Preparation, Characterization and Intracellular Targeting of Biomolecule-Gold Nanoparticle Complexes

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Title: Preparation, Characterization and Intracellular Targeting of Biomolecule-Gold Nanoparticle Complexes
Author: Xie, Huan
Advisors: Daniel L. Feldheim, Committee Chair
Abstract: This dissertation described preparation, characterization and intracellular localization of peptide-modified gold nanoparticles as multifunctional nuclear targeting vectors. The vectors have the potential advantages for intracellular diagnostics and therapeutic delivery. Gold nanoparticles were used in this study as carriers to combine multiple-functional cell-translocating peptides and antisense oligonucleotides. Two proteins, bovine serum albumin (BSA) and streptavidin (SA), have been systematically studied for crosslinking peptides or oligonucleotides with gold nanoparticles. Protocols for modifying gold nanoparticles with BSA-peptide conjugates are described within. The resulting constructs were characterized using a number of techniques including UV-visible absorbance spectroscopy and fluorescence spectroscopy in order to quantify peptide-BSA binding isotherms, exchange rates, critical flocculation concentrations, and the composition of mixed peptide-BSA monolayers on gold nanoparticles. The abilities of BSA-peptide/gold complexes to target the nucleus of several cell lines (HeLa, 3T3-NIH and HepG2) were explored. Four peptide/nanoparticle complexes were investigated, particles modified with the adenovirus nuclear localization signal (NLS), the adenovirus receptor-mediated endocytosis (RME) peptide, the adenovirus fiber containing the RME and NLS and the NLS for SV 40. The translocation of the gold nanoparticles was identified by video-enhanced color differential interference contrast (VEC-DIC) microscopy. The strong affinity between SA and biotin was employed to conjugate SA with biotinylated peptide and oligonucleotide. Systematical studies have been done to characterize the complexes, including SA adsorption on nanoparticles, biotinylated DNA adsorption on SA-coated nanoparticles, stabilities in solution, exchange rate in cell growth media and the composition of mixed SA-biotin peptide and SA-biotin DNA monolayers on gold nanoparticles. VEC-DIC microscopy characterized the translocation of the complexes in HeLa cells. In further experiments, gold nanoparticle was conjugated with the adenovirus RME peptide and peptide nucleic acid-biotin leash DNA duplex and delivered to enhanced green fluorescence protein modified HeLa cells. Antisense function of the complexes was investigated by fluorescence microscopy and reverse transcription-polymerase chain reaction assay. The preliminary results demonstrated that the complexes had antisense function but the efficiency was low. Suggestions for future improvements were discussed.
Date: 2004-10-21
Degree: PhD
Discipline: Chemistry
URI: http://www.lib.ncsu.edu/resolver/1840.16/5617


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