Graduation Year

2023

Document Type

Thesis

Degree

M.S.

Degree Name

Master of Science (M.S.)

Degree Granting Department

Graduate School

Major Professor

Subhra Mohaptra, Ph.D.

Committee Member

Sivakumar Panguluri, Ph.D.

Committee Member

Manas Biswal, Ph.D.

Keywords

BDNF, Drug delivery, Nanotechnology, PAMAM, TBI

Abstract

Background: Traumatic Brain Injury (TBI) is a major contributor to death and disability due to motorvehicle accidents, sports, physical abuse, and battlefield injuries. The primary insult to the brain leads to inflammation, vascular dysfunction, and oxidative stress in the brain as well as in the eye. This leads to loss of Retinal Ganglion Cells (RGCs) and downregulation of Brain derived Neurotopic Factor (BDNF). BDNF is a neurotrophic factor that binds to Tropomyosin Receptor Kinase B (TrkB) receptor to promote cell growth, survival, and differentiation. Current treatment strategies do not promote neuronal regeneration. Therefore, novel treatments are needed to restore vision following TBI. Dendrimers are nanoscale, branched polymers that have been widely used to deliver drugs due to their ability to cross the blood-brain barrier (BBB) and blood-retina barrier (BRB). β-cyclodextrin (β-CyD) has a hydrophobic cavity that can encapsulate drugs. We aim to induce BDNF expression in ARPE-19 cells by complexing BDNF plasmid to PAMAM-β-cyclodextrin conjugate. Methods: In this study, we conjugated β-CyD to Polyamidoamine (PAMAM) dendrimers and used the PAMAM-β-cyclodextrin nanoparticles to deliver BDNF plasmid in retinal pigmented epithelial cell line, ARPE-19. Two different NP (N): plasmid (P) ratios – 2:1 and 5:1 were tested. Results: BDNF levels were downregulated in TBI mouse retina. Conjugation of β- CyD to PAMAM increased transfection efficiency compared to PAMAM alone. BDNF plasmid was complexed with PAMAM-β-CyD conjugated in different N/P ratios. Higher N/P ratios increase cytotoxicity due to positive charges of PAMAM disrupting the negatively charged cell membrane in the PAMAM-BDNF treatment group. Vice-versa was observed in BDNF complexed PAMAM- β-CyD treatment groups which might be because β-CyD can interact with phospholipids in the cell membrane. N/P ratio 5:1 increased transfection efficiency and Bdnf mRNA levels compared to 2:1 due to complete condensation of DNA around the PAMAM as a result of higher positive surface charges. Conclusion: BDNF complexed PAMAM-β-CyD conjugate are able to transfect ARPE-19 cells and upregulate Bdnf mRNA levels. This study using ARPE-19 cells can be further validated for ocular drug delivery to the posterior segment in-vivo animal models of TBI. For future studies, the hydrophobic cavity of β-CyD can be utilized to encapsulate drugs to decrease inflammation, oxidative stress, and intraocular pressure in the eye. The nanoparticles-mediated gene therapy may be a promising approach for gene therapy for retinal degeneration post-TBI, in the future.

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