Engineered peptides on Gold Nanostructures for cell targeting as SERRS biosensors for cancer diagnostics

Due to their optical properties, facile surface chemistry and biocompatibility, Gold Nanoparticles (AuNP) have attracted an increasing interest in the last years for Nano-biotechnological applications. Their targeted delivery to malignant tumours, in particular, has become a powerful tool in cancer nanomedicine exploiting AuNP as promising platform for imaging, using for example SERS spectroscopy, diagnosis and therapy. In order to fully exploit their potentialities, AuNP have to be conjugated with active targeting ligands which could improve their pharmacokinetic and pharmacodynamic profiles. Among many targeting agents, such as antibodies, proteins and small molecules, peptides have achieved great success in cancer nanotechnology for their low toxicity and immunogenicity as well as for their relatively low synthetic costs. To achieve an efficient cancer cell targeting a convenient design of the targeting ligands is necessary to optimize the association of the nanostructures with the receptor, reducing the interactions with healthy tissues. However, the effect of the organization of the targeting units on the nanostructures surface has never been studied in detail. In this PhD thesis the engineering of peptide targeted gold nanostructures, as SERRS biosensors for colorectal and liver cancer diagnostics, is described. The targeting activity of the nanostructures was studied both in vitro on cancer cells and in vivo on murine tumour models. Experimental results were combined to Molecular Dynamics (MD) calculations and spectroscopic analysis to investigate the role of the peptide organization on the nanostructures surface for the receptor recognition. In Chapter 2 methodologies used to synthesize and characterize peptides as well as gold nanostructures are described. Chapters 3-6 are focused on colorectal cancer targeting achieved by conjugating gold nanostructures with peptides specific for the Epidermal Growth Factor receptor (GE11 peptide) and αvβ3 Integrins (RGD peptide), two receptors overexpressed in cancer cells and tissues. In Chapters 3 and 4 the exposure of the targeting units on the nanostructures surface for achieving an efficient association with the receptor is investigated with the help of molecular dynamics. To achieve this goal, several peptide analogues were conveniently synthesized to present the targeting unit in different arrangements on the nanostructure surface and the targeting properties of the resulting nanosystems were assessed. Since among many ligands those engineered by conjugating the peptide to polyethylene glycol (PEG) showed the best targeting activity, the role of the polymer was investigated in dept by studying the targeting activity of nanostructures coated with a mixed monolayer of PEG and peptides (Chapter 5). For in vivo applications of peptide functionalized nanoparticles, the stability to proteolysis of the targeting units is an important issue that was evaluated by incubation of the nanostructures with different proteolytic enzymes and in serum (Chapter 6). Chapter 7 concerns liver cancer cells targeting achieved by functionalizing gold nanostructures with properly designed analogues of the peptide PreS1, known as specific ligand of the Squamous Cell Carcinoma Antigen 1 which is overexpressed in hepatoma and hepatoblastoma. The arguments developed in the thesis are introduced in the Chapter 1.