Investigating theranostic potentials of heteroatom-doped graphene quantum dots in targeting alpha-synuclein aggregation in Parkinson’s disease

Parkinson’s Disease is the second-most prevalent age-related neurodegenerative disorder globally. The abnormal aggregation of alpha-synuclein (ɑ-syn) is a prominent hypothesis explaining the degeneration of midbrain dopaminergic neurons. Currently, there is a need for advancement in diagnostic and therapeutic approaches targeting ɑ-syn aggregation, particularly in terms of dosing and selectivity of aggregation inhibitors and detection agents. Graphene quantum dots (GQDs) are biocompatible nanoparticles that have demonstrated potential as both therapeutic and diagnostic agents (theranostics) for ɑ-syn aggregation. Nevertheless, enhancements in their design are necessary to improve targeting of ɑ-syn aggregation and monitoring. This thesis aims to expand current understanding of GQDs’ theranostic potential against ɑ-syn aggregation, through a detailed study of anti-aggregation and optical detection capabilities of multiple heteroatom-doped GQDs (nitrogen, sulfur, phosphorous, and boron) through biochemical, and biophysical methods, and in in vitro models. In addition, a structure-activity-relationship study is performed to gain insights into the relationship between GQD design and anti-ɑ-syn aggregation efficacy. Firstly, nitrogen was selected as a heteroatom dopant to be incorporated into the structure of GQDs. Accordingly, different nitrogen-containing precursors were used in the synthesis of six different nitrogen-doped GQDs (N-GQDs) and characterized. One of the N-GQDs was conjugated on its surface with curcumin, a well-known polyphenol known to possess anti-aggregative effects against ɑ-syn, to explore synergistic inhibitory effects together with its conjugated N-GQD. Through high-throughput fluorescence-based assays involving the wild-type, A53T and A30P mutant variants of ɑ-syn, SDS-PAGE, and mass spectrometry, four N-GQDs (N-GQD-1, CUR@N-GQD, N-GQD-2, and N-GQD-6) were selected as candidates owing to the promise they showed in the initial experiments in inhibiting ɑ-syn aggregation. The hit N-GQDs were further validated through TEM studies, additional fluorescence disaggregation assays, cell viability, and in vitro immunofluorescence experiments to assess their potential in not only inhibiting ɑ-syn aggregation, but in effecting fibril disaggregation, biocompatibility, and in vitro efficacy in a mutant ɑ-syn-overexpressing SH-SY5Y line. While N-GQD-1 and CUR@N-GQD exhibited milder inhibitory effects on ɑ-syn aggregation, N-GQD-2 and N-GQD-6 were recognized as more effective inhibitors. Following the positive outcomes from several of the N-GQDs, GQDs doped with alternative heteroatoms, such as boron, phosphorous, sulfur, and nitrogen-sulfur, were synthesized and characterized. Utilizing assays similar to those used to shortlist hit N-GQDs, two GQDs – sulfur- and phosphorus-doped – were selected as they demonstrated the highest aggregation inhibition capacity. While the sulfur- and phosphorus-doped GQDs showed inhibitory potential in TEM studies and SDS-PAGE, their efficacies in reducing formation of large insoluble phosphorylated serine 129-positive aggregates in vitro were relatively weak. Nevertheless, the formation of smaller soluble fibrils was diminished. Subsequent SAR analyses further indicated that the effectiveness of GQDs in inhibiting or decelerating ɑ-syn aggregation was associated with the percentage of sp2 carbon content, as well as the sp2/sp3 ratios. To probe the diagnostic capabilities of theranostic GQDs, two N-GQDs which exhibited excellent optical properties both in cell-free as well as in vitro studies, were investigated. These N-GQDs, particularly the curcumin conjugated N-GQD, were revealed to both fluorescently detect ɑ-syn fibrils in cell-free systems, but also in vitro. CUR@N-GQD exhibited greater sensitivity as well as selectivity in detecting only fibrillar ɑ-syn, excluding monomers. Moreover, CUR@N-GQD showed selectivity for ɑ-syn over Aβ. Overall, this novel study explored the theranostic potential of a variety of GQDs in their ability to both inhibit, as well as detect ɑ-syn aggregation. This investigation paves the way for the potential usage of GQD candidates in further preclinical studies, to evaluate their efficacies in vivo. Moreover, it provides valuable insights into the proper design of GQDs to appropriately target ɑ-syn aggregation.