Developing Methods Towards Quantifying and Enhancing the Endosomal Escape of DNA-Gold Nanoparticle Conjugates 公开

Abstract

This thesis is divided into five chapters. The first chapter provides background for development of spherical nucleic acids (SNAs) and focuses on a particular type: DNA-gold nanoparticle (DNA-AuNP) conjugates. The introduction provides detail into the design, commercial availability, therapeutic applications, and intracellular uptake of DNA-AuNPs. It includes a brief summary of our contribution to the field via the development of deoxyribozyme-gold nanoparticle (Dz-AuNP) conjugates. Additionally, the introduction discusses an ongoing debate as to whether DNA-AuNPs, as well as other SNAs, achieve significant cytosolic access or primarily remain sequestered in endosomes. The second chapter describes work to develop a method to quantify and assess the intracellular location of these particles through a split green fluorescent protein (GFP) complementation assay. In our hands, the assay proved to be insensitive for the desired intracellular applications. The third chapter describes the ongoing investigations of a ratiometric assay that utilizes two fluorescent dyes, fluorescein (FAM) and ATTO647N, to develop a standard curve of their pH-dependent fluorescence intensity ratio. This chapter contains extensive findings of the in-development assay with the goal of determining the intracellular and environmental pH of these probes. The fourth chapter describes work toward the synthesis and characterization of known endosomal enhancing peptides. Chapter 4, also, discusses the potential effects of conjugating endosomal disrupting peptides to pH-sensing AuNPs. Finally, Chapter 5 contains an overall discussion and conclusion of this thesis. Future directions for this project and the implications our findings are detailed here. 

Table of Contents

Chapter 1: Introduction .......................................................................................................... 1

1.1. Nanomaterials as Therapeutics .........................................................................................2

1.2. DNA-Gold Nanoparticle Conjugates ..................................................................................3

1.3. Commercially Available and Clinically in-Development SNAs ..........................................4

1.4. DNAzyme-AuNP Conjugates ..............................................................................................6

1.5. Endocytosis and Fate of DNA-AuNP Conjugates ............................................................... 7

1.6. Overview of Goals .................................................................................................................... 11

Chapter 2: Quantifying Endosomal Escaping Using a Split GFP Complementation Assay...... 12

2.1. Background and Design............................................................................................................ 13

2.2. Methods..................................................................................................................................... 15

2.3. Results ....................................................................................................................................... 17

2.3.1. Confirmation of GFPβ11 Synthesis ...................................................................................... 17

2.3.2. Preliminary Validations of Split GFP Complementation Assay............................... 18

2.3.3. Validation of Split GFP Complementation on AuNPs............................................... 20

2.4. Discussion................................................................................................................................... 21

2.5. Conclusions ............................................................................................................................... 22

Chapter 3: Quantifying Endosomal Escaping Using a Ratiometric pH-Sensitive Assay ......... 24

3.1. Background and Design............................................................................................................ 25

3.2. Methods..................................................................................................................................... 28

3.3. Results ....................................................................................................................................... 31

3.3.1. Confirmation and Characterization of FAM-/ATTO647N-DNA AuNPs................... 31

3.3.2. In Solution Manipulations and the Effect on Fluorescence..................................... 34

3.3.3. In Cell Manipulations and the Effect on Fluorescence............................................. 37

3.3.4. pH-Sensing AuNPs in Cells............................................................................................ 39

3.4. Discussion................................................................................................................................... 42

3.5. Conclusions................................................................................................................................ 47

Chapter 4: Steps Toward Screening Endosomal Escape Enhancing Peptides.......................... 48

4.1. Background and Design............................................................................................................ 49

4.2. Methods..................................................................................................................................... 51

4.3. Results........................................................................................................................................ 53

4.3.1. Confirmation of AMP Syntheses................................................................................. 53

4.3.2. Confirmation of Linking an AMP to DNA ................................................................... 55

4.4. Discussion................................................................................................................................... 57

4.5. Conclusions................................................................................................................................ 58

Chapter 5: Conclusions.................................................................................................................... 59

Supplemental Information; Miscellaneous Methods.................................................................. 62

References................................................................................................................................................ 63 

About this Honors Thesis

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School	Emory College
Department	Chemistry
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Chemistry, Biochemistry Nanotechnology
关键词	Chemistry
Committee Chair / Thesis Advisor	Khalid Salaita, Emory University
Committee Members	Cherry Wongtrakool, Emory University Stefan Lutz, Emory University

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