Date of Award

Spring 5-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Pharmaceutical Sciences

First Advisor

Keykavous Parang

Second Advisor

Rakesh Kumar Tiwari

Third Advisor

Sun Yang

Fourth Advisor

Aftab Ahmed

Abstract

Amphipathic Hybrid Cyclic-Linear Peptides as a Drug Delivery System by Jonathan A. Moreno Peptides

[RW]4 and [R5W4] have been previously identified as molecular transporter Cell Penetrating Peptide (CPP) and antimicrobial agents, respectively. These peptides consisted of alternating and non-alternating arginine (R) as positively charged residues and tryptophan (W) as hydrophobic residues. Herein we report the synthesis of two hybrid amphipathic cyclic-linear cell-penetrating peptides [RW]4K-AH135 (1) and [R5W4]K-AH135 (2) composed of [RW]4 and [R5W4] and a linear peptide AH-135 (Ac)NH-RWLRRWLRWWRR-COOH. The hybrid peptides were assessed for their antimicrobial activity and capacity to deliver small and large molecules, including proteins. Remarkably, (2) exhibited superior efficiency as an anticancer drug delivery system. Consequently, nonacetylated (2) was fluorescently labeled with FITC using a short β-alanine linker, resulting in [R5W4]K-AH135(βA)-F' (3), to investigate its mechanistic uptake. Similarly, Doxorubicin (DOX) was conjugated to Peptide (2) through a β-alanine, glutarate linker, resulting in [R5W4]K–RRWWRLWRRLWRβA–DOX (4). Antiproliferative assays compared the efficacy of the peptide-DOX conjugate (PDC) to free DOX and its peptide-DOX physical mixtures in cancer cell lines. Conjugate (4) exhibited higher cytotoxicity against cancer cells at concentrations of 5 μM and 8 μM compared to free DOX at 5 μM. After 72 h of incubation, the conjugate inhibited the viability of ovarian adenocarcinoma (SK-OV-3) cells by 63%, and breast cancer cells (MDA-MB-231 and MCF-7) by 74% and 66%, respectively, in comparison to the free drug 31%, 41%, and 48% in each cell line. The PDC showed lower toxicity towards healthy myocardial cells (H9C2) with 80% cell viability compared to 60% for free DOX. It exhibited higher activity against DOX- vi resistant cells (MES-SA/MX2) with only 13% cell viability compared to 84% for the free DOX treatment. Mechanistic studies revealed that DOX transport occurred through direct translocation and caveolae-mediated endocytosis. Analytical HPLC monitored the stability of the DOX conjugate in various media, and the glutarate linker was found to undergo acid-catalyzed hydrolysis, releasing its payload with a half-life of 14 h. Intracellular release experiments demonstrated approximately 100% release of free DOX from the conjugate within 72 h. Conjugate 4 markedly enhances cancer cell apoptosis by 7-fold compared to DOX alone and induces a 1.7-fold increase in G2/M phase cell cycle arrest.

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Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.

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