Title: “A Lipid-Coated Calcium Phosphate Nanoparticle for Delivery of siRNA to the Brain”
Tyrone Porter, PhD – ME (Advisor)
Allison Dennis, PhD – BME (Chair)
Mary Dunlop, PhD – BME
Mark Grinstaff, PhD – BME
Neil Ganem, PhD – Pharmacology & Medicine
Glioblastoma multiforme (GBM) is the most common and most lethal primary brain cancer, killing approximately 16,000 patients in the US each year. Despite innovations in drugs and targeted therapies, the mortality rate for primary brain cancer has remained unchanged for over 2 decades, largely due to the impermeability of the blood brain barrier (BBB) and the poor response of GBM to most chemotherapeutics. Small interfering RNA (siRNA) has demonstrated clinical success in modulating the expression of oncogenic genes, and is emerging as an attractive therapeutic against primary brain cancer. Delivery of siRNA, particularly to brain cancer, is challenging due to enzymatic degradation of the oligonucleotide and the presence of the blood-brain barrier, which tightly regulates transport in and out of the brain. These challenges can be addressed by using nanoparticles which are gaining prominence in cancer therapy for their capacity to package a broad spectrum of drugs, extend circulation times, and modify the surface with a variety of targeting moieties. This thesis introduces a lipid-coated calcium phosphate (LCaP) nanoparticle that merges RNA transfection technology with nanomedicine.
LCaPs were synthesized and the physiochemical properties were characterized. By altering the calcium to phosphate ratio, LCaP features such as size, surface charge, and siRNA loading could be tailored. LCaPs were then used to deliver siRNA in vitro and demonstrated successful and sustained knockdown of a reporter gene stably transfected in a cancer cell line. In addition, siRNA against the epidermal growth factor receptor variant III (EGFRvIII), a mutation specific to GBM, was delivered via LCaPs and demonstrated dose-dependent protein knockdown and a subsequent reduction in cell viability, indicating the potential of EGFRvIII as a therapeutic target. In order to improve BBB penetration, LCaPs were decorated with the targeting peptide, Angiopep-2, which binds to the low density lipid receptor related protein 1 that facilitates molecular transport across the BBB. BBB penetration was evaluated as a function of Angiopep-2 density via a BBB model incorporating human-derived induced pluripotent cells differentiated into brain endothelial cells along with immortalized human cancer cells. The BBB platform enabled more physiologically relevant evaluation of LCaP penetration and could be used as a screening platform for other nanoparticles or GBM therapeutics.