University of Groningen Amphiphilic DNA and its application in biomedicine Li, Hongyan

University of Groningen

Amphiphilic DNA and its application in biomedicine

Li, Hongyan

DOI:

10.33612/diss.125274906

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Publication date: 2020

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Li, H. (2020). Amphiphilic DNA and its application in biomedicine. University of Groningen. https://doi.org/10.33612/diss.125274906

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Amphiphilic DNA and its

Application in Biomedicine

Amphiphilic DNA and its Application in Biomedicine Hongyan Li

PhD thesis

University of Groningen May 2020

Zernike Institute PhD thesis series 2020-08 ISSN: 1570-1530

ISBN: 978-94-034-2631-0 (printed version) ISBN: 978-94-034-2630-3 (electronic version)

The research described in this thesis was performed in Polymer Chemistry and Bioengineering group at Zernike Institute for Advanced Materials, University of Groningen, the Netherlands. This work was financially supported by the Chinese Scholarship Council (CSC), the University of Groningen, the Netherlands Organi-zation for Science Research (NWO) and European Research Council (ERC).

Cover design: Hongyan Li Printed by: ProefschriftMaken

Amphiphilic DNA and its

Application in Biomedicine

PhD thesis

to obtain the degree of PhD at the University of Groningen

on the authority of the Rector Magnificus Prof. C. Wijmenga

and in accordance with the decision by the College of Deans. This thesis will be defended in public on

Friday 22 May 2020 at 16:15 hours

by

Hongyan Li

born on 11 March 1987 in Shaanxi, China

Supervisor

Co-supervisor

Assessment Committee

Those things that hurt, instruct.

Contents

1 DNA Nanotechnology Enters Cell Membranes 1

1.1 Introduction . . . 2

1.2 Synthesis of DNA Amphiphiles . . . 3

1.3 Nanoscale Assemblies from DNA Amphiphiles . . . 6

1.3.1 Micelles from DNA Amphiphiles . . . 7

1.3.2 Liposomes from DNA Amphiphiles. . . 11

1.4 Interactions of DNA Amphiphiles with Cell Membranes . . . 16

1.4.1 Anchoring DNA Amphiphiles on Cell Membranes. . . 17

1.4.2 Factors Influencing the Interaction between DNA Amphiphiles and Cell Membranes. . . 18

1.4.3 Stability of the Complex between DNA Amphiphiles and Cell Membranes. . . 19

1.4.4 Characteristics of DNA Amphiphiles Interacting with Cell Membranes. . . 20

1.5 Applications . . . 21

1.5.1 Drug Delivery. . . 21

1.5.2 Immunotherapy . . . 23

1.5.3 Gene Silencing . . . 24

1.5.4 Sensing the Extra and Intracellular Environment . . . 25

1.5.5 Cell Capture and Assembly . . . 28

1.5.6 Complex DNA Nanostructures on and in the Cell Membrane. 29 1.6 Conclusions and Perspective . . . 31

1.7 Thesis Motivation and Overview . . . 33

Bibliography . . . 35

2 Amphiphilic DNA Anchoring to Membranes 45 2.1 Introduction . . . 46

CONTENTS

2.2 Results and Discussions . . . 48

2.2.1 Lipid DNA Anchoring to Liposome Membrane. . . 48

2.2.2 Lipid DNA Anchoring to Cell Membrane . . . 54

2.3 Conclusion . . . 59

2.4 Experimental Section . . . 60

2.4.1 Materials . . . 60

2.4.2 Lipid DNA Synthesis and Characterization. . . 61

2.4.3 Liposome Preparation . . . 63

2.4.4 FRET Assay . . . 64

2.4.5 Surface Anchored U4T Quantification . . . 64

2.4.6 Calculation of U4T on Liposome . . . 64

2.4.7 Characterization of Lipid DNA Anchored Liposome. . . 65

2.4.8 Cell Culture . . . 65

2.4.9 Evaluation of Toxicity . . . 66

2.4.10 Anchoring Lipid DNA to Cell Membrane . . . 66

2.4.11 Lipid DNA Stability on Cell Membrane . . . 66

2.4.12 Hybridization on MDA-MB-468 cell. . . 67

Bibliography . . . 67

3 Fast, Efficient, and Targeted Liposome Delivery Mediated by DNA 71 3.1 Introduction . . . 72

3.2 Results and Discussions . . . 73

3.2.1 Enhanced Cellular Uptake by DNA Hybridization . . . 73

3.2.2 Specific Delivery by DNA Hybridization . . . 75

3.2.3 Mechanism of Liposome Cellular Uptake . . . 77

3.2.4 After Endocytosis. . . 80 3.2.5 Cellular Toxicity. . . 81 3.3 Conclusion . . . 82 3.4 Experimental Section . . . 83 3.4.1 Materials . . . 83 3.4.2 DNA Used. . . 83

3.4.3 Confocal Microscopy Sample Preparation . . . 85

3.4.4 Flow Cytometry Measurements . . . 86

CONTENTS

3.4.5 Uptake of Calcein and PI Loaded Liposomes. . . 86

3.4.6 Proliferation Assay . . . 87

3.4.7 Specific Delivery . . . 88

3.4.8 Liposome Uptake at Low Temperature . . . 88

3.4.9 Macropinocytosis of HeLa Cells. . . 88

3.4.10 Proof of Endocytosis Inhibition . . . 89

Bibliography . . . 89

4 DNA Hybridization as a General Method to Enhance Nanostructure Uptake 93 4.1 Introduction . . . 94

4.2 Results and Discussions. . . 95

4.2.1 DNA Tetrahedron Characterization, Cellular Uptake, and Stability . . . 95

4.2.2 Cellular Uptake of Gold Nanoparticles . . . 99

4.2.3 Cellular Uptake of Polystyrene Nanoparticles . . . 100

4.3 Conclusions. . . 101

4.4 Experimental Section . . . 102

4.4.1 Materials . . . 102

4.4.2 DNA Used. . . 102

4.4.3 Cell Culture . . . 103

4.4.4 Formation of DNA Tetrahedron Structures. . . 104

4.4.5 AuNPs Synthesis, Conjugation and Characterization . . . 104

4.4.6 Confocal Microscopy Measurements . . . 105

4.4.7 Flow Cytometry Measurements . . . 106

4.4.8 DNA Tetrahedron Nanostructure Degradation in Cell Media. 106 4.4.9 PSNPs Conjugation and Characterization . . . 107

4.4.10 Dark-field Imaging. . . 107

Bibliography . . . 108

5 the Core of Immunostimulatory Nanoparticle Makes a Difference 111 5.1 Introduction . . . 112

5.2 Results and Discussions. . . 114

5.2.1 Nanoparticle Characterization . . . 114

CONTENTS 5.2.2 Activation of DCs in Vivo. . . 117 5.3 Conclusion . . . 120 5.4 Experimental Section . . . 120 5.4.1 Materials . . . 120 5.4.2 Mice. . . 120 5.4.3 DNA Used. . . 121

5.4.4 Micelle Preparation and Characterization . . . 121

5.4.5 AuNPs Preparation and Characterization. . . 121

5.4.6 Liposome Preparation and Quantification . . . 122

5.4.7 TEM Measurement. . . 123

5.4.8 DLS Measurement . . . 123

5.4.9 Zeta Potential Measurement. . . 123

5.4.10 Surface Coverage Calculation . . . 123

5.4.11 in Vivo Treatment. . . 124

5.4.12 Analysis of Spleen DCs. . . 124

5.4.13 Flow Cytometry Analysis. . . 125

5.4.14 ELISA . . . 125 5.4.15 Statistical Analysis . . . 125 Bibliography . . . 126 Summary 129 Samenvatting 133 Acknowledgement 137 List of Publications 141 x