Porphyrinic-nanoplatforms: controlled intracellular generation of reactive oxygen species in human mesenchymal stem cells

Reactive Oxygen Species (ROS) are known as important intracellular signaling molecules. These are also well known for their role in oxidative stress and cellular damage, leading to their involvement in several pathologies. Despite the widespread postulation of ROS mechanisms, little is actually known about the immediate response in living cells to the generation of these highly reactive compounds. The development of nanoplatforms incorporating photosensitizers would permit the generation of ROS at specific sub-cellular locations and determine the in situ cellular response. The work presented in this thesis describes the development of porphyrinic nanoplatforms for the controlled generation of ROS and investigates their impact on the surface marker expression of human Mesenchymal Stem Cells (hMSCs). Surface tailoring of polyacrylamide nanoparticles with alkyne and amine functionalities were exploited to achieve stable reactive chemical groups for further conjugation. Nanoplatforms surface was also modulated with trimethylammonium functionalities for the development of nanosystems for sub-cellular targeting and facilitated uptake. Physicochemical characterization of alkyne and alkyne/trimethylammonium functionalised constructs showed sizes in the range of 40 nm with a positive surface charge. Alkyne/trimethylammonium nanosystemswere found to be stable over long periods of time, whilst amino functionalized nanosystems were found to be prone to aggregation. Mechanisms of conjugation were exploited to create covalent linkage of porphyrinic photosensitizers to mono and dually functionalised constructs. Conjugation through "click chemistry" allowed stable coupling with alkyne and alkyne/trimethylammonium nanosystems. To overcome aggregation associated with amino functionalised nanoplatforms, porphyrin conjugated monomers were synthesised which resulted in stable polyacrylamide nanoparticles. The developed conjugated nanosystems showed final sizes in the range 40-100 nm, while conjugates with surface charges greater than + 20 mV have led to sizes higher than 100 nm. The effect of surface charge on cellular delivery was investigated and nanosystems with a surface charge in the range + 13 mV to + 18 mV proved optimal in terms of cell delivery and viability. It was found that highly charged nanosystems (above + 20 mV) remained attached to the cellular membrane and had a negative effect on cell viability. In addition, intracellular co-localisation studies showed preferential mitochondrial targeting of the delivered nanosystems. Production of ROS in nanoparticle treated hMSCs was achieved by exposure to light at wavelength of 575 nm. For porphyrin conjugated nanosystems a single light dosage resulted in a "blast zone" in the irradiated area where significant production of hydrogen peroxide was also observed. Titration of the amount of porphyrin conjugated at the surface of nanoparticles resulted in systems with different levels of ROS production. Control of ROS generation allowed development of a nanoplatform that was used to expose cells to repeated exposure of ROS over a time period of 100 minutes. The surface marker expression of hMSCs treated with porphyrin conjugated nanosystems was investigated. In the absence of light the surface marker expression of hMSCs was maintained, positive for CD29 and CD105 and negative for CD34 and CD45. Increased generation ROS in hMSCs did not produce alterations in the surface marker expression of cells, and over two generations of treated cells (light and nanoparticles) no changes were detected in surface marker expression. The developed nanoplatforms have the potential to be applied as a tool to investigate the cellular mechanisms and metabolism associated with different levels of oxidative stress. In addition, these nanosystems could also represent an innovative platform for theranostic applications (drug delivery/diagnostic).