W24 - Targeted CNS Delivery of β-Galactosidase via ApoE-Functionalized Polymersomes in a Murine Model

Saturday, October 11, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

AD

Ashlyn Denny

Student Researcher
Clemson University, United States

Co-Author(s)

Noah Arnold

Phd candidate
Clemson University Chemical and Biomolecular Engineering
Seneca, South Carolina, United States

Primary Investigator(s)

Jessica Larsen

Associate Professor
Clemson University, South Carolina, United States

Co-Author(s)

Amanda L. Gross, MS, PhD

Assistant Research Professor
Auburn University Veterinarian Medicine School
Auburn University, Alabama, United States

Introduction: : Lysosomal storage disorders (LSDs) are genetic diseases that result in decreased or limited functioning enzyme within the lysosome of a person’s cells. The standard of care for these diseases is enzyme replacement therapy (ERT) where healthy enzymes are administered to patients regularly. However, ERT has limitations in neuropathic LSDs. GM1 gangliosidosis is one such disease that is currently untreatable by ERT due to the brain involvement and the presence of the blood brain barrier (BBB). The BBB prevents the passage of > 98% of small molecule drugs and is even more restrictive for large biomolecules like enzymes. Healthy enzymes administered through ERT are excluded from passive diffusion through the BBB. In GM1, the missing enzyme is β-galactosidase (β-gal), which results in the buildup of GM1 gangliosides, which are lipids that continue to accumulate in the lysosome and result in cell death. In our lab, we have developed poly (ethylene glycol)-b-poly (lactic acid) (PEG-b-PLA) polymersomes that can encapsulate β-gal and be tagged with Apolipoprotein E (ApoE). This cholesterol ligand binds to low density lipoprotein receptors (LDLR) and induces BBB transcytosis. In GM1, where neuroinflammation is prevalent, these receptors are upregulated on the BBB endothelium. We hypothesize that ApoE tagged β-gal loaded polymersomes have the potential to induce active transport across the BBB resulting in efficient delivery of β-gal to neural lysosomes. To continue our studies, we have begun using a GM1-affected mouse model to help us get more information on the biodistribution, pharmacokinetics, and overall therapeutic efficacy of the polymersome system.

Materials and

Methods: : PEG-b-PLA polymersomes were used as the primary vehicle for delivering β-gal to GM1-affected mice. To create these nanoparticles, the block co-polymer PEG-b-PLA was dissolved in DMSO (dimethyl sulfoxide) with a PEG-N-hydroxysuccinimide linker for ApoE attachment and then injected into an aqueous solution. To confirm successful tagging, DLS (dynamic light scattering) registered superficial and width changes on polymersomes. A Lowry assay was used to confirm changes in protein content of polymersome samples indicating the presence of ApoE. Using Cy5 (Cyanine 5) and β-gal, which was fluorescently tagged, encapsulation efficiency (EE) of the polymersomes was determined. For the GM1 mouse models, heterozygote β-gal knockout mice were bred to produce homozygous mutant pups with minimal ability to produce β-gal. The mice in this project were genotyped using Polymerase Chain Reaction (PCR), where the DNA was obtained from ear punches and tissue digestion assays. GM1-affected mice were subjected to a tail vein injection of polymersome-encapsulated β-gal to increase their deficient enzyme levels. This was verified with enzyme assays for β-gal and use of the X-gal substrate to qualitatively analyze its spatiotemporal distribution. Biodistribution of the polymersomes was analyzed by intravenous tail vein injection of Cy5-loaded polymersomes. Using IVIS (In Vitro Imaging System) and blood draws at 15 minutes, 1 hour, 4 hours, and 24 hours, plasma was collected for UV-vis analysis to create a profile for neural distribution of polymersomes and compare untagged and ApoE-tagged performance. The mice were sacrificed 24 hours post-injection, and immunofluorescence, NeuN staining, and IVIS of major organs were performed.

Results, Conclusions, and Discussions:: The polydispersity index for both our tagged and untagged polymersomes indicated that the polymersomes were approximately monodispersed. After ApoE tagging, the diameter increased while the surface charge became less negative (Figure 1A). The maintained negative charge was expected due to our system being a PEGylated structure. Cy5 dye was encapsulated at 1.7E-5 ± 6.8E-7 mg/mg of polymersome for pharmacokinetic analysis in vivo (Figure 1A). Fluorescently tagged β-gal was also encapsulated at 0.01 ± 0.008 mg β-gal/mg polymersome for therapeutic delivery in vivo (Figure 1A). After confirming via PCR, mutants were injected with ApoE-tagged and untagged polymersomes encapsulated with the Cy5 dye to determine biodistribution and pharmacokinetic efficacy. ApoE tagging enabled detectable Cy5 fluorescence in the brains of teil vein injected mice (Figure 1B). The untagged polymersomes presented much less Cy5 in the brain (Figure 1B). Blood serum profiling showed that mice injected with ApoE tagged polymersomes exhibit less blood serum fluorescence. Mice that were injected with β-gal loaded polymersomes were analyzed for therapeutic effect. Our NanoDIVERT approach resulted in an increase in β-gal enzyme activity in all areas of mutant brains compared to untreated mutant brains and spinal cord (Figure 1C). The β-gal enzyme activity in all neural sections was raised to wild type levels (Figure 1C). Further, X-gal distribution indicated a notable increase in β-gal distribution throughout various brain regions. Blood serum enzyme assays also confirm that specific activity of β-gal is increased over the first 24 hours post-injection to that of wild type levels. We hypothesize that, by 24 hours, β-gal either resides in tissue or is cleared by the mouse’s waste mechanisms.
PEG-b-PLA polymersomes tagged with ApoE demonstrate distinct potential in neural delivery of missing enzymes in vivo. To prove its effectiveness, ApoE-tagged, polymersome-mediated delivery of Cy5 dye to the brains of GM1-affected mice was performed and showed increased fluorescence in brains compared to untagged controls. Furthermore, examination of therapeutic efficacy with β-gal loaded polymersomes led to higher levels of β-gal activity in the brain compared to untreated mutant mice.

Acknowledgements and/or References (Optional): :