Undergraduate Researcher Clemson University, United States
Introduction: Lysosomal storage disorders (LSDs) are a category of genetic diseases that affect the lysosome’s ability to break down naturally occurring cell waste. One of these disorders, GM1 Gangliosidosis, is a rare, neurologically based LSD that causes a lack of the enzyme beta galactosidase, or β-gal, due to a mutation in the GLB1 gene. This eliminates the cell’s ability to digest cell waste, specifically GM1 gangliosides, in the neurons. Because of this, the disorder is extremely neurodegenerative, and all patients with the infantile form of the disorder don’t live past early childhood, hence the urgency to find a cure. Since enzyme replacement therapy can’t effectively treat LSDs affecting the brain, polymersomes are being tested to see if they can be used as a vehicle to deliver β-gal across the blood brain barrier (BBB). Apolipoprotein (ApoE) exhibits a high affinity for Low Density Lipoprotein Receptors (LDLRs), specifically LRP-1, which are highly expressed in the BBB of GM1 patients. In this project, we bind ApoE to the surface of the polymersomes to enable transport across the BBB in a murine model of GM1 gangliosidosis, β-gal⁻/⁻ mice.
Materials and
Methods: Solvent injection was used to formulate polymersomes from the block copolymer PEG-b-PLA. Dynamic Light Scattering (DLS) was used to determine the size, monodispersity and zeta potential of the particles. Dialysis was run to test the encapsulation efficiency of cyanine 5 (Cy5) dye in the ApoE-tagged and untagged polymersomes. Mice were bred to obtain the 𝛽-gal-/- phenotype. Their DNA was collected, and polymerase chain reaction and gel electrophoresis were performed to genotype the mice to identify homozygous mutants. Using the dye-loaded polymersomes, tail vein injections were performed on the 𝛽-gal-/- mice with wild type age matched controls. Additionally, mutant mice were tested with either ApoE-tagged or untagged polymersomes to observe the biodistribution with a focus on the brain. With each observation, the mice were put in an In Vivo Imaging System (IVIS) to test for fluorescence throughout their bodies, and a tail stick was performed to collect blood. At the 24-hour mark, the mice organs were harvested and put into IVIS for a better view of fluorescence, and urine was collected. A free dye tail vein injection using Cy5 was done on one wild type mouse and one 𝛽-gal-/- mouse, using the same procedure above, to test for a leaky BBB. Blood and urine analyses were performed using a Take-3 plate and UV/Vis.
Results, Conclusions, and Discussions: DLS and Zeta potential results showed that the ApoE tagged polymersomes were larger in diameter than the untagged polymersomes. The charge of the PEG-PLA polymersomes was more negative than that of ApoE tagged polymersomes. PEG-PLA polymersomes encapsulated 1.7*10⁻⁵ mg Cy5/mg of polymersome, while ApoE-tagged PEG-PLA polymersomes encapsulated 1.3*10⁻⁵ mg Cy5/mg of polymersome. Tail vein injections in 𝛽-gal-/- mice resulted in significant fluorescence in the brains of mutant mice (Figure 1), with ~20% increased neurologic uptake with the addition of the ApoE tag. To test for BBB integrity due to previous results showing that untagged polymersomes also caused an increase in fluorescence in mutant brains, a free dye injection on two mice, a 𝛽-/- mouse and a wild type mouse, proved extravasation in the BBB of mutants. On the IVIS image, the brain of the 𝛽-/- mouse showed much more fluorescence than the wild type mice, which showed little to none. This model clearly presents with leaky BBB, which is consistent with late-stage GM1 gangliosidosis affected patients. However, the ApoE-tagged PEG-PLA polymersome showed more delivery via higher radiant efficiency in the brain than the untagged PEG-PLA polymersomes. PEG-b-PLA polymersomes were successfully synthesized and characterized by Zetasizer parameters, including diameter, polydispersity index (PDI), and zeta potential. ApoE attachment was confirmed by an increase in particle diameter and PDI, as well as a decreased zeta potential. Knockout mice were successfully bred to obtain homozygous mutant mice at a rate of approximately 8 percent per breeding group. Cy5 dye was successfully encapsulated in both untagged and tagged polymersome systems. Pharmacokinetic study injections successfully showed the uptake of polymersomes in the brains of mutant mice. Mutant mice exhibited greater fluorescence when treated with ApoE-tagged polymersomes due to the targeting ability of ApoE. Mutant mice showed a lack of BBB integrity as free dye injections could enter the brain. This shows promise in the future that more effective treatment is possible for GM1 gangliosidosis affected patients and that they can live fuller and longer lives.
