Nanotools for cancer research and therapy

Illes, Bernhard (2020): Nanotools for cancer research and therapy. Dissertation, LMU München: Fakultät für Chemie und Pharmazie

Abstract

Cancer remains one of the most dominant diseases and is responsible for almost 10 million deaths worldwide each year. To combat it, both a deeper understanding of its mechanisms and novel treatment options are necessary. In this work we present a new tool for cancer research and a variety of novel drug delivery systems. Firstly, it is known that tumor progression and invasion are strongly influenced by the mechanical properties of the extracellular matrix. A key regulator of translating mechanical cues from the extracellular surroundings is the mechanotransduction protein Yes-associated protein (YAP). Although there is evidence that YAP plays a role in both tumor progression and metastasis, it is unclear as of yet, if YAP activation on its own is enough to trigger the onset of invasion. To investigate YAP’s role in cancer invasion, we designed a research tool that is based on optogenetics. This tool – a photo-activatable YAP (optoYAP) - allows for spatio-temporal control of YAP’s activation. As YAP is only active in the nucleus, the tool facilitates its activation via nuclear translocation upon an optical trigger. After activation, optoYAP induces downstream signaling for several hours. Additionally, its activation induces growth, leading to increased proliferation in two-dimensional and increased spheroid size in three-dimensional cultures. When applied to cancer spheroids, activation of optoYAP lead to invasion of the surrounding collagen matrix. Strikingly, site-selective activation of optoYAP in cancer spheroids exclusively leads to invasion from the activated site. This strongly hints at YAP activation being enough to trigger the onset of invasion. Secondly, conventional chemotherapy involves systemic administration of toxic drugs at high dosage, which leads to severe adverse effects on the patient’s health. One possible solution to prevent these negative effects is the reduction of the drug dosage through the direct delivery of anti-cancer drugs to the tumor. For this purpose, we designed a delivery system that is a synergistic combination of metal-organic framework (MOF) nanoparticles and exosomes for drug delivery, which takes advantage of the beneficial properties of each compound. This approach enables highly efficient drug loading of exosomes, by successfully coating drug-loaded MOF nanoparticles with exosomes via lipid fusion. The resulting exosome-coated nanoparticles show an efficient release of their cargo after cell uptake with no premature leakage. Loading with a chemotherapeutic agent as biologically active cargo results in a steep decrease of cell viability. The proposed release mechanism is mainly based on the decomposition of the MOF nanocarrier. This, in combination with possible endogenous exosomal release mechanisms, allows for drug escape from the endosome. The combination of the chemical tunability of the MOF core and the cell-derived exosome shell provides a promising drug delivery platform for biomedical applications. Thirdly, effective treatment of cancer is frequently hindered by resistances, which often require combination therapy for successful treatment. The main challenge drug carriers, e.g. liposomes, face in combination therapy, is the effective loading and retention of desired drug ratios. The potential of nanoparticle-based biomedicine for theranostic applications has increasingly come into the spotlight, as these systems offer potential solutions for the challenges, which limit success of conventional therapies. We present an effective, biocompatible and controlled drug delivery system with on-demand release abilities, and its usage as a versatile and powerful class of nanocarriers. To directly address the challenge of cancer resistance that results in the need for combination therapy, we made use of the previously developed liposome coated MOF nanoparticles. These hybrid nanoparticles combine the advantages of liposomes with the easy and efficient loading process of MOFs. Liposome coated MOF nanoparticles were successfully synthesized via the fusion method. Once loaded, the nanoparticles exhibit no premature leakage and an efficient release. We demonstrate their successful loading with both single and multiple drugs at the same time, which makes them a potential candidate for use in combination therapy. Fourthly, we report the synthesis of a novel biocompatible and multifunctional drug delivery system. It is entirely build out of covalently crosslinked organic molecules. We crosslinked β-cyclodextrin structures with rigid organic linker molecules to obtain β-CD nanoparticles. These small, thermally stable and highly water-dispersable nanoparticles possess an accessible pore system. Covalently labeling them with dye molecules allowed for effective tracking of them in in vitro cell experiments. The β-CD nanoparticles show an incredibly fast cell-uptake within only 30 minutes in HeLa cells, which is based on rapid sugar-mediated cell-uptake kinetics. Furthermore, the particles were successfully loaded with various cargo molecules and possess pH-responsive release behavior. Both nuclei staining with Hoechst 33342 dye as cargo and effective cell killing with doxorubicin as cargo was demonstrated in live-cell experiments. This nanocarrier system is a promising platform for the development of novel adaptable and highly biocompatible theranostic systems. Lastly, another potential approach to overcome the inherent toxicity of conventional chemotherapeutics is the use of calcium phosphate and citrate. While both have been discussed as very promising, non-toxic anti-cancer agents, their development as therapeutics and specifically their successful administration has been hindered by the strict regulatory mechanisms of the cell. Here, we present the successful development of a novel administration system for the combinatorial administration of calcium, phosphate, and citrate. They are administered as colloidal nanoparticles (CPC) that can selectively kill cancer cells without the need for any inherently toxic drugs. The presented particles show no toxicity before the endosomal release nor after their degradation. This highly selective toxicity of CPCs was used to successfully treat two different aggressive pleural tumors in mice. After only two local applications tumor size was decreased by about 40% and 70%, respectively. The only sign of adverse effects was a slight pleural thickening effect after up to 8 applications during long term studies. These results are a breakthrough for the successful application of calcium and citrate in chemotherapeutics and a promising start for further refinement. In summary, we have developed a novel tool to photocontrol YAP activity and induce growth in cells and potentially trigger the onset of invasion. Additionally we developed and characterized a variety of novel drug delivery systems: liposome coated MIL-88A nanoparticles proved suitable for drug delivery and showed high cell uptake and an efficient release in vitro, both for single and combination drug therapy approaches. We also developed a β-cyclodextrin based nanoparticle with fast cell uptake and successful delivery in vitro. Lastly, we created liposome coated calcium phosphate-citrate nanoparticles that proved to be highly selective towards cancer cells and showed strong anti-cancer effects both in vitro and in vivo.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 540 Chemie
• Fakultäten: Fakultät für Chemie und Pharmazie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 1. Dezember 2020
• 1. Bericht­erstatter:in: Engelke, Hanna