Live-cell imaging elucidates cellular interactions of gene nanocarriers for cancer therapy

170 Pages·2013·4.96 MB·English

by  Frauke Martina Mickler

DISSERTATION ZUR ERLANGUNG DES DOKTORGRADES
DER FAKULTÄT FÜR CHEMIE UND PHARMAZIE
DER LUDWIG-MAXIMILIANS-UNIVERSITÄT MÜNCHEN

Live-cell imaging elucidates cellular interactions
of gene nanocarriers for cancer therapy

Frauke Martina Mickler (geb. König)
aus
Braunschweig, Deutschland

2013
Erklärung

Diese Dissertation wurde im Sinne von §7 der Promotionsordnung vom 28. November 2011
von Herrn Prof. Dr. Christoph Bräuchle betreut.

Eidesstattliche Versicherung

Diese Dissertation wurde eigenständig und ohne unerlaubte Hilfe erarbeitet.

München, den 10.6.2013 Frauke Martina Mickler

Dissertation eingereicht am: 11.06.2013

1. Gutachter: Prof. Dr. Christoph Bräuchle
2. Gutachter: PD Dr. Manfred Ogris
Mündliche Prüfung am: 18.07.2013
Summary

The nanocarrier-mediated delivery of therapeutic transgenes into human target cells is a promising
approach to treat life-threatening diseases such as cancer.
For effective gene delivery, the nanocarrier has to meet a series of challenging requirements. First,
high capacity loading of the genetic material and high stability of the formed nanoparticles in the blood
circulation is required. Next, the gene carrier must specifically bind target cells of interest, e.g. cancer
cells, and enter them. After uptake, trafficking towards the cell nucleus and destabilization of
endosomal membranes has to be realized, followed by DNA release from particles and DNA import
into the nucleus. Furthermore, ideal gene nanocarriers should be non-toxic and non-immunogenic and
allow cheap and reproducible manufacturing.
In this work polymeric nanocarriers were studied that contained different functionalities to sense their
environment and adapt dynamically to overcome cellular barriers for gene delivery. Highly-sensitive
fluorescence microscopy was applied as a tool to dissect the interactions of functionalized gene
nanocarriers on the single-cell level in real-time. To study the effects of polymer design on DNA
condensation, cell binding and internalization, live-cell imaging experiments were combined with
biological assays, new experimental setups and tailor-made image analysis routines. The influence of
polyethylene glycol (PEG) shielding and receptor targeting on particle uptake was examined in detail
and microscopy-based assays were applied to study endosomal release and nuclear import of
biomolecules.
The results from live-cell experiments with PEGylated polymer particles demonstrate that fine-tuning of
the PEG length is important to reduce non-specific interactions and maximize specific receptor-
mediated uptake of targeted particles. The data additionally reveals that the applied particle dose can
significantly affect the uptake characteristics. A second study with bioreducible PEGylated PDMAEMA
polyplexes demonstrates that reversible PEG shielding is a promising approach to enhance the
transfection efficiency of gene nanocarriers.
Furthermore, a study on EGF receptor targeted polyplexes is presented. Applied polyplexes were
equipped either with natural full-length EGF or the alternative peptide ligand GE11. Presented data
demonstrates that the ligands induce two distinct endocytosis mechanisms for particle uptake. The
full-length EGF triggers accelerated endocytosis due to its dual active role in receptor binding and
signaling. For GE11 an alternative EGFR signaling-independent, actin-driven pathway is proposed.

V
SUMMARY

In addition to optimization of the targeting ligand itself, a method is introduced that can be used to
determine the optimal ligand density on the particle surface for efficient particle internalization.
Furthermore the setup of a microfluidic device is reported in this thesis that can be applied to screen
the interactions of nanoparticles with cells and physiological surfaces. Experimental results on the
cellular adhesion of targeted and untargeted polyplexes under flow conditions are presented.
In an additional study the gene delivery potential of novel four-arm PEG dendrimer hybrids as well as
sequence-defined polymers from solid phase assisted synthesis was investigated using live-cell
imaging. The results indicate a clear advantage of the four-arm construct in comparison to a two-arm
dendrimer construct. Successful ligand installation and EGF receptor-mediated uptake of sequence-
defined polymers was confirmed.
Furthermore, endosomal destabilization in cells was monitored by a calcein release assay proving the
positive effect of histidine incorporation on endosomal escape of gene vectors.
Finally, successful nuclear import of biomolecules with nuclear localization sequences was visualized
after direct microinjection into the cytoplasm.
In conclusion, our results demonstrate that the rational design of “intelligent” nanocarriers can lead to
more specific, more efficient and safer gene delivery into cancer cells. Fluorescence live-cell imaging
provides detailed insight into the cellular interactions of nanocarriers and can support the development
of improved gene vectors for clinical application.

VI
CONTENTS

Contents

Summary ............................................................................................................................................ V
1 Introduction ....................................................................................................................................... 1

2 Principles of gene therapy ................................................................................................................ 5
2.1 Gene delivery systems ................................................................................................................. 6
2.2. Cancer therapy ............................................................................................................................ 8
2.2.1 Strategies for tumor targeting ................................................................................................ 9
2.3 In vivo barriers for gene carriers ................................................................................................. 12
2.4 Cellular interactions of gene carriers .......................................................................................... 15
2.4.1 Attachment to the cell surface ............................................................................................. 15
2.4.1.1 Tumor-associated cell surface receptors .......................................................................... 15
2.4.2 Endocytosis pathways and intracellular trafficking ............................................................... 19
2.4.3 Endocytosis-independent pathways .................................................................................... 22
2.4.4 Endosomal escape .............................................................................................................. 23
2.4.5 Decondensation and transport to the nucleus ..................................................................... 24
2.4.6 Transgene expression ......................................................................................................... 25
2.4.7 RNA interference ................................................................................................................. 26
2.4.8 Toxicity ................................................................................................................................ 27
2.5 From in vitro studies towards clinical application ........................................................................ 28

3 Fluorescence microscopy .............................................................................................................. 31
3.1 Resolution and contrast .............................................................................................................. 32
3.2 Principles of fluorescence ........................................................................................................... 33
3.3 Fluorescence labeling ................................................................................................................. 35
3.3 Special considerations for live-cell imaging ................................................................................ 36
3.4 Wide-field and confocal scanning microscopy ............................................................................ 37

4 Surface shielding of gene vectors ................................................................................................. 39
4.1 Interplay between PEG shielding and receptor targeting -
live-cell imaging of integrin-targeted polyplex micelles………......... ................................................. 40
4.1.1. Particle design .................................................................................................................... 40
4.1.2. Coincubation of RGD(+) and RGD(-) micelles at low concentration ................................... 41
4.1.3 Colocalization analysis of coincubated micelles at low concentration ................................. 42
4.1.4 Coincubation of micelles at high dose ................................................................................. 43
4.1.5 Colocalization analysis at high dose .................................................................................... 45
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CONTENTS

4.1.6 Quantification of micelle uptake by flow cytometry .............................................................. 46
4.1.7 Identification of the uptake pathway .................................................................................... 46
4.1.8 Luciferase reporter gene expression ................................................................................... 50
4.1.9 Discussion ........................................................................................................................... 51
4.2 Reversible PEG shielding for improved intracellular DNA release.............................................. 54
4.2.1 Particle design ..................................................................................................................... 54
4.2.2 Live-cell imaging of particle uptake and trafficking to late endosomes ................................ 55
4.2.3 Luciferase reporter gene expression ................................................................................... 57
4.2.4 DNA release ........................................................................................................................ 58
4.2.5 Discussion ........................................................................................................................... 59

5 Receptor targeting of gene vectors ............................................................................................... 61
5.1 Tuning nanoparticle uptake: Natural and artificial EGFR targeting ligand mediate two distinct
endocytosis mechanisms ................................................................................................................. 63
5.1.1 Particle design ..................................................................................................................... 63
5.1.2 Uptake kinetics determined by quenching assay ................................................................. 64
5.1.3 Live-cell imaging of polyplex uptake .................................................................................... 65
5.1.4 Uptake pathway ................................................................................................................... 69
5.1.5 Receptor signaling activation ............................................................................................... 70
5.1.6 Correlation between receptor signaling and uptake kinetics ................................................ 70
5.1.7 Effect of serum starvation .................................................................................................... 72
5.1.8 Discussion ........................................................................................................................... 73
5.2 Influence of ligand density and dual targeting ............................................................................ 76
5.2.1 Particle design ..................................................................................................................... 76
5.2.2 Quantifying the uptake efficiency from confocal images ...................................................... 76
5.2.3 Discussion ........................................................................................................................... 79
5.3 Receptor targeting under flow..................................................................................................... 81
5.3.1 Microfluidic set-up ................................................................................................................ 81
5.3.2 Influence of PEG shielding on polyplex adhesion under flow .............................................. 84
5.3.3 EGF receptor-targeted polyplexes under flow ..................................................................... 85
5.3.4 Discussion ........................................................................................................................... 86

6 Improved scaffolds for gene and drug delivery ............................................................................ 89
6.1 Internally functionalized dendrimers with PEG core for gene therapy ........................................ 90
6.1.1 Particle design ..................................................................................................................... 90
6.1.2 DNA binding and gene transfection ..................................................................................... 91
6.1.3 Discussion ........................................................................................................................... 92
6.2 Intrinsically functionalized dendrimers for drug delivery ............................................................. 94
6.2.1 Particle design ..................................................................................................................... 94
6.2.2 Live-cell imaging of loaded and unloaded dendrimers ......................................................... 95
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CONTENTS
6.2.3 Discussion ........................................................................................................................... 97
6.3 Sequence defined scaffolds from solid phase supported synthesis............................................ 98
6.3.1 Particle design ..................................................................................................................... 98
6.3.2 EGF ligand induces cell binding and uptake of STP polyplexes .......................................... 98
6.3.3 Comparing gene transfer efficiency of EGF-PEG-STP and EGF-PEG-PEI polyplexes ....... 99
6.3.4 Discussion ......................................................................................................................... 100

7 Endosomal escape and nuclear import ....................................................................................... 103
7.1 Histidine as endosomal escape agent ...................................................................................... 104
7.1.1 Particle design ................................................................................................................... 104
7.1.2 Uptake efficiency and gene transfer .................................................................................. 105
7.1.3 Endosomal escape monitored by calcein release assay ................................................... 106
7.1.4 Discussion ......................................................................................................................... 107
7.2 Visualizing nuclear localization sequence (NLS) mediated import............................................ 109
7.2.1 Micromanipulators for direct cytoplasmic delivery ............................................................. 109
7.2.2 NLS mediated import of microinjected proteins ................................................................. 109
7.2.3 Discussion ......................................................................................................................... 110

8 Conclusion ..................................................................................................................................... 111

9 Experimental Methods .................................................................................................................. 113
9.1 Particle preparation ...... ........................................................................................................... 113
9.1.1 DNA labeling.......................................................................................................................113
9.1.2 Integrin-targeted polyplex micelles with different PEG lengths .......................................... 113
9.1.3 Reversibly shielded PDMAEMA polyplexes....................................................................... 114
9.1.4 Receptor-targeted PEG-PEI polyplexes ............................................................................ 115
9.1.5 Dendrimer hybrids for gene and drug delivery ................................................................... 116
9.1.6 Sequence defined STP Polymers from solid phase synthesis ........................................... 117
9.2 Cell culture................................................................................................................................ 118
9.3 Single-cell imaging ................................................................................................................... 118
9.3.1 Particle addition ................................................................................................................. 118
9.3.2 Particle quenching ............................................................................................................. 119
9.3.3 Markers of cellular compartments ...................................................................................... 119
9.3.4 Dead cell staining .............................................................................................................. 119
9.3.5 Inhibition of endocytic pathways ........................................................................................ 120
9.3.6 Receptor signaling assays ................................................................................................. 120
9.3.7 Fixation of cells .................................................................................................................. 120
9.3.8 Calcein Release Assay ...................................................................................................... 120
9.3.9 GFPnuc expression ........................................................................................................... 121
9.4 Bulk cell assays ........................................................................................................................ 121
9.4.1 Flow cytometry .................................................................................................................. 121
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CONTENTS

9.4.2 Luciferase reporter gene expression ................................................................................. 121
9.4.3 Western Blotting ................................................................................................................ 122
9.5 Microscopical setup .................................................................................................................. 122
9.5.1 Wide-field fluorescence microscopy .................................................................................. 122
9.5.2 Spinning disk confocal microscopy ................................................................................ 123
9.6 Microfluidic setup ...................................................................................................................... 123
9.6.1 SAW system .................................................................................................................... 123
9.6.2 Syringe pump device ......................................................................................................... 123
9.7 Micromanipulator for cytoplasmic injection ............................................................................... 124
9.8 Data analysis ............................................................................................................................ 125
9.8.1 Image calibration and editing ............................................................................................. 125
9.8.2 Particle counting ................................................................................................................ 125
9.8.3 Particle tracking ................................................................................................................. 125
9.8.3 Colocalization analysis ...................................................................................................... 126
9.8.4 Nano_In_Cell_3D .............................................................................................................. 126

Appendix ........................................................................................................................................... 127
Bibliography ..................................................................................................................................... 131
List of Abbreviations ........................................................................................................................ 151
List of Publications .......................................................................................................................... 155
Curriculum Vitae ............................................................................................................................... 157
Acknowledgements .......................................................................................................................... 159

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