Dissertation

Peptides: From Synthesis to Biomedical Application in Two Types of Cancer

Luna González, Omar Fernando

2024

Overview

The aim of this work was to synthesize peptides that would perform two functions in the potential treatment of two types of cancer.The first group of peptides was used as the antigenic component of a nanovaccine formulation that represents an immunotherapeutic approach to treating pancreatic ductal adenocarcinoma. In some cases, the peptides were modified at the N-terminus through palmitoylation and PEGylation, with the objective of enhancing their immunogenic potential. Additionally, they were synthesized as single epitopes or as multi-epitope constructs derived from tumor-associated antigen proteins. The peptides were formulated in poly(lactic-co-glycolic) acid-based nanoparticles, and the resulting nanoformulation was tested in a mouse model to assess its immunogenic activity. Two multiepitope peptides demonstrated a markedly positive response in vitro. These were the PalmitoylPLTVAEVQKLLGPHVKKALPLDLLLFLKKSLLFLLFSL-NH2 peptide and the HKVYLRVRPLLKKSYGVLLWEIKKRFVPDGNRI-NH2 peptide. These findings suggest that long multi-epitope constructs are the most effective alternative for use as nanovaccine components, as single epitope peptides were demonstrated to lack immunogenicity. However, preliminary in vivo assays of the two multi-epitope peptides exhibited minimal activity against the tumor in a mouse model. Additional validation is necessary through the repetition of these assays.The second group of peptides served as targeting units in a quatsome nanovesicle delivery system that is designed to carry a therapeutic nucleic acid for the treatment of neuroblastoma. The peptides were initially synthesized with fluorescein as a probe. In parallel, a small molecule ligand, a thiolated paminobenzylguanidine derivative, was also synthesized, labeled and evaluated in conjunction with the targeting peptide moieties to determine their internalization capability in a neuroblastoma cell line.A sequence targeting the GD2 receptor in neuroblastoma cells (H-WHWRLPSGGGC-NH2) and the thiolated p-aminobenzylguanidine derivative, demonstrated the greatest capacity to internalize into these cells and were therefore selected for the development of a conjugation methodology in quatsome nanovesicles using a thiol-maleimide click reaction. The methodology was successfully developed, and the optimal conditions were identified as a pH of 7.5, a reaction time of two hours, the presence of a reducing agent and a clean-up methodology of size exclusion chromatography in Sephadex G50 and aqueous elution followed by mild acidic elution. This allowed for the separation of nanovesicles from unreacted ligands and the indirect estimation of the conjugated targeting moiety in the nanovesicle. The methodology yielded conjugation estimates of 50% to 65% for both the GD2- binding peptide and the thiolated p-aminobenzylguanidine derivative. Furthermore, this formulation was demonstrated to have the capacity to deliver a nucleic acid to a neuroblastoma cell line. However, a switch of the PEGyl moiety carrying the maleimide function from PEG2000 to PEG1000 is required to achieve quantitative internalization.Furthermore, a study was conducted to evaluate the suitability of five different carbodiimides for use in solid-phase peptide synthesis, the methodology employed for the production of all peptide compounds in this research. The objective of this comparative study was to identify an optimal alternative to N,N'-diisopropylcarbodiimide (DIC) that can prevent the formation of the toxic compound hydrogen cyanide, which can occur when the reaction is conducted in the presence of oxyma. The study demonstrated that 1-tert-butyl-3-ethylcarbodiimide is an effective alternative to DIC. It exhibited comparable synthetic performance in the production of two peptide models and an antigenic peptide, while reducing the occurrence of hydrogen cyanide by threefold compared to DIC.

• Publisher: ProQuest Dissertations & Theses
• Subject: Biomedical engineering / Oncology
• ISBN: 9798314834732