Study and development of nanocomposites containing gold nanoparticles and biomolecules: synthesis and application in nanomedicine

Author(s)

Valeria Spolon Marangoni

• Document type: Master's Dissertation
• Press: São Carlos.
• Institution: Universidade de São Paulo (USP). Instituto de Física de São Carlos (IFSC/BT)
• Defense date: 2012-02-09

Examining board members

Valtencir Zucolotto; Adriana Raffin Pohlmann; Laudemir Carlos Varanda

• Advisor: Valtencir Zucolotto

Abstract

The convergence between biotechnology and nanotechnology has led to the development of new hybrid nanocomposites that conjugate the bio-recognization properties of biomaterials and the unique electronic, optic and catalytic properties of the nanoparticles. Despite the recent advances in the development of nanobiocomposites, the biomedical applications of these materials are still limited, among other factors, by the low efficiency of functionalization and biocompatibility. The present study was aimed at developing proteinconjugated nanoparticles for application in nanomedicine. Our main focus were the understanding and characterization of the interactions between proteins and gold nanoparticles (AuNPs), which was accomplished using two distinct systems, viz.: Jacalin-functionalized AuNPs, and Becen1-functionalized AuNPs. In the former, the interest is due the capability of the protein Jacalin of recognize the disaccharide (Galβ1-3GalNAc), largely expressed in some tumors cells. AuNPs were synthesized in the presence of the polyamido amine generation 4.0 (PAMAM G4) and conjugated with a Jacalin target with the fluorescein isothiocyanate (FITC). The excess of protein was removed by centrifugation and the complex formation was confirmed by Transmission Electron Microscopy (TEM), Dynamic Light Scattering (DLS), UV-VIS Absorption and Vibrational Spectroscopy (FTIR). The interactions between AuNP-PAMAM G4 and Jacalin seemed to be driven by an entropic process with moderate affinity and complex formation, as revealed by Isothermal Titration Calorimetry (ITC) and quenching fluorescence measurements. Furthermore, Circular Dichroism (CD) analyses revealed that protein maintained its secondary structure upon conjugation with the nanoparticles. In vitro tests revealed that the AuNPs/Jacalin complexes presented higher affinity and cytotoxicity against human cervical cancer cell (HeLa) compared to healthy mouse fibroblasts (L929). These results are relevant, since the AuNP-PAMAM G4/Jacalin-FITC complex may be used for biomedical applications including cancer treatment and diagnostics. The second nanocomplex, comprising AuNPs and BeCen1, was chosen due to the ability of BeCen 1 to polymerize in the form of nanometric filaments as a function of temperature. The AuNPs were formed in the presence of the protein using diluted formic acid as reducing agent and the excess of protein was removed by Molecular Exclusion Chromatography. CD analysis showed a decrease in the -helix structures confirmed by FTIR, which may be related to the interaction between the AuNPs and the amide groups of the protein. Light scattering measurements revealed an increase in the turbidity of the dispersions upon increasing the temperature, indicating a change in the arrangement of the AuNPs. Such BeCen-1 induced alignment was confirmed by TEM images. The latter results point to the possibility of fabrication of novel thermoresponsive nanobiocomposites, which are of great relevance for nanodevices applications. (AU)

• FAPESP's process: 09/11679-9 - Study and Development of Nanocomposites containing Metallic/Magnetic Nanoparticles-Biomolecules Conjugated: Synthesis and Application in Nanomedicine.
• Grantee: Valeria Spolon Marangoni
• Support Opportunities: Scholarships in Brazil - Master