(732h) Enzyme Replacement Therapy Extended to the Brain through Nano-Polymersomes

Approximately 1 in 5,000 to 8,000 children are born annually with a lysosomal storage disease (LSD). Current treatment of LSDs, called enzyme replacement therapy (ERT), involves weekly to monthly intravenous(IV) infusions of missing endogenous enzymes. While ERT has proven effective in LSDs without central nervous system (CNS) involvement, the brain has remained untreatable due to the presence of the blood-brain barrier (BBB). The BBB is made up of endothelial cells with tight junctions, possessing a width between connections of 0.2 nm in size, which prevents passage of 98% of small molecule drugs, including enzymes, from the blood into the brain. Patients with neuropathic LSDs, which account for 50-70% of diseases in this category, present with severe CNS degeneration, ataxia, and premature death, with no treatment available on the market. The disease of focus, GM1 gangliosidosis, causes premature death between the ages of two and four with no hope available for parents of patients. Encapsulating enzyme into polymeric vesicles, called polymersomes, facilitates the transport of this missing enzyme into the brain through IV injections, effectively extending ERT therapy to the brain for the first time.

Polyethylene glycol-b-poly(lactic acid) (PEGPLA) has been proven to self-assemble into vesicles via both passive mixing and the solvent injection, using dimethyl sulfoxide (DMSO) and water. The mixing enzyme in GM1 gangliosidosis, β-galactosidase (βgal), is fluorescently tagged with reactive Alexa Fluor 488 at an efficiency of 13 dye molecules per mole of protein, with a finally protein concentration of 0.35 mg/mL attainable. Fluorescently tagged βgal is dissolved in stirring water prior to injection of DMSO and PEGPLA. Apolipoprotein E (ApoE) and CF350 attachment is facilitated through the introduction of homobifunctional PEG during injection. Polymersome diameter is characterized by dynamic light scattering (DLS). Self-assembly was stopped by freezing prior to lyophilization, with mannitol as a lyoprotectant. βgal loaded polymersomes were released in vitro in buffers of pH 7.4 and 4.8. Attachment was confirmed with DLS and Lowry assays. An immortalized feline fibroblast line, GM1/SV3, was used in in vitro studies to confirm particle uptake and treatment efficacy.

Previous studies indicated that incorporating 2 wt%/v mannitol during formation led to PSDs 54 ± 0.8% smaller after lyophilization. Therefore, 2 wt%/v mannitol was used to aid in the formation of brain-deliverable polymersomes encapsulated with βgal. PEGPLA polymersomes loaded βgal at 0.08 ± 0.03 mg βgal/mg polymersome with activity maintained at a value of 452 ± 82 a.u. DLS data indicates that polymersomes with βgal had diameters statistically similar to control. Release of βgal was doubled over 6 hours when loaded polymersomes are placed into citrate buffer (pH=4.1) when compared to tris buffer (pH=7.4), indicating increased enzyme release expected in the lysosome. First order kinetics of release were followed for three hours when βgal was released from polymersomes in acidic buffer prior to a plateau in mass released, indicating the potential for sustained long term release from polymeresomes. Activity of βgal was maintained after release. CF350 Amine was attached to polymersome surface, with an increase in diameter from 147.2 ± 24 nm to 210.5 ± 8.6 nm (Fig 1A). Fluorescence microscopy confirmed attachment (Fig 1C), with 79 ± 21% of polymersomes labeled with blue fluorescence. Furthermore, our carriers were capable of both encapsulating Alexa Fluor 488 and attaching CF350 at a high efficiency, with 87 ±12% of polymersomes presenting with both colors in flow cytometry. Attachment of ApoE was confirmed with an increase in polymersome diameter to 194.4 ± 3.5 nm and an increase in protein content from 0.06 ± 0.11 mg/mL to 1.03 ± 0.71 mg/mL. GM1SV3 cells, an immortalized cell line from felines with GM1 gangliosidosis, present low density lipoprotein receptors (LDLR) on the surface, as shown in confocal microscopy, indicating that they are a good model for ApoE mediated delivery through the BBB. ApoE increases the uptake of polymersomes into GM1/SV3 fibroblasts when compared to unlabeled control polymersomes.

This work highlights our ability to create polymersomes capable of protecting βgal in the blood stream, shown by limited release in a pH 7.4 environment, targeting the nanocarrier to the BBB, shown by the facilitated attachment of ApoE, and after transcytosis, releasing βgal in the lysosome of neural cells, shown by extended release in a pH 4.1 enviornment and cell studies in GM1SV3 cells. This combination of ERTand nanotechnology, demonstrating the capability of our carrier to transport enzymes to the brain while maintaining their activity, will create a paradigm shift in the treatment of CNS disease, providing treatment for currently untreatable and fatal diseases like GM1 gangliosidosis.