(64e) Novel Nanoparticle-Mediated Treatment for Fatal Neurodegenerative Diseases

Delivery of therapeutics to the brain through noninvasive administration is an impossible task due to the presence of the blood-brain barrier (BBB). The BBB serves as the first level of defense for the brain, selectively permitting the passage of necessary ions and molecules while preventing the transport of 98% of small molecule therapeutics. Patients with lysosomal storage disorders, which occur when a patient is not producing one or more necessary lysosomal enzymes, require central nervous system treatment. Lack of enzymatic activity causes buildup of storage materials in neural cells, leading to loss of body control and premature death. Specifically, in GM1 gangliosidosis, patients are missing β-galactosidase. GM1 gangliosidosis is fatal in infancy, with no clinically available treatment and no hope for parents of diagnosed children. Currently explored treatment methods for GM1 gangliosidosis involve cranial injections of adeno-associated viral vectors in various neural locations in a feline model. Although success has been demonstrated, intravenous (IV) delivery of the missing enzyme would be ideal. We are designing and characterizing the first nanoparticle-mediated treatment of GM1 gangliosidosis through the use of self-assembled polymersomes with high physiological stability and tunable release for IV delivery. When coupled with an endothelial cell receptor, delivery through the BBB and into the lysosome of neural cells will occur, effectively treating patients without invasive surgery.

Polyethylene glycol-b-poly(lactic acid) (PEGPLA) copolymer has been proven to self-assemble, forming vesicle structures using both dimethyl sulfoxide (DMSO) and water. Transmission electron microscopy (TEM) images confirmed vesicle formation. Molecules to be encapsulated were dissolved in stirring water prior to injection of DMSO and PEGPLA. Particle size distributions (PSDs) were determined using dynamic light scattering (DLS). Self-assembly was stopped by liquid nitrogen prior to lyophilization, with mannitol and inulin explored as novel lyoprotectants. Studies indicated that incorporating 2 wt%/v mannitol during formation led to PSDs 54 ± 0.8% smaller after lyophilization. Because of this and mannitol’s ability to disrupt the BBB, 2 wt%/v mannitol was used to aid in the formation of brain-deliverable polymersomes encapsulated with Alexa Fluor 488.

Alexa Fluor 488 was loaded into lyophilized polymersomes, which were placed in buffers of pH 7.4, 4.8, and 1 to monitor release. Amine-reactive homobifunctional PEG was introduced during formation to mitigate the attachment of CF 350 Amine, a blue fluorescent ligand, to the polymersome surface. Attachment was confirmed with fluorescent microscopy, XPS, and DLS techniques and monitored with absorption spectroscopy of NHS leaving groups. DLS data indicates that polymersomes loaded with Alexa Fluor 488 in water had diameters of 191 ± 64.7 nm, with no statistical difference in size compared to control. Solvent, block copolymer concentration, injection speed, and needle gauge were varied. At PEGPLA concentrations of 2, 20, and 50 µmol/mL, polymersomes formed at diameters of 139 ± 27.2 nm, 382 ± 21.5 nm, and 1282 ± 663 nm respectively. 2 µmol/mL PEGPLA led to 74% of polymersomes with a diameter less than 200 nm, the goal size for potential delivery through the BBB using receptor mediated transcytosis. Because of this, all loading, release, and attachment studies were done using polymersomes formed with a concentration of 2 umol/mL PEGPLA. 44% of Alexa Fluor 488 added to lyophilized polymersomes was loaded. When placed in pH 7.4 buffer, 37% of the loaded mass of Alexa Fluor 488 was released in 12 hours, compared to 69% in pH 4.8 buffer, indicating increased drug release expected in the lysosome.

Attachment of CF 350 was shown using fluorescence microscopy and increasing concentration of NHS leaving groups after the formation of an amide bond in only 5 minutes, indicating attachment. DLS data also indicated a shift in PSD upon ligand attachment using a mass of 1 mg NHS-PEG(2000)-NHS during polymersome formation. Upon ligand attachment, the overall average polymersome hydrodynamic diameter increased from 138.9 ± 1.58 nm to 170.3 ± 1.50 nm. The amount of polymersomes formed with sizes 100 nm or less decreased from 15.4% to 4.04%, while the amount of polymersomes formed with sizes between 100 and 200 nm increased from 58.8% to 72.4%. XPS data confirmed the presence of nitrogen groups on the surface of self-assembled polymersomes prior to washing.

This work highlights tremendous control over size formed, release location, and ligand attachment possible when utilizing PEGPLA polymersomes as an IV drug delivery vehicle. Varying PEGPLA concentration had the largest impact on polymersome size, with control of this parameter limiting expensive and necessary separation techniques currently used with drug delivery nanotechnologies. Attachment of a ligand with an available amine group proves the capability of polymersomes with amine-functionalized surface groups to bind targeting ligands appropriately. Results are promising towards the goal of creating the first clinical treatment for GM1 gangliosidosis, a disorder impacting the central nervous system, using a combination of enzyme replacement therapy and nanotechnology methods to cross the BBB.