ligand DDAC
2.2.2 Synthesis of Zn Coordinative Cationic Polymers
2.2.3 Gel Retardation Assays
2.2.4 Polyplex Size and Zeta Potential Measurements
2.2.5 In Vitro Gene Transfection
2.2.6 Cytotoxicity Assays
2.2.7 Cellular Uptake of Polyplexes
2.2.8 Statistical Analysis
3 Notes
4 Conclusion
References
21 Virus-Mimetic DNA-Ejecting Polyplexes for Cancer Gene Delivery
1 Overview
2 Materials
2.1 Synthesis of the Polymers
2.2 Preparation of the Fluorescent Dye-Labeled Polymers and DNA
2.3 Fabrication of Polymer/DNA Polyplexes
2.4 Size and Zeta Potential Measurements
2.5 Transmission Electron Microscope Measurements
2.6 Agarose Gel Retardation Electrophoresis
2.7 Esterase-Responsive Activities of the Polymer
2.8 Esterase-Responsive Activities of the Polyplexes
2.9 Cell Line
2.10 In Vitro Gene Transfection
2.11 Subcellular Distribution and Colocalization
2.12 Effects of Inhibitors on Cellular Uptake
3 Protocols
3.1 Synthesis of the Polymers
3.2 Preparation of the Fluorescent Dye-Labeled Polymers
3.3 Preparation of the Fluorescent Dye-Labeled DNA
3.4 Fabrication of Polymer/DNA Polyplexes
3.5 γPGA-Coating Polyplexes
3.6 Size and Zeta Potential Measurements
3.7 Transmission Electron Microscope Imaging
3.8 Agarose Gel Retardation Electrophoresis
3.9 Esterase-Responsive Activities of the Polymer
3.10 Esterase-Responsive Activities of the Polyplexes
3.11 Gene Transfection
3.12 Subcellular Distribution and Colocalization
3.13 Effects of Inhibitors on Cellular Uptake
4 Notes
5 Conclusion
References
22 Rattle-Structured Rough Nanocapsules with In Situ-Formed Gold Nanorod Cores for Complementary Gene/Chemo/Photothermal Thera...
1 Overview
2 Protocol
2.1 Materials
2.1.1 Synthesis of Rough Hollow Silica Nanoparticles (HSNs)
2.1.2 Synthesis of Rattle-Structured Rough Au@HSN
2.1.3 Synthesis of CD-PGEA
2.1.4 Preparation of Rough Au@HSN-PGEA (AHPs)
2.1.5 BET Characterization of Material
2.1.6 Cytotoxicity Assay
2.1.7 In Vitro Gene Transfection Assay
2.1.8 Cellular Internalization
2.1.9 SAHP/p53 Stability Characterization
2.1.10 Complementary Gene/Chemo/Photothermal Therapy In Vitro and In Vivo
2.1.11 Photoacoustic Imaging of AHPs In Vitro and In Vivo
2.1.12 Photothermal Effect of AHP In Vitro and In Vivo
2.1.13 Sorafenib Loading
2.1.14 NIR-Triggered Sorafenib Release
2.1.15 Effects of NIR-Triggered Drug Release on HCC Cells
2.2 Methods
2.2.1 Synthesis of Rough Hollow Silica Nanoparticles (HSNs)
2.2.2 Synthesis of Rattle-Structured Rough Au@HSN
2.2.3 Synthesis of CD-PGEA
2.2.4 Synthesis of AHP
2.2.5 BET Characterization of Material
2.2.6 Cytotoxicity Assay
2.2.7 In Vitro Gene Transfection Assay
2.2.8 Cellular Internalization
2.2.9 Photothermal Effect of AHP In Vitro and In Vivo
2.2.10 SF Loading
2.2.11 NIR-Triggered SF Release
2.2.12 Effects of NIR-Triggered Drug Release on HCC Cells
2.2.13 Evaluation of SAHP/p53 Stability
2.2.14 Complementary Gene/Chemo/Photothermal Therapy In Vitro and In Vivo
2.2.15 PA Imaging of AHPs In Vitro and In Vivo
3 Discussion
3.1 Note
4 Conclusion
References
23 Preparation and Evaluation of Boronate-Linked Nanoassembly for Efficient Gene Delivery
1 Introduction
2 Materials
2.1 Synthesis of EHDO-Modified Oligoethylenimine (OEI-EHDO)
2.2 Synthesis of Cholest-5-en-3-Ol(3b)-,(3-Boronophenyl)Carbamate (Chol-PBA)
2.3 Nanoassembly Preparation
2.4 Determination of Grafting Degree
2.5 Determination of Critical Micelle Concentration (CMC)
2.6 Visual Inspection on Nanoassembly in the Presence of Nile Red Dye
2.7 Variation of Nanoassembly at Different pHs
2.8 Preparation of Nanoassembly/DNA Complexes
2.9 Agarose Gel Retardation Assay
2.10 Determination of Particle Size, Zeta Potential, and Morphology
2.11 Stability Study of Nanoassembly and its Complexes
2.12 Acid-Triggered Unpacking of Nanoassembly/DNA Complexes in the Presence of Heparin
2.13 Cell Culture
2.14 Amplification and Purification of Plasmid DNA
2.15 In Vitro Gene Transfection
2.16 Flow Cytometry
2.17 Confocal Laser Scanning Microscopy (CLSM)
2.18 NH4Cl-Associated Interference of Endosomal Acidification Procession
2.19 In Vitro Cytotoxicity Assays
2.20 In Vivo Animal Study
3 Methods
3.1 Preparation and Characterization of Boronate-Linked Nanoassembly
3.1.1 Synthesis of EHDO-Modified Oligoethylenimine (OEI-EHDO)
3.1.2 Synthesis of Cholest-5-en-3-Ol (3b)-,(3-Boronophenyl)Carbamate (Chol-PBA)
3.1.3 Nanoassembly Preparation
3.1.4 Determination of Grafting Degree
3.1.5 Determination of Critical Micelle Concentration (CMC)
3.1.6 Visual Inspection on Nanoassembly in the Presence of Nile Red Dye
3.1.7 Variation of Nanoassembly at Different pHs
3.1.8 Preparation of Nanoassembly/DNA Complexes
3.1.9 Agarose Gel Retardation Assay
3.1.10 Particle Size and Zeta Potential Measurements
3.1.11 Observation of the Morphology by TEM
3.1.12 Stability Study
3.1.13 Acid-Triggered Unpacking of Nanoassembly/DNA Complexes in the Presence of Heparin
3.2 Cell Culture
4 Maintain Cells at 37 C in a Humidified Atmosphere Containing 5% CO2
4.1 Amplification and Purification of Plasmid DNA
5 Determine the Purity and Concentration of DNA by UV Absorbance at 260-280 Nm
5.1 In Vitro Gene Transfection
5.2 Flow Cytometry
5.3 Confocal Laser Scanning Microscopy (CLSM)
5.4 NH4Cl-Associated Interference of Endosomal Acidification Procession
5.5 In Vitro Cytotoxicity Assays
5.6 In Vivo Animal Study
5.7 Statistical Analysis
6 Notes
7 Conclusion
References
24 Preparation and Evaluation of Virus-Inspired Nanogenes for Host-Specific Transfection
1 Introduction
2 Materials
2.1 Cell Culture and Amplification of Plasmid DNA
2.2 Preparation of Cracked Cancer Cell Membrane (CCCM)
2.3 Cancer Cell Membrane Protein Characterization
2.4 Agarose Gel Retardation Assay
2.5 Optimization of Membrane Weight Ratio Within Gd/DNA@CCCM
2.6 Preparation of CCCM-Coated Gd/DNA (Gd/DNA@CCCM)
2.7 Transmission Electron Microscopy (TEM) Observation
2.8 Protein Adsorption Assay
2.9 Stability of Gd/DNA@CCCM in the Presence of Heparin and/or DNase
2.10 Stability of Gd/DNA@CCCM under 10% Serum Conditions
2.11 In Vitro Targeting Recognition Toward Homotypic Cancer Cells
2.12 In Vitro Macrophage Uptake Study
2.13 Luciferase Assay
2.14 Green Fluorescent Protein Assay
2.15 Cytotoxicity Assay
2.16 In Vivo Gene Transfection Study
2.17 In Vitro and In Vivo MR Imaging
3 Methods
3.1 Preparation and Characterization of Virus-Inspired Nanogenes
3.1.1 Cell Culture
3.1.2 Amplification of Plasmid DNA
3.1.3 Preparation of Cracked Cancer Cell Membrane (CCCM)
3.1.4 Cancer Cell Membrane Protein Characterization
3.1.5 Agarose Gel Retardation Assay
3.1.6 Preparation of CCCM-Coated Gd/DNA (Gd/DNA@CCCM)
3.1.7 Transmission Electron Microscopy (TEM) Observation
3.1.8 Protein Adsorption Assay
3.1.9 Stability Study
3.2 In Vitro Homotypic Targeting Study
3.3 In Vitro Macrophage Uptake Study
3.4 Luciferase Assay
3.5 Green Fluorescent Protein Assay
3.6 Cytotoxicity Assay
3.7 In Vivo Gene Transfection Study
3.8 In Vitro and In Vivo MR Imaging
3.9 Statistical Analysis
4 Notes
5 Conclusion
References
25 Calcium Carbonate-Based Nanoparticles for Gene Delivery
1 Overview
2 Protocol
2.1 Materials
2.1.1 CaCO3 NPs: Vascular Endothelial Growth Factor-C siRNA (He et al. 2008)
2.1.2 Coprecipitation for the Synthesis of CaCO3-Plasmid DNA (Chen et al. 2011)
2.1.3 KALA-Modified CaCO3-DNA (Zhao et al. 2012)
2.1.4 Alginate-CaCO3 Hybrid (Zhao et al. 2012)
2.1.5 Nanostructured CaCO3-DNA (Chen et al. 2012)
2.1.6 CaCO3 Particles: Plasmid pEGFP-C1-p53 (Kong et al. 2012)
2.1.7 Protamine Sulfate CaCO3-DNA Nanoparticles (Wang et al. 2014)
2.1.8 KALA-Protamine Sulfate-CaCO3-DNA Nanoparticles (Wang et al. 2014)
2.1.9 Calcium Carbonate-Calcium Phosphate-DNA NPs (Zhao et al. 2014)
2.1.10 Polyethyleneimine-CaCO3-DNA Nanoparticles (Chen et al. 2016)
2.1.11 Lipid-Coated Calcium Carbonate/Phosphate Hybrid (LCC) NPs (Wu et al. 2017)
2.1.12 MnO2-CaCO3-ICG/siRNA Nanoplatform (Liu et al. 2019)
2.2 Methods
2.2.1 CaCO3 NPs: Vascular Endothelial Growth Factor-C siRNA
Preparation of CaCO3 NPs
Preparation of CaCO3-DNA Complex
CaCO3-DNA Protection Analysis
In Vitro Transfection
CaCO3-siRNA Transfection for Gastric Cell Line SGC-7901
2.2.2 Coprecipitation for the Preparation of CaCO3-Plasmid DNA
Formulation of CaCO3-DNA Complex
In Vitro Transfection
2.2.3 KALA-Modified CaCO3-DNA
Preparation of CaCO3-KALA-DNA
In Vitro Transfection
2.2.4 Alginate-CaCO3 Hybrid
Preparation of Aginate-CaCO3-DNA/DOX
Evaluation of Encapsulation Efficiency and Amount Loaded of DOX and DNA
In Vitro Release of Drug
In Vitro Cell Inhibition Analysis
2.2.5 Nanostructured CaCO3-DNA
Preparation of CaCO3-DNA NPs
Calculation of Encapsulation Efficiency and Loading Content
Calculation of in Vitro Drug Release
In Vitro Cell Inhibition Evaluation
Cell Apoptosis Study
2.2.6 CaCO3 Particles: Plasmid pEGFP-C1-p53
Preparation of CaCO3 Particles
Plasmid pEGFP-C1-p53 Construction
Preparation of Plasmid-Loaded CaCO3
In Vitro Transfection
Cell Viability, Cytotoxicity, and Apoptosis Assay
Identification of p53 Gene Expression
2.2.7 Protamine Sulfate CaCO3-DNA Nanoparticles
Preparation of PS-CaCO3-DNA NPs
Encapsulation Efficiency of DNA
In Vitro Luciferase Plasmid Transfection
Cell Viability Assay
2.2.8 KALA-Protamine Sulfate-CaCO3-DNA Nanoparticles
Synthesis of KALA-PS-CaCO3-DNA NPs
In Vitro Transfection and Cell Viability
2.2.9 Calcium Carbonate-Calcium Phosphate-DNA NPs
Synthesis of CaCO3-CaP-DNA NPs
DNA Encapsulation Efficiency
In Vitro Transfection and Gene Expression
Cellular Uptake Assay
2.2.10 Polyethyleneimine-CaCO3-DNA Nanoparticles
Preparation of CaCO3 NPs
Preparation of PEI-ACa-DNA NPs
In Vitro Transfection of NPs
p53 Gene Expression and Cell Growth Inhibition
2.2.11 Lipid-Coated Calcium Carbonate/Phosphate Hybrid (LCC) NPs
Preparation of LCC NPs with Various Ratios of C/P
Encapsulation of siRNA
Colloidal Stability Assay
PD-L1 Expression
2.2.12 MnO2-CaCO3-ICG/siRNA Nanoplatform
Synthesis of MnO2 NPs
Preparation of MnO2-CaCO3-ICG/siRNA Nanoplatform
Biocompatibility and Cellular Uptake
3 Discussion
4 Conclusion
References
26 A Codelivery System of Anticancer Drug Doxorubicin and Tumor-Suppressor Gene p53 Based on Polyphosphoester for Lung Cancer ...
1 Introduction
2 Materials
3 Methods
3.1 Preparation of pH-Responsive Doxorubicin Derivative DOX-hyd-N3
3.2 Preparation of Alkynyl-Containing Block Copolymer Precursor mPEG-b-PBYP
3.2.1 Preparation of Cyclic Phosphoester Monomer BYP
Preparation of 2-Chloro-1,3,2-Dioxaphospholane (CP)
Preparation of 2-Chloro-2-Oxo-1,3,2-Dioxaphospholane (COP)
Preparation of 2-(but-3-yn-1-yloxy)-2-Oxo-1,3,2-Dioxaphospholane (BYP)
3.2.2 Preparation of Diblock Copolymer Precursor mPEG-b-PBYP
3.3 Preparation of pH-Responsive Doxorubicin Prodrug mPEG-b-PBYP-hyd-DOX
3.4 Preparation of Polycationic Carrier mPEG-b-PBYP-g-DAE
3.5 Characterization of Chemical Structure and Molecular Weights
3.6 Measurement of Critical Aggregation Concentration (CAC) of Copolymers
3.7 Preparation and Characterization of Hybrid Micelles
3.8 In Vitro Drug Release
3.9 Gel Retardation Assay
3.10 Cell Culture and Gene Supplier
3.11 In Vitro Cytotoxicity Assay
3.12 In Vitro Transfection
3.13 Measurement of Cellular Uptake
3.14 Intracellular Release of DOX and p53 Genes
4 Notes
5 Conclusion
References
27 Preparation and Evaluation of siRNAsome as siRNA and Drug Delivery System
1 Introduction
2 Materials
2.1 Synthesis and Characterization of siRNA-SS-PNIPAM
2.1.1 Synthesis of 2-(2-Pyridyldithio)Ethylamine Hydrochloride
2.1.2 Synthesis of Poly(N-Isopropylacrylamide) (PNIPAM)
2.1.3 Synthesis of PNIPAM-SS-Py
2.1.4 Synthesis of siRNA-SS-PNIPAM
2.1.5 Characterization of siRNA-SS-PNIPAM
Ultraperformance Liquid Chromatography (UPLC) Analysis
Lower Critical Solution Temperature (LCST) of siRNA-SS-PNIPAM
2.2 Formation and Characterization of siRNAsome
2.3 Drug Encapsulation by siRNAsome
2.4 Reduction Responsiveness of siRNAsome
2.4.1 Size, Morphology Change, and siRNA Release
2.4.2 In Vitro DOXHCl Release
2.5 In Vitro Evaluation of siRNAsome
2.5.1 Flow Cytometry Assay
2.5.2 Confocal Laser-Scanning Microscopy Assay
2.5.3 In Vitro Cytotoxicity Assay of siRNAsome
2.5.4 Real-Time Quantitative PCR Analysis
2.6 In Vitro and In Vivo Evaluation of DOXHCl-Loaded siRNAsome
2.6.1 MTT Assay
2.6.2 In Vivo Antitumor Assay in Subcutaneous MDR MCF-7 Tumor Model
3 Methods
3.1 Synthesis and Characterization of siRNA-SS-PNIPAM
3.1.1 Synthesis of 2-(2-Pyridyldithio)Ethylamine Hydrochloride (Wang et al. 2016)
3.1.2 Synthesis of Poly(N-isopropylacrylamide) (PNIPAM, 19 kDa, Fig. 2(i)) (Li et al. 2010)
3.1.3 Synthesis of PNIPAM-SS-Py (Fig. 2(ii)) (Xu et al. 2009)
3.1.4 Synthesis of siRNA-SS-PNIPAM (Fig. 2(iii))
3.1.5 Characterization of siRNA-SS-PNIPAM
Ultraperformance Liquid Chromatography (UPLC) Analysis
Lower Critical Solution Temperature (LCST) of siRNA-SS-PNIPAM
3.2 Formation and Characterization of siRNAsome
3.3 Drug Encapsulation by siRNAsome (Table 1)
3.4 Reduction Responsiveness of siRNAsome
3.4.1 Size, Morphology Change, and siRNA Release
3.4.2 In Vitro DOXHCl Release
3.5 In Vitro Evaluation of siRNAsome
3.5.1 Flow Cytometry Assay (Fig. 5a)
3.5.2 Confocal Laser-Scanning Microscopy Assay (Fig. 5b)
3.5.3 In Vitro Cytotoxicity Assay of siRNAsome (Fig. 5c)
3.5.4 Real-Time Quantitative PCR Analysis (Fig. 5d)
3.5.5 MTT Assay (Fig. 6a)
3.5.6 In Vivo Antitumor Assay in Subcutaneous MDR MCF-7 Tumor Model (Fig. 6b)
4 Notes
5 Conclusion
References
28 Development of Cationic Lipid-Assisted PEG-b-PLA Nanoparticle for Nucleic Acid Therapeutics
1 Overview
2 Protocol
2.1 Materials
2.1.1 Preparation of siRNA-Loaded CLAN Systems
2.1.2 Determination of Encapsulation Efficiency, Drug Release, Particle Size, and Morphology
2.1.3 Cellular Uptake
2.1.4 Cell Transfection
2.1.5 Cell Apoptosis
2.1.6 Effects on the Anticancer Activity of Breast Tumor-Bearing Mice
2.2 Methods
2.2.1 Preparation of siRNA-Loaded CLAN Systems (Notes 1, 2, 3, 4, 5, 6)
2.2.2 Encapsulation Efficiency (Note 7)
2.2.3 Drug Release
2.2.4 Particle Size (Note 8)
2.2.5 Determination of the Particle Morphology under TEM
2.2.6 Cellular Uptake (Notes 9, 10)
2.2.7 Cell Transfection (Notes 11, 12, 13)
2.2.8 Effects on the Apoptosis of HepG2 Cells (Note 14)
2.2.9 Effects on the Anticancer Activity of Breast Tumor-Bearing Mice (Note 15)
3 Discussion
4 Notes
5 Conclusion
References
Index