Current diagnostic and therapeutic nanocarriers, including liposomes, micelles, and polymeric- and protein-based nanoparticles, are designed to have key functional properties such as: (i) longevity in the bloodstream, leading to accumulation of therapeutic cargos in neoplastic areas with leaky vasculatures; (ii) targeting of specific pathological sites through surface modification with targeting ligands; (iii) stimuli-responsive characteristics for controlled drug release under specific conditions. While some of these drug delivery systems have advanced into clinical stages, other nanocarriers remain under development to overcome issues with effective delivery such as lack of target-ability and fast clearance from circulation. Self-assembling peptides have recently shown great potential as nanocarrier materials for drug and gene delivery, owing to their safety, efficiency, and targeting capabilities. An amino acid pairing strategy enables us to design self/co-assembling peptides with multiple functionalities to fulfill drug delivery requirements.
This thesis focuses on functionalization and characterization of self/co-assembling peptides as nanocarriers for hydrophobic anticancer drug delivery. Diethylene glycol (DEG) conjugation and protein binding are the two modification strategies used in this thesis to impart longevity and target-ability upon the peptide-based delivery system. The studies include: (i) characterization of self-assembling properties of the diethylene glycol (DEG)-conjugated amino acid pairing peptide AAP8, (ii) investigation of the self/co-assembling features of a model ionic-complementary peptide (EAR8-II) in complex with the hydrophobic drug pirarubicin, and the anticancer activity of the complex, (iii) the interactions between peptide-drug complexes and serum proteins from the thermodynamic viewpoint, (iv) quantification of the effect of protein binding to the peptide-based delivery system on immune responses and biocompatibility, and (v) exploration of the targeting capability of albumin-bound peptide-drug complexes towards lung cancer cells.
Uncontrollable aggregation of AAP8 was the first issue to address in order to develop a promising platform for the peptide-based delivery system. Diethylene glycol (DEG), a short segment of polyethylene glycol (PEG), was conjugated to AAP8 either at one or both terminals, and then self-assembling and drug encapsulation properties of both functionalized AAP8s were characterized to evaluate the effect of DEG-modification. The results illustrated a significant reduction in uncontrollable aggregation, and the formation of uniform fibular nanostructures. In addition, DEG conjugation provided the peptide with safer features towards immune cells by reducing cellular toxicity to macrophages. Moreover, DEG-functionalization improved hydrophobic drug stabilization, as demonstrated by sustained cytotoxic efficacy against lung carcinoma cells over a relatively long time compared to the non-functionalized AAP8.
Protein binding strategy was the second approach to utilize the peptide-based delivery system with more biocompatibility and target-ability features. EAR8-II was studied as a model ionic-complementary peptide with high capability of pirarubicin encapsulation and anticancer activities against different cancer cells. Albumin as a most abundant protein in serum was selected to assess its binding affinity to the delivery system, and evaluate its binding effect on immune responses and anticancer activities.
The results showed a central role of albumin in the in vitro delivery of peptide-drug complexes to target lung cancer cells based on the following characteristics: (a) Non-covalent binding of albumin to the complex through hydrogen bonding and Van der Waals interactions. The interaction was confirmed by physicochemical methods such as fluorescence quenching and isothermal titration calorimeter (ITC). (b) Shielding properties of albumin for the complex against macrophages and blood components (erythrocytes and complement protein C5b-9). In the presence of albumin, phagocytosis and cytokine expression level of macrophages and hemolytic activity of the peptide-drug complex reduced significantly due to the smaller particle size of the albumin-bound complexes compared to unprotected ones. (c) Targeting the lung cancer cells, possibly because of the inhibition of the albumin-binding protein SPARC (secreted protein, acidic and rich in cysteine). SPARC is a glycoprotein over expressed in lung cancer cells with high affinity to albumin. The results from in vitro SPARC expression in A549 cells, a type of human non-small cell lung carcinoma (NSCLC), showed a significant drop by the albumin-bound complex at the mRNA level evaluated by qRT-PCR. This effect can be explained by transporting the albumin-bound complex into the cell surface, binding to the SPARC proteins, and so inhibiting the SPARC expressions.
This work lays out a foundation for modification and characterization of the self/co-assembly peptide-based nanocarriers for hydrophobic anticancer drug delivery, especially to improve longevity and target-ability properties.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:OWTU.10012/8352 |
| Date | 24 April 2015 |
| Creators | Sadatmousavi, Parisa |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Thesis or Dissertation |
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