Defense Date

3-19-2020

Graduation Date

Spring 5-8-2020

Availability

Immediate Access

Submission Type

thesis

Degree Name

MS

Department

Pharmaceutics

School

School of Pharmacy

Committee Chair

Devika Soundara Manickam

Committee Member

Rehana Leak

Committee Member

Wilson Meng

Committee Member

Younsoo Bae

Keywords

Gene delivery, transfection efficiency, extracellular vesicles, non-viral vectors, cell proliferation, paracellular permeability, blood brain barrier

Abstract

Stroke is a major cause of death in the United States. We aim to develop safe and efficient approaches for the treatment of stroke. Regenerative therapies have been identified as the major need for curing stroke [1]. We explored two different carriers (poly (ethylene glycol)5k-poly (L-aspartate-diethylenetriamine)48 (PEG-DET) / Pluronic P84 (P84) and exosomes (EXOs) / microvesicles (MVs)) to deliver brain-derived neurotrophic factor (BDNF) plasmid DNA (pDNA), a functional gene encoding a neurotrophin. In the first project, our data showed that DNA nanoparticles (NPs) with 0.03% P84 was a promising formulation to deliver Luciferase pDNA into immortalized human cerebral microvascular endothelial cells (hCMEC/D3 cells) efficiently. A lucifer yellow apparent permeability (LY Papp) method was developed to determine that DNA NPs with 0.03% P84 was a safe formulation for TJ integrity. However, this formulation did not result in increased BDNF secretion compared to untreated cells. To determine if natural carriers such as extracellular vesicles can deliver BDNF pDNA into hCMEC/D3 cells, we isolated EXOs and MVs from the hCMEC/D3 cell line. We used a standard differential ultracentrifugation method to extract EXOs and MVs from hCMEC/D3 cell medium and characterized EXOs and MVs by particle size measurement and western blotting. Although the transfection efficiency of extracellular vesicles (EVs) was low, the plain EXOs and MVs at a certain EV protein level (6 µg) resulted in relatively higher cellular ATP levels in healthy cells, which indicated that EXOs and MVs had a great potential to provide extra nutrients and energy for injured cells.

Language

English

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