Lanthanum-mediated Biomimetic Aminoacylation

Methods are being developed to produce “designer proteins” from unnatural amino acids that are added into specific locations by the ribosome using an altered mRNA. To date, over seventy unnatural amino acids have been incorporated at specific sites in proteins by in vitro biosynthetic methods using chemically acylated-tRNAs and in vivo protein mutagenesis based on orthogonal tRNA/aminoacyl-tRNA synthetase pairs. Lanthanum-mediated aminoacylation of cis-diols provides a general and selective method for the one-step preparation of aminoacyl-tRNA. The nature of this biomimetic process was studied for the reaction of ribonucleosides and nucleotides with N-t-Boc-protected aminoacyl ethyl phosphates. Successful aminoacylation was also achieved with unprotected aminoacyl ethyl phosphates. This method was extended for the aminoacylation of tRNA and analyzed by reversed-phased HPLC and MALDI-MS. These results will provide an insight to the ultimate goal of lanthanum-mediated direct acylation of tRNA and its applications in in vitro site-specific incorporation of unnatural amino acids.MAS

Development of Gold Nanoparticle-based Nanoformulations for Cancer Radiotherapy

One of the major clinical challenges in radiotherapy (RT) is dose-limiting toxicity to surrounding normal tissues. Gold nanoparticles (AuNPs) have emerged as a promising approach to overcoming this challenge by combining the radiation dose enhancement effects of Au with the unique features of nanoparticles that enable tumour-selective delivery of AuNPs. Theoretical and experimental studies have demonstrated that AuNPs have the potential to improve radiotherapeutic efficacy. These improvements are the result of physical enhancement of the local radiation dose, and/or sensitization of cells via biological and chemical pathways. To further enhance the radiosensitization effects of AuNPs, this thesis aims to design and develop AuNP-based nano-formulations that (1) combine AuNPs with an agent with anti-cancer properties (pentamidine or cisplatin), or (2) target the nucleus for physical dose enhancement, and evaluate their in vitro radiation enhancement effects. Overall, this work demonstrates the promising potential of the newly developed nano-formulations to improve the radiation enhancement effects of AuNPs. The combination of AuNPs and pentamidine demonstrated enhanced radiosensitization effects relative to AuNPs alone by promoting cellular uptake and via inhibition of post-IR DNA repair. As well, cisplatin prodrug-conjugated AuNPs combined the AuNP-induced production of reactive oxygen species with persistent DNA damage imparted by cisplatin to result in superior radiosensitization in 2D monolayers and growth inhibition in 3D multicellular tumour spheroids. Finally, nuclear-targeted Au-liposomes formed by encapsulation of AuNPs in pH-sensitive liposomes showed significant radiation enhancement effects whereas no significant effects were observed with untargeted Au-liposome, demonstrating the role of nuclear targeting in physical dose enhancement. Based on these findings, future research is warranted to evaluate the in vivo efficacy and safety of these nano-formulations, and to optimize the nuclear-targeted Au-liposomes to fully elucidate the impact of nuclear localization on physical dose enhancement.Ph.D.2020-07-10 00:00:0

Functionalization of Cellulose Nanocrystals with PEG-Metal-Chelating Block Copolymers via Controlled Conjugation in Aqueous Media

Elongated nanoparticles have recently been shown to have distinct advantages over their spherical counterparts in drug delivery applications. Cellulose nanocrystals (CNCs) have rodlike shapes in nature and have demonstrated biocompatibility in a variety of mammalian cell lines. In this report, CNCs are put forward as a modular platform for the production of multifunctional rod-shaped nanoparticles for cancer imaging and therapy. For the first time, PEGylated metal-chelating polymers containing diethylenetriaminepentaacetic acid (DTPA) (i.e., mPEG-PGlu­(DPTA)₁₈-HyNic and PEG-PGlu­(DPTA)₂₅-HyNic) are conjugated to CNCs to enable the chelation of radionuclides for diagnostic and therapeutic applications. The entire conjugation is based on UV/vis-quantifiable bis-aryl hydrazone-bond formation, which allows direct quantification of the polymers grafted onto the CNCs. Moreover, it has been shown that the mean number of polymers grafted per CNC could be controlled. The CNCs are also fluorescently labeled with rhodamine and Alexa Fluor 488 by embedding the probes in the polymer corona. Preliminary evaluation in a human ovarian cancer cell line (HEYA8) demonstrated that these CNCs are nontoxic and their penetration properties can be readily assessed in multicellular tumor spheroids (MCTSs) by optical imaging. These findings provide support for biomedical applications of CNCs, and further in vitro and in vivo studies are warranted to evaluate their potential as imaging and therapeutic agents for cancer treatment