Amphipathic Hybrid Cyclic-Linear Peptides as a Drug Delivery System

<p>Amphipathic Hybrid Cyclic-Linear Peptides as a Drug Delivery System by Jonathan A. Moreno Peptides</p> <p>[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 (<strong>1</strong>) and [R₅W₄]K-AH135 (<strong>2</strong>) 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, (<strong>2</strong>) exhibited superior efficiency as an anticancer drug delivery system. Consequently, nonacetylated (<strong>2</strong>) was fluorescently labeled with FITC using a short β-alanine linker, resulting in [R₅W₄]K-AH135(βA)-F' (<strong>3</strong>), 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 (<strong>4</strong>). 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 (<strong>4</strong>) 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.</p>

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Pharmaceutical Sciences

Year dc:date.available

2024

Author dc:creator

• Moreno, Jonathan

Contributors dc:contributor

• Keykavous Parang
• Rakesh Kumar Tiwari
• Sun Yang
• Aftab Ahmed

• Drug Delivery
• Molecular Transport
• Cell-penetrating peptides
• Peptide therapeutics
• Peptide-drug conjugate
• Amino Acids, Peptides, and Proteins
• Medicinal and Pharmaceutical Chemistry

Repository record dc:identifier

https://digitalcommons.chapman.edu/pharmaceutical_sciences_dissertations/33

OAI identifier oai:identifier

oai:digitalcommons.chapman.edu:pharmaceutical_sciences_dissertations-1034