Sauer, Anna Magdalena (2011): Live-cell imaging of drug delivery by mesoporous silica nanoparticles: Drug loading, pore sealing, cellular uptake and controlled drug release. Dissertation, LMU München: Faculty of Chemistry and Pharmacy |
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Abstract
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.
Item Type: | Theses (Dissertation, LMU Munich) |
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Keywords: | live-cell imaging, mesoporous silica, nanoparticles, drug delivery, controlled releas |
Subjects: | 500 Natural sciences and mathematics 500 Natural sciences and mathematics > 540 Chemistry and allied sciences |
Faculties: | Faculty of Chemistry and Pharmacy |
Language: | English |
Date of oral examination: | 18. October 2011 |
1. Referee: | Bräuchle, Christoph |
MD5 Checksum of the PDF-file: | 7ed802a55aa4d4977ff05bfea991dc9b |
Signature of the printed copy: | 0001/UMC 19983 |
ID Code: | 13822 |
Deposited On: | 10. Jan 2012 10:34 |
Last Modified: | 24. Oct 2020 03:13 |