Poster O5 - Engineering Elastin-Like Polypeptides to Facilitate Intranasal Delivery of a Large Alzheimer's Disease Therapeutic

Introduction: Alzheimer’s disease (AD) is a devastating condition currently affecting more than 6 million Americans and costs the US $300 billion annually. AD is a progressive disease leading to significant memory loss and cognitive disability, affecting the financial and emotional well-being of the patient and their family. Two major hallmarks of AD are the increased prevalence of reactive oxygen species (ROS) in the brain and the improper cleavage of amyloid proteins which form plaque deposits and decrease synaptic connectivity and viability of neurons. Many promising treatments fail to enter the brain due to the blood-brain barrier (BBB), which only allows small serum components to permeate into brain tissue. Vescalagin (VSG) is a large polyphenolic tannin that has shown promising preliminary data for sequestering ROS and reversing plaque formation. However, due to its size it cannot be delivered through the BBB or with conventional oral or intravenous methods. The intranasal (IN) delivery route is a promising delivery method as there is no BBB between the nasal mucosa and brain tissue, but is limited by mucociliary clearance which displaces therapeutics to the gut. To mitigate this, we developed and screened libraries of thermo-gelling elastin-like polypeptides (ELPs), which are recombinantly fabricated peptides with tunable and temperature-depending phase separation and gelling. ELPs can possess specific chemistries for retention and subsequent delivery of a drug and contain peptide sequences that facilitate cell interactions. In this project, we synthesized novel ELPs and studied their ability to complex and transport VSG through an in vitro nasal mucosa model.

Materials and

Methods: Recursive Directional Ligation (RDL) was used to select the ELP genes of interest and ligate them together to create unique sequences with varying chemistries as described in previous work in the lab (Nelson DW, et al., SynBio. 2024). ELPs with varying chemistries were combined with VSG in PBS and subjected to a “pulldown” assay to assess VSG binding efficiency of each ELP. An in vitro nasal mucosa model was then developed using trans-well inserts to culture RPMI 2650 human nasal epithelial cells. This model was developed so that an air-liquid interface facilitated mucus production. After establishing the model, VSG permeation was tested with and without ELPs bearing various cell-interactive chemistries. PrestoBlue, 2,2diphenyl-1-picrylhydrazyl (DPPH), and lactate dehydrogenase (LDH) assays were used to assess the therapeutic efficacy, permeation of VSG, and ELP cytotoxicity.

Results, Conclusions, and Discussions: ELPs were engineered to bind VSG, a promising Alzheimer’s disease therapeutic, upon gelation as well as form stable microgels that can facilitate nasal epithelial uptake. A unique plasmid mutagenesis scheme was used to create a library of ELPs that were then used to screen polyphenol interactions and ELP microgel cell-interactive properties. This informed the decision of which ELPs to test for optimal VSG binding and minimize mucous clearance. Through multiple pulldown assays, it was determined that ELPs bearing phenolic amino acids bound the most polyphenol. Therefore, ELPs with this optimal drug binding ability were chosen and used to develop diblock ELP structures with various cell interactive peptides that would resist bulk hydrogel formation and maintain a microgel structure. When testing these ELPs in the developed in vitro nasal mucosa model, it was found that ELPs with a cell-penetrating peptide allowed for greater VSG permeation than ELPs with an epithelial adhesive peptide or ELPs with no interactive peptide.

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