Live-cell imaging of drug delivery by mesoporous silica nanoparticles

117 Pages·2011·4.69 MB·English

by  Anna Magdalena Sauer

Dissertation zur Erlangung des Doktorgrades der Fakultät Chemie und Pharmazie
der Ludwig-Maximilians-Universität München
Live-cell imaging
of drug delivery by
mesoporous silica nanoparticles
Drug loading, pore sealing, cellular uptake and controlled drug
release
Anna Magdalena Sauer
aus
Assis, Brasilien
2011
Erklärung
Diese Dissertation wurde im Sinne von §13 Abs. 3 bzw. 4 der Promotionsordnung vom 29. Januar
1998 (in der Fassung der sechsten Änderungssatzung vom 16. August 2010) von Herrn Prof. Dr.
Christoph Bräuchle betreut.
Ehrenwörtliche Versicherung
Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe erarbeitet.
München, den 31. August 2011
Anna Magdalena Sauer
Dissertation eingereicht am 31.08.2011
1. Gutachter Prof. Dr. Christoph Bräuchle
2. Gutachter Prof. Dr. Thomas Bein
Mündliche Prüfung am 18.10.2011
Summary
In order to deliver drugs to diseased cells nanoparticles featuring controlled drug release are de-
veloped. Controlled release is of particular importance for the delivery of toxic anti-cancer drugs
that should not get in contact with healthy tissue. To evaluate the effectivity and controlled drug-
release ability of nanoparticles in the target cell, live-cell imaging by highly-sensitive fluorescence
microscopy is a powerful method. It allows direct real-time observation of nanoparticle uptake into
the target cell, intracellular trafficking and drug release. With this knowledge, existing nanoparticles
can be evaluated, improved and more effective nanoparticles can be designed. The goal of this work
was to study the internalization efficiency, successful drug loading, pore sealing and controlled drug
release from colloidal mesoporous silica (CMS) nanoparticles. The entire work was performed in
close collaboration with the group of Prof. Thomas Bein (LMU Munich), where the nanoparticles
were synthesized.
To deliver drugs into a cell, the extracellular membrane has to be crossed. Therefore, in the first
part of this work, the internalization efficiency of PEG-shielded CMS nanoparticles into living HeLa
cells was examined by a quenching assay. The internalization time scales varied considerably from
cell to cell. However, about 67% of PEG-shielded CMS nanoparticles were internalized by the cells
within one hour. The time scale is found to be in the range of other nanoparticles (polyplexes,
magnetic lipoplexes [1, 2]) that exhibit non-specific uptake.
Besides internalization efficiency, successful drug loading and pore sealing are important parameters
for drug delivery. To study this, CMS nanoparticles were loaded with the anti-cancer drug colchicine
and sealed by a supported lipid bilayer using a solvent exchange method (additional collaboration
with the group of Prof. Joachim Rädler, LMU). Spinning disk confocal live-cell imaging revealed
that the nanoparticles were taken up into HuH7 cells by endocytosis. As colchicine is known to ex-
hibit toxicity towards microtubules, the microtubule network of the cells was destroyed within 2 h of
incubation with the colchicine-loaded lipid bilayer-coated CMS nanoparticles. Although successful
drug delivery was shown, it is necessary to develop controlled local release strategies.
To achieve controlled drug release, CMS nanoparticles for redox-driven disulfide cleavage were syn-
thesized. The particles contain the ATTO633-labeled amino acid cysteine bound via a disulfide
linker to the inner volume. For reduction of the disulfide bond and release of cysteine, the CMS
nanoparticles need to get into contact with the cytoplasmic reducing milieu of the target cell. We
showed that nanoparticles were taken up by HuH7 cells via endocytosis, but endosomal escape seems
to be a bottleneck for this approach. Incubation of the cells with a photosensitizer (TPPS2a) and
photoactivation led to endosomal escape and successful release of the drug. In addition, we showed
that linkage of ATTO633 at high concentration in the pores of silica nanoparticles results in quench-
ing of the ATTO633 fluorescence. Release of dye from the pores promotes a strong dequenching
effect providing an intense fluorescence signal with excellent signal-to-noise ratio for single-particle
imaging. With this approach, we were able to control the time of photoactivation and thus the time
of endosomal rupture. However, the photosensitizer showed a high toxicity to the cell, due to its
v
presence in the entire cellular membrane.
To reduce cell toxicity induced by the photosensitizer and to achieve spatial control on the endoso-
mal escape, the photosensitizer protoporphyrin IX (PpIX) was covalently surface-linked to the CMS
nanoparticles and used as an on-board photosensitizer (additional collaboration with the groups of
Prof. Joachim Rädler and Prof. Heinrich Leonhardt, both LMU). The nanoparticles were loaded
with model drugs and equipped with a supported lipid bilayer as a removable encapsulation. Upon
photoactivation, successful drug delivery was observed. The mode of action is proposed as a two-
step cascade, where the supported lipid bilayer is disintegrated by singlet oxygen in a first step
and the endosomal membrane ruptures enabling drug release in a second step. With this system,
stimuli-responsive and controlled, localized endosomal escape and drug release is achieved.
Taken together, the data presented in this thesis show that real-time fluorescence imaging of CMS
nanoparticles on a single-cell level is a powerful method to investigate in great detail the processes
associated with drug delivery. Barriers in the internalization and drug delivery are detected and can
be bypassed via new nanoparticle designs. These insights are of great importance for improvements
in the design of existing and the synthesis of new drug delivery systems.
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Contents
Summary v
1 Introduction 1
2 Principles of nanomedical drug delivery 5
2.1 Uptake and trafficking of nanoparticles in cells . . . . . . . . . . . . . . . . . . . . . 5
2.1.1 Accumulation at the target tissue . . . . . . . . . . . . . . . . . . . . . . . . . 6
2.1.2 Cellular internalization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
2.1.3 Intracellular trafficking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
2.1.4 Endosomal release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
2.2 Nanoparticle designs for drug delivery . . . . . . . . . . . . . . . . . . . . . . . . . . 10
2.2.1 Polymeric nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
2.2.2 Lipid-based nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
2.2.3 Viral nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
2.2.4 Inorganic nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
3 Colloidal mesoporous silica (CMS) nanoparticles 13
3.1 Mesoporous silica materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
3.2 Synthesis of CMS nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
3.2.1 Outer-shell functionalized CMS . . . . . . . . . . . . . . . . . . . . . . . . . . 14
3.2.2 Core-shell functionalized CMS . . . . . . . . . . . . . . . . . . . . . . . . . . 15
3.2.3 Template extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
3.3 CMS nanoparticles as drug delivery vehicles . . . . . . . . . . . . . . . . . . . . . . . 16
3.3.1 Drug loading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
3.3.2 Pore sealing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
3.3.3 Cancer cell targeting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
3.3.4 Stimuli-responsive release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
3.4 Biocompatibility of CMS nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . 22
3.4.1 Size, surface properties and concentration . . . . . . . . . . . . . . . . . . . . 22
3.4.2 Degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
4 Fluorescence live-cell imaging 25
4.1 Principles of fluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
4.2 Bleaching and quenching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
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Contents
4.3 Wide-field and spinning disk confocal microscopy . . . . . . . . . . . . . . . . . . . . 27
4.4 Living cancer cells in fluorescence microscopy . . . . . . . . . . . . . . . . . . . . . . 29
5 Experimental methods and data analysis 31
5.1 Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
5.2 Cell culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
5.3 Preparation of
Contents
List of abbreviations 77
Bibliography 81
Acknowledgments 103
List of publications 105
Curriculum Vitae 107
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