Date of Award

Spring 5-2023

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular Science and Nanotechnology

First Advisor

Teresa Ann Murray

Abstract

Cell-penetrating peptides (CPP) possess the ability to transport different cargos efficiently across the cell membrane. In this study, the ability of a CPP to cross the blood-brain barrier (BBB) and stably carry therapeutic components across the plasma membrane was assessed. The therapeutic efficacies were determined by the ability of the KAFAK conjugated anti-inflammatory peptide drug to inhibit the progression of secondary neuronal damage in a traumatic brain injury (TBI) model at 7-day post-injury (DPI) by inhibiting the production of inflammatory cytokines. A related study with minocycline and PgP-rolipram was conducted to improve the outcome in TBI models at 14 DPI. Similarly, the ability of halloysite nanotubules (HNTs) to cross the BBB was investigated. Improving the BBB-crossing carrier library is the primary objective of this study, which seeks to identify reliable carrier molecules that can transport therapeutic compounds to the brain to treat brain injury and disorders.

Initially, two peptides were synthesized: (1) an anti-inflammatory peptide (AIP-1) that specifically target the MK2 pathway (mitogen-activated protein kinase) to regulate the inflammatory response (control), and (2) KAFAK, a CPP conjugated to AIP-1. The intracellular delivery, therapeutic efficacy, and cytotoxicity of these peptides were compared in rodent primary BBB cells (primary brain microvascular endothelial cells (BMVEC's)) and four cell lines (macrophages (RAW, J774), neuronal cells (SHEP-1), and hepatocytes (HEP G2)). KAFAK did not induce cell toxicity at concentrations below 250 µM in primary BMVEC cells and below 1000 µM in the cell lines. Subsequently, the CPP conjugate was validated in vivo for its uptake and localization in the brain and its therapeutic efficacy in a TBI mouse model following non-invasive intranasal administration for six days. The results showed that KAFAK was primarily localized in the olfactory bulbs. Some diffused throughout the cortex, and it reduced cytokine (IL-1β, IL-6, and TNF-α) production in TBI mice as compared to vehicle-treated mice.

Halloysite nanotubules (HNTs), naturally available nanoparticles, were also explored for their ability to penetrate the BBB. HNTs were loaded with rhodamine isothiocyanate (RITC) to determine their uptake and localization in the brain at 4, 24, and 48 hours after a single noninvasive intranasal administration. Another group of mice received HNTs loaded with diazepam intranasally for six days and was evaluated for behavioral changes versus mice that received HNTs alone. Fluorescence from RITC was observed in the brain tissue of mice treated intranasally with HNTs loaded with RITC but not in mice treated with RITC alone. The intensity of fluorescence decreased over time, and no HNTs-associated behavioral changes were observed. Mice treated with HNT-diazepam exhibited diazepam-associated behavioral changes, indicating that HNTs could penetrate the BBB and release the drug into the brain. In summary, HNTs and CPPs have demonstrated that they have the potential to transport drugs across the BBB.

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