Poster J6 - Polyelectrolyte Complex Micelles for the Targeted Delivery of Aptamers to Inhibit TXNDC5 for the Treatment of Atherosclerosis

Friday, October 25, 2024

3:30 PM - 4:30 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Taylor Elliott (she/her/hers)

Undergraduate Student
University of Chicago
New York, New York, United States

Co-Author(s)

HK

Ha Ram Kim

Graduate Student
University of Chicago, United States
KY

Kai-Chien Yang

Assistant Professor
National Taiwan University, United States

Primary Investigator(s)

MT

Matthew V. Tirrell

D. Gale Johnson Distinguished Service Professor Emeritus
UChicago Pritzker School of Molecular Engineering, United States

Co-Author(s)

YF

Yun Fang

Professor
University of Chicago Department of Medicine, United States

Introduction: Atherosclerosis is the most common form of cardiovascular disease and a leading cause of human mortality. Current therapies for atherosclerosis are suboptimal, focusing on controlling systemic risk factors like hypercholesterolemia and hypertension. Atherosclerosis has been found to develop in regions of vasculature curvature where disturbed blood flow patterns induce low sheer stress levels on the endothelial cells lining the arteries. This activates mechanosensing pathways that trigger localized inflammation and leaky vasculature. Therefore there is a need for precision delivery of therapeutics to these regions of vasculature curvature where inflammation and atherosclerotic plaques preferentially form. Here, we devise a multifunctional nanomedicine system that efficiently targets inflamed vascular endothelium for nucleic acid drug delivery. Our system delivers aptamers, a class of synthetic single-stranded DNA with unique 3D structures for enhanced target affinity and specificity. We utilize the binding specificity and efficacy of aptamers to inhibit TXNDC5, a key endoplasmic reticulum protein in the inflammatory mechanosensing pathway. Aptamers are widely underexplored as therapeutics, and nanocarriers have been shown to be essential in protecting small molecule drugs from degradation or rapid clearance in biological systems. Here, we demonstrate that the integration of aptamers and nanomaterials can effectively and specifically treat atherosclerosis.

Materials and

Methods: We have engineered polymeric micelle nanoparticles, or polyelectrolyte complex micelles (PCMs). PCMs form spontaneously when two oppositely charged polyelectrolytes interact under aqueous conditions. In our experiments, we take advantage of the fact that nucleic acids are negatively charged and can therefore be complexed with a cationic polymer to form a PCM. We chemically conjugate the cationic polymer to a neutral polymer chain to protect the PCM from degradation followed by a peptide that targets VCAM-1 – a membrane ligand specifically expressed on endothelial cells in disturbed flow regions. These modifications make PCMs an effective delivery platform for aptamers engineered to inhibit the activity of TXNDC5. We have validated the stability, structure, and encapsulation efficiency of the PCMs through dynamic light scattering (DLS), TEM, and a nucleic acid-staining gel assay, respectively. We subsequently transfected the PCMs into endothelial cells to determine uptake and therapeutic effect in vitro. We mimic biological conditions by using a cone and plate flow system to expose endothelial cells to different types of flow which mirrors the blood patterns in human aortic arteries. PCM uptake into the endothelial cells is analyzed qualitatively with immunofluorescence imaging using fluorescently labeled PCMs and quantitatively by extracting DNA and performing qPCR to measure the relative concentration of delivered aptamers. We assess the therapeutic effect at both the transcriptional and translational levels using RNA extraction / RT-qPCR and western blotting techniques to examine differential expression levels in key genes and proteins downstream of TXNDC5.

Results, Conclusions, and Discussions: We have successfully formulated aptamer-encapsulating PCMs and treated them in endothelial cells. DLS and TEM show that the PCMs are stable, monodisperse, and of a sufficiently small diameter. We have validated that naked aptamers have a therapeutic effect but the PCM delivery system allows the effective concentration of the therapeutic to be reduced. We have shown via qPCR that PCM-encapsulated aptamers possess superior uptake into endothelial cells, particularly when compared to a non-targeted control PCM. Immunofluorescence imaging additionally shows the effective release of the aptamer from the PCM once inside the cell – and their relative position suggests the aptamers are able to bind to TXNDC5. Initial experiments have been conducted under static conditions which, as expected, show little differential expression levels of our genes of interest. We are now conducting experiments with the flow device to determine the therapeutic effect of PCMs under flow conditions. Previous research shows the device effectively produces differential expression of these genes under different flow patterns. We have replicated this with qPCR and western blotting. Ongoing studies are focused on assessing the effect of naked versus PCM-encapsulated aptamers utilizing the flow system.

These findings demonstrate the ability of aptamers to be effective therapeutics by harnessing their nucleic acid structure and unique targeted binding properties. PCMs are targeted to regions of inflamed vasculature and enhance aptamer uptake and effectiveness in endothelial cells, which enables lower effective doses. Current work will further assess the ability of aptamer-induced TXNDC5 inhibition to affect downstream atherogenic and atheroprotective proteins.

Prior research has elucidated the mechanosensing mechanisms by which endothelial cells initiate inflammatory responses to disturbed blood flow, including the role of the protein TXNDC5. Additionally, it has been demonstrated that TXNDC5 knockout reduces atherosclerotic plaques. However, this approach utilized PEI nanoparticles, which were limited by their inability to target the specific athero-prone sections of the vasculature. As targeted nanocarriers, PCMs provide a solution, as they have the capacity for region-specific TXNDC5 inhibition. Aptamer PCMs additionally yield novel insights into the potential of aptamers as effective therapeutics and their uses in nucleic acid drug delivery.

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