Live-cell imaging of drug delivery by mesoporous silica nanoparticles

In order to deliver drugs to diseased cells nanoparticles featuring
controlled drug release are developed. 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) 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 exhibit 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 synthesized. 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 quenching 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
presence in the entire cellular membrane. To reduce cell toxicity
induced by the photosensitizer and to achieve spatial control on
the endosomal 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.