Bachelor of Science
Drug delivery, Polymers, Receptor-targeting, RAFT polymerization, Blood-brain barrier, Central nervous system, Protein engineering, Drug delivery systems, Drug receptors
The blood-brain barrier is the major bottleneck in drug transport to the central nervous system (CNS). Its selective permeability has previously allowed only a very select few of therapeutics to reach the CNS, and most neurotherapeutics that manage to be transported successfully across the barrier are ejected back out via efflux transport systems. The disease of interest, and the motivation behind this thesis project, is Alzheimer’s Disease. Alzheimer’s is a devastating disease known to happen in patients above 60 years old, some in their 40’s and newly identified cases in children. It is theorized that mutations in the Amyloid Precursor Protein (APP) lead to its own misfolding and aggregation in the brain causing plaques that destroy signal transduction. While there has been a surge in development of potential therapeutics for Alzheimer’s, many of them are subject to the scrutiny of the blood-brain barrier.
The question asked in this thesis is how to go about bypassing the barrier to deliver therapeutics for CNS disorders like Alzheimer’s. There are conventional drug delivery systems such as oral, inhalation, injection, adsorption and so on. However, these conventional methods often require that a patient take multiple dosages or injections to achieve therapeutic effects and they cannot actively target the drug’s target in the body. The advent of controlled drug delivery methods aims at solving this issue by developing ways to achieve sustained and extended release of any drug. One attractive, novel method is the use of antibodies to carry a drug. Antibodies allow a drug to selectively reach a target, giving it active-targeting properties unseen in conventional delivery methods. However, antibodies are limited to the amount of drug they can carry. To design a solution for this, there has been a surge of research done on the use of polymeric micelles. Polymeric micelles, or even linear polymers, aim at encapsulating and carrying a higher concentration of any drug and can be designed such that the polymer degrades with time and releases the drug at a controlled rate. However, micelles themselves are not active-targeting systems. How do we bridge the gap between delivering high dosages at a controlled rate but also have these systems actively target their receptors?
The goal of this thesis is to develop a novel drug-delivery design that use a linear, brush polymer functionalized with highly-tunable and reactive azlactone groups (PVDMA) synthesized by a controlled method called RAFT polymerization. The focus of this project is to conjugate a novel fibronectin-based protein, called 𝛼-TfR, that actively targets transferrin receptors (TfR) found on the blood-brain barrier to PVDMA polymer. This novel conjugation system is a potential solution to active targeting and controlled drug release to the CNS.
We have found that 𝛼 -TfR is eluted off of the size-exclusion chromatography column at about 38 minutes, and we obtained a final protein concentration of 15 𝜇𝑀. We also found the PVDMA is insoluble in water or PBS and thus we needed to functionalize it with Jeffamine, a polyetheramine that makes the polymer more water-soluble. We characterized the functionalized PVDMA using FTIR spectroscopy. The more Jeffamine we added, the more water-soluble the polymer became. We also found that functionalized PVDMA successfully conjugates to bovine serum albumin and lysozyme used as control proteins using SDS-PAGE protein gel analysis. Ultimately, the functionalized PVDMA will be tested with 𝛼 -TfRprotein and we will use mass spectrometry techniques to quantitatively determine the ratios of protein to polymer, and gel permeation chromatography to determine the molecular weight of PVDMA.
Jumai'an, Eugenie Amjad, "Conjugation of engineered proteins to Azlactone-functionalized polymers for drug-delivery applications" (2017). Theses, Dissertations, and Projects. 1827.