Date on Master's Thesis/Doctoral Dissertation

5-2023

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Interdisciplinary and Graduate Studies

Degree Program

Interdisciplinary Studies (Individualized Degree), PhD

Committee Chair

O'Toole, Martin

Committee Co-Chair (if applicable)

Gerber, Erin

Committee Member

Gerber, Erin

Committee Member

Klinge, Carolyn

Committee Member

Kopecheck, Jonathan

Committee Member

Soucy, Patricia

Author's Keywords

Nanoparticles; oligonucleotides; glioblastoma; RNA interference

Abstract

Glioblastoma (GBM) brain tumors are highly aggressive gliomas due to genetic and cellular heterogeneity. Current GBM treatment consists of surgical resection of the tumor combined with radio- or chemo-therapies. While these treatments have increased the life expectancy for GBM patients up to 20 months, they have had little effect on the 5-year survival rate. The complex cellular and genetic composition of the tumor makes current treatments less effective long term. One approach to developing more effective GBM treatments is to customize nanoparticle-based drug delivery systems that can directly target the aberrant gene expression patterns within a particular GBM tumor. Delivery systems that include oligonucleotide therapies are ideal for this approach due to their ease of synthesis and ability to tailor the oligonucleotide base sequence to allow the targeting of specific gene sequences and proteins. Two therapeutically relevant classes of oligonucleotides, DNA aptamers (e.g., AS1411) and RNA-interfering anti-sense microRNAs (anti-miRs), have exhibited anti-GBM properties. However, their use as standalone therapies is hindered by instability within in vitro and in vivo environments and the inability to cross some biological barriers. The conjugation of individual oligonucleotides to the surface of gold nanoparticles (GNPs) has been shown to help overcome these difficulties to make AS1411 and anti-miRs viable therapeutics for GBM; however, the conjugation of both oligonucleotides to GNPs has not been investigated. This dissertation presents a novel GNP therapy incorporating AS1411 and an anti-miR as a multi-faceted therapy against GBM. Anti-miR-21 (A21) targets miR-21, a microRNA implicated in the increased aggressiveness of GBM tumors. U-87 MG GBM cells treated with GNPs coated with AS1411 and the polymer poly (ethylene glycol) (PEG/AS1411 GNPs) displayed decreased cellular metabolic activity and growth with altered cell morphology. The GNPs were optimized based on the PEG to AS1411 ratio to maximize these bioactive effects against GBM cells. An optimal ratio of 1:3 PEG to AS1411 conjugated GNPs exhibited ~ 72% inhibition of cellular metabolic activity, reduced growth by 75%, and profoundly affected cellular morphology. The effects of AS1411 were enhanced upon conjugation to GNPs. Additionally, using a base-substituted analog of AS1411 showed that the effects on U-87 MG cells were specific to the sequence of AS1411. In addition, this dissertation details the synthesis of AS1411 GNPs coated with A21 (PEG/AS1411/A21 GNPs) and investigates the benefit of A21 addition to PEG/AS1411 GNPs. Treatment of U-87 MG cells with PEG/AS1411/A21 GNPs retained the effects seen from PEG/AS1411 GNPs and significantly lowered the motility rate of U-87 MG cells. PEG/AS1411/A21 GNPs showed additional effects on gene expression patterns of U-87 MG cells due to A21 addition. PEG/AS1411/A21 GNPs reduced miR-21 expression 3-fold in U-87 MG cells. The ability of PEG/AS1411/A21 GNPs to influence the expression of miR-21-associated proteins PTEN and STAT3 within U-87 MG cells was also investigated. The in vivo performance of PEG/AS1411 GNPs, with or without A21, was investigated within a mouse orthotopic xenograft model of GBM. Both GNP types were shown to cross the blood-brain barrier and influence tumor progression. Mice treated with PEG/AS1411 GNPs ultimately survived longer (47 days post-tumor implantation) than untreated mice (37.5 days)

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