W18 - PEG-b-PLA Polymersomes Encapsulate siRNA Towards the Treatment of Amyotrophic Lateral Sclerosis (ALS)

Saturday, October 11, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

SN

Sierra Niskanen

Undergraduate student
Clemson University, United States

Primary Investigator(s)

Jessica Larsen

Associate Professor
Clemson University, South Carolina, United States

Co-Author(s)

Kati Lorenz

Graduate student
Clemson University
Taylors, South Carolina, United States

Introduction: : Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease that presents as muscle atrophy and weakness. A key hallmark pathology of this disease is the aggregation of a protein called TDP-43. This protein is typically found in the nuclei of cells, but in almost all ALS patients, it aggregates in stress granules in the cytoplasm. siRNA is a short double-stranded RNA molecule that plays a role in RNA interference. RNA interference can be used as a therapeutic for upregulated proteins. There have also been studies on the effect of RNA interference on stress granule formation. One of these studies demonstrated that a connection exists between RNA-induced silencing complex (RISC), and stress granules, suggesting that stress granule formation was suppressed because of RNA interference. In this project, we are developing a model of ALS using SH-SY5Y cells and exploring the delivery of siRNA towards TDP-43 using polymersomes. Polymersomes are used to deliver siRNA because polymersomes are highly stable in biological environments, can encapsulate hydrophilic drugs like siRNA into their core, and can be engineered to target cells that need treatment to decrease off-target effects.

Materials and

Methods: : The first aim of this study was to optimize the encapsulation and delivery of siRNA in polymersomes. The polymersomes were formed using polyethylene glycol (PEG)-b-polylactic acid (PLA) through a solvent injection method, then frozen and lyophilized. Polymersomes were characterized for their size, polydispersity index and zeta potential using dynamic light scattering (DLS). SiGLO was encapsulated in polymersomes as a model siRNA with encapsulation efficiency and loaded content characterized via dialysis. SH-SY5Y cells were treated at various concentrations with PEG-b-PLA polymersomes loaded with siGLO. Flow cytometry was used to determine the uptake of polymersomes. For the second aim, an ALS model in SH-SY5Y cells was developed. The cells were treated with various concentrations of palbociclib to arrest the cells in G1 to better mimic neuronal ALS cells, followed by treatment with sodium arsenite to induce oxidative stress and form stress granules. To confirm stress granule formation, immunofluorescent staining was performed for TDP-43 and TIA-1. Once a model was established, stressed cells were treated with siRNA specific to the TDP-43 mRNA and additional immunostaining was performed to compare with the ALS stressed model.

Results, Conclusions, and Discussions:: The first aim of the study resulted in optimized encapsulation of siRNA in PEG-b-PLA polymersomes. The polymersomes have an average diameter of 117.8 ± 13.5 nm, which can penetrate cells and deliver a large payload of drugs. The average zeta potential of the polymersomes was -21.8 ± 4.97 mV, displaying a stable surface charge. The polydispersity index was calculated to be 0.058 ± 0.018, which indicates the monodispersity of the polymersomes. Encapsulation efficiency was also analyzed for two green, fluorescent controls: fluorescein and siGLO. The fluorescein was encapsulated at an efficiency of 70.1 ± 8.04% whereas the siGLO was encapsulated at a rate of 98.5 ± 1.26%. Importantly, siGLO was loaded at a content of 1.30E-5 ± 1.63E-7 mg siGLO/mg polymersomes. Flow cytometry was used to determine if the assimilation of the loaded polymersomes was efficient into the cells (Figure 1). 100 nM of siGLO was delivered to ~20% of treated cells, while a slightly higher dose of 125 nM was delivered to ~65% of treated cells, indicating efficient delivery via polymersomes.
The second aim was to create an ALS model of TDP-43 aggregates in SH-SY5Y cells. The cells were arrested in G1, then stressed using sodium arsenite. Immunofluorescent staining was performed before and after the cells were stressed. Anti-TDP43 and anti-TIA1 were used as the primary antibody tags with AF 488 and Texas Red as the secondaries, respectively. Before the cells were stressed, it was found that TDP-43 was mostly concentrated in the nucleus of the cells, and TIA-1 was mostly not present due to this protein being associated with stress granules. After stressing the cells, the results showed TDP-43 increased in the cytoplasm as well as associating with TIA-1, which had higher levels. Future works aim to treat the cellular model being developed by treating the stressed cells with siRNA loaded polymersomes that would target the TDP-43 mRNA.

Acknowledgements and/or References (Optional): :