Document Type

Thesis

Degree Name

Master of Science (MSc)

Department

Health Science

Faculty/School

Faculty of Science

First Advisor

Sarah Poynter

Advisor Role

Primary Investigator

Abstract

Background: Long dsRNA is a potent inducer of innate immune responses through the activation of pattern recognition receptors. Synthetic dsRNA molecules, including polyinosinic-polycytidylic (PIC), have demonstrated considerable therapeutic potential as broad spectrum immunostimulatory molecules. The effectiveness of dsRNA treatments is by limited its relatively small size, negative charge, and susceptibility to nuclease. Nanoparticle-based delivery systems offer a promising strategy to improve cellular delivery while enhancing immune activation. Nanodendrix (NDX) is a naturally derived, biodegradable nanoparticle that has emerged as a potential carrier capable of facilitating dsRNA delivery. This study investigated the ability of NDX to enhance the delivery and immunostimulatory activity of PIC in murine RAW 264.7 and human THP-1 macrophage cells.

Methods: Cellular uptake of fluorescently labelled PIC was assessed using fluorescence microscopy, while metabolic and proliferative activity was evaluated using alamarBlue and DAPI-mediated cell counting assays respectively. Innate immune activation was characterized by quantifying transcript levels of genes in the interferon and pro-inflammatory pathways using qRT-PCR, ROS production with DCFDA fluorescence, and assessing IRF reporter activity in RAW 264.7 reporter cells. Responses were compared between serum-free (basal) conditions and complete culture media (10% fetal bovine serum) to determine the influence of serum on nanoparticle-mediated delivery.

Results: NDX substantially enhanced cellular association of PIC in both macrophage cell lines under basal media conditions. In contrast, uptake of both treatments was markedly reduced in complete media, demonstrating a significant influence of serum on nanoparticle-mediated delivery. Gene expression analysis revealed cell dependent immune responses. In RAW 264.7 cells, PIC and PIC-NDX induced robust expression of the innate genes for CXCL10 and ISG15. In THP-1 macrophages, NDX-mediated delivery markedly increased IFIT1 and CXCL10 expression under basal conditions compared to PIC alone, whereas responses were largely abolished in complete media. ROS activity was enhanced with PIC-NDX treatment with specific concentrations in full media. IRF-activation was present with PIC and PIC-NDX stimulation, however NDX presence significantly lowered the response.

Conclusions: Collectively, these findings demonstrate that NDX effectively enhances macrophage delivery of long dsRNA under optimized culture conditions and modulates downstream innate immune responses in a cell-type dependent manner. This work advances our understanding of nanoparticle-mediated dsRNA delivery in professional immune cells and supports the continued development of nanoparticle vehicles for immunotherapeutic applications as antivirals, vaccine adjuvants, and cancer immunotherapies.

Convocation Year

2026

Convocation Season

Fall

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Available for download on Wednesday, August 30, 2028

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