Morisco, Anna (2009) TARGETING PEPTIDES CONTAINING SUPRAMOLECULAR AGGREGATES TOWARD CANCER CELLS. [Tesi di dottorato] (Inedito)

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Abstract

Multimodal supramolecular compounds that combine imaging and therapeutic capabilities are the focus of intensive research for applications in the growing field of nanomedicine. These compounds offer the prospect to increase diagnostic accuracy and therapeutic effectiveness, while minimizing side effects from treatment. Nanoparticles with a micellar or liposomal structure obtained by assembling amphiphilic compounds are promising candidates for such multifunctional therapeutic platforms. Selective target of these nanoparticles for cancer cells can be obtained by labeling supramolecular aggregates with peptides able to recognize transmembrane receptors overexpressed in many human tumors. Examples of peptide that can be used to drive selectively the nanoparticle on the cancer cells are the octreotide (analog of the somatostatin), [7-14] bombesin and CCK8 peptides. To reach this result, new amphiphilic monomers were designed, synthesized and structurally characterized. Amphiphilic monomers are functionalized with polydentate chelating agent (such as DOTA, DTPA and DTPAGlu) able to coordinate radioactive or paramagnetic metals for diagnostic applications, or with bioactive molecules able to recognize the cellular target. These molecules are able to auto assemble or to aggregate in mixed micelles or liposomes in aqueous solution. All monomers were synthesized in solid-phase following an Fmoc strategy. The aggregates formulated are investigated by physical chemical methods like Small Angle Neutron Scattering (SANS) and Dynamic Light Scattering (DLS), relaxivity measurement and spectroscopy techniques (fluorescence and UV-Vis). These studies have allowed selecting the best systems for shape and size with relation to relaxivity parameters and aggregation stability. To validate the specificity and the citotoxicity of the delivery systems in vitro and in vivo studies were also performed. The monomers containing the chelating agent, (C18)2DOTA, (C18)2DTPA and (C18)2DTPAGlu are able to auto assemble. Moreover in mixed aggregates they showed to drive the aggregation process. Aggregates fuctionalized with octreotide were investigated as promising target-selective contrast agent for MRI (Magnetic Resonance Imaging). The high relaxivity values, associated with the supramolecular aggregates, confirm the obtainment of a more rigid contrast agent able to carry numerous gadolinium ions on target cells. In vitro binding assays on bombesin labeled liposomes allowed to select (C18)2DOTA/(C18)2-L5-bombesin as the lead compound. In these supramolecular aggregate, the peptidic monomer contains the more appropriate spacer of the length between the peptide and the hydrophobic moiety. In vivo studies confirmed the specificity towards to the receptor of the bombesin labeled liposome with respect to the liposome exposing a scrambled peptide sequence.Aggregates fuctionalized with CCK8 peptide were investigated as target-selective drug delivery systems. The drug delivery of chemioterapeutics (i.e. doxorubicin) was studied on A431 cells overexpressing CCK receptors. The loading and release of the drug were carried out in (C18)2DOTA/(C18)2-L5-CCK8 liposome. The results obtained show the specificity and the efficacy of the system on cellular target, compared with control aggregate. In conclusion this research has permitted the achievement of new supramolecular systems containing a bioactive molecule and a chelating agent able to target tumor cells, as possible of diagnostic and therapeutic.

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