Biocompatible multitasking nanoparticles for spatiotemporally resolved monitoring and photoinactivation of cancer cells and antibiotic resistant bacteria.
Final Report Abstract
We developed a straightforward strategy to disperse highly insoluble photosensitizers in aqueous environments, without major synthetic efforts and keeping their photosensitizing abilities unaffected. A layered nanoclay was employed to adsorb and to solubilize a highly efficient yet hydrophobic Si(IV) phthalocyaninate in water. The aggregation of the photoactive dye was correlated with its photophysical properties, particularly with the ability to produce cytotoxic singlet oxygen. The resulting hybrid nanomaterial is able to selectively photoinactivate Gram-positive antibiotic-resistant pathogens, due to local interactions between the bacterial membranes and the negatively charged nanodiscs. Nanototoxicity assays confirmed its innocuousness towards eukaryotic cells, showing that it constitutes a new class of “phototriggered magic bullet” for the inactivation of pathogens in phototherapy, as well as in the development of coatings for self-disinfecting surfaces. In a further step, we explored a supramolecular array based on an adamantane-functionalized phthalocyaninate and cyclodextrin vesicles. The results indicate that the host–guest interaction between the photosensitizer and the vesicles significantly prevents the formation of inactive aggregates, and enhances the photosensitizing ability. This supramolecular assembly constitutes a biocompatible photoactive platform for the design of phototherapeutic agents. Moreover, a modular building-block strategy based on axially substituted Si(IV) phthalocyanines was developed. The photosensitizing module possesses a sterically demanding architecture that avoids hydrolysis of the substituents and H-aggregation of the photoactive macrocycle. Two axial azide units render this module a versatile building block for the decoration with a plethora of solubilising moieties or targeting units, such as clickable (poly)saccharides and biotin. The cycloaddition followed by deprotection yielded targeted photosensitizers bearing two axial substituents that provide solubility in water and target the photosensitizer to the bacteria. Besides constituting highly efficient photosensitizers, they also display a significant fluorescence, which facilitates the monitoring of the pathogens. The photobiological evaluation indicated that even S. aureus USA300, a meticillin resistant Gram-positive bacterium (MRSA), is fully photoinactivated. Finally, we implemented novel microscopy methods for Frequency Domain Fluorescence Lifetime Imaging Microscopy and Fluorescence Polarization Anisotropy Microscopy that enabled us to robustly quantify the state of biosensors in single living cells. An important feature of these methods is that they permitted us to enlarge the dynamic range of the technique, thereby allowing a large and diverse population of cells to be imaged in a short time. Using these methods, we investigated the activation of caspases upon perturbation with reactive oxygen species, either localized or ubiquitous. For this purpose, we developed photoactive and biocompatible surfaces where cells can attach and grow, acting as photogenerators of reactive oxygen species upon illumination with visible light. By monitoring simultaneously the phenotype and molecular state of the cells, we found that similar phenotypes were associated to different cellular processes. We also showed that apoptosis was not induced with the surface-bound photosensitizer, as the cells were rather protected from apoptosis upon external reactive oxygen species generation, despite the apparent apoptosis-like blebbing. To further investigate the molecular pathways, we developed three sensors to monitor the activities of the effector caspase 3, as well as of two initiator caspases, namely caspase 8 and caspase 9. Due to their spectral properties, they can be simultaneously measured in single cells. We showed that while the variance in response time to the stimuli is in the order of hours, the timing of subsequent activations is very well timed within minutes. In summary, our methodologies facilitate the in vitro design and evaluation of photosensitizers for the treatment of cancer and infectious diseases with the aid of functional fluorescence microscopy.
Publications
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flatFLIM: enhancing the dynamic range of frequency domain FLIM. Opt. Express 2012, 20, 20730
Schuermann, K. C.; Grecco, H. E.
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A soft supramolecular carrier with enhanced singlet oxygen photosensitizing properties. Soft Matter 2013, 9, 2453
Voskuhl, J.; Kauscher, U.; Gruener, M.; Frisch, H.; Wibbeling, B.; Strassert, C. A.; Ravoo, B. J.
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Self-assembled benzophenone bis-urea macrocycles facilitate selective oxidations by singlet oxygen. Journal of Organic Chemistry 2013, 78, 5568
Geer, M. F.; Walla, M. D.; Solntsev, K. M.; Strassert, C. A.; Shimizu, L. S.
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Structural and photosensitizing features of phthalocyanine-zeolite hybrid nanomaterials. Photochemistry and Photobiology 2013, 89, 1406
Grüner, M.; Siozios, V.; Hagenhoff, B.; Breitenstein, D.; Strassert, C. A.
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Photofunctional surfaces for quantitative fluorescence microscopy: Monitoring the effects of photogenerated ROS at single cell level with spatiotemporal resolution. ACS Applied Materials and Interfaces 2015, 7, 5944
Stegemann, L.; Schuermann, K.; Strassert, C. A.; Grecco, H. E.
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Photophysical efficiency-boost of aqueous aluminium phthalocyanine by hybrid formation with nano-clays. Chemical Communications 2015, 51, 13534
Staniford, M. C.; Lezhnina, M. M.; Gruener, M.; Stegemann, L.; Strassert, C. A.; Kucziusd, R.; Bleicherd, V.; Kynast, U. H.
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Selective inactivation of resistant Gram-positive pathogens with a light-driven hybrid nanomaterial. ACS Applied Materials & Interfaces 2015, 7, 20965
Grüner, M. C.; Tuchscherr, L.; Löffler, B.; Gonnissen, D.; Riehemann, K.; Staniford, M.; Kynast, U. H.; Strassert, C. A.
