Publication Date

2018-05-14

Document Type

Honors Project

Degree Name

Bachelor of Science

Department

Engineering

Advisors

Sarah J. Moore

Keywords

Protein engineering, Targeted drug delivery, Azlactone, Transferrin, Blood-brain barrier, Alzheimer's disease, Drug targeting, Polymerization

Abstract

The treatment of diseases of the central nervous system (CNS) is hindered by the blood brain barrier (BBB), a selective barrier that keeps toxins and other harmful substances that are circulating in the blood vessels away from brain tissue. Targeted drug delivery systems need to be created to enhance the treatment of diseases rooted in the CNS, such as Alzheimer’s disease and other neurodegenerative diseases. Synthetic polymers and protein engineering can effectively be used to create such targeted drug delivery systems. A novel approach to overcome the limitations related to drug delivery across the BBB is to hijack natural transport systems that are native to this barrier. Essential molecules such as iron are allowed through the barrier and delivered to the brain by a process called receptor mediated-transcytosis. A transport protein, transferrin (Tf), is recognized by receptors present on the surface of the BBB, and allowing Tf to deliver its cargo to the brain. Proteins can be engineered to have similar functions as these transport proteins, targeting receptors on the BBB that trigger their internalization and transport across the BBB. Conjugation of engineered proteins to drug-loaded polymeric structures can be used to facilitate target-specific drug delivery to the brain, making it possible to treat challenging neurodegenerative diseases that currently lack an effective treatment method due to limits in drug transport to the brain tissue. In this thesis project, reactive, azlactone functionalized polymers [i.e., poly(2-vinyl 4,4-dimethylazlactone)] were synthesized and functionalized with hydrophilic side chains to improve their solubility in aqueous solutions. These reactive polymers were then conjugated to model proteins: lysozyme (lys), bovine serum albumin (BSA), and holo-transferrin (HTf). SDS-PAGE protein gel analysis and fluorescence imaging were used to verify that polymer protein conjugates (PPCs) were successfully made. The conjugates were then used to perform internalization studies using MCF-7 cells, a breast cancer cell line that expresses the transferrin receptor on its surface. Confocal microscopy images showed that HTf PPCs were successfully internalized by cells. Cells incubated with BSA PPCs showed no signs of internalization. In continuing to develop the PPCs toward clinical applications, model proteins should be replaced by proteins engineered to target the transferrin receptor in a way that reduces potential toxicities associated with using the native transferrin ligand. Proteins previously engineered to target the transferrin receptor were successfully induced using a bacterial expression system. Further troubleshooting is needed to successfully isolate and purify these engineered proteins. In future work, these proteins that target the CNS will be characterized and conjugated to PVDMA to develop a more effective targeted drug delivery system.

Rights

2018 Analia Jazmin Vázquez Cegla.Access limited to the Smith College community and other researchers while on campus. Smith College community members also may access from off-campus using a Smith College log-in. Other off-campus researchers may request a copy through Interlibrary Loan for personal use.

Language

English

Comments

72 pages : color illustrations. Includes bibliographical references (pages 64-67)

Share

COinS