Synthesis and Biomodification of Gold Nanoparticles for Multimodal Cancer Therapeutics
Drezek, Rebekah A.
Doctor of Philosophy
Gold nanoparticles (AuNPs) have potential for medical and biological applications due to their optical properties, including scattering and absorbance, and ease of surface modification. AuNPs can transfer energy from absorbed light into heat, which has been utilized for photothermal therapy (PTT). Hollow gold nanoshells (HGNs) are of particular interest because they are easy to synthesize by using a sacrificial template, and can be tuned to the desired plasmon wavelength by altering the thickness of the gold layer. Also, HGNs are normally synthesized in the 40-80 nm diameter ranges, which is the optimal size for cellular endocytosis and effective biodistribution in tumors. However, HGNs are difficult to track in vivo due to limited optical penetration depth with light. Therefore, we designed a novel HGN that incorporated several small iron oxide nanoparticles (IONP) as an improved contrast agent for T2 magnetic resonance imaging (MRI). Traditionally, gold-coated iron oxide complexes are either not tuned in the near infrared region for maximum optical penetration depth or are greater than 100 nm in diameter, which can hinder cellular and tumor retention. This design produces nanoparticles in the sub-100 nm range while still having an absorbance peak in the near infrared range. Therefore, these magnetic HGNs have PTT capabilities and were able to debulk tumors and improve survival in a murine model. In addition, AuNPs are attractive as nanocarriers because they are inert, non-toxic, and can be readily endocytosed by dendritic cells and other phagocytic mononuclear cells enabling AuNPs for the delivery of cancer vaccines. For antigen-based delivery, we designed novel gold-based nanovaccines (AuNVs) using a simple self-assembling bottom-up conjugation method to generate high-peptide density delivery and effective immune responses with limited toxicity. AuNVs were synthesized using a self-assembling conjugation method and optimized using DC-to-splenocyte interferon-γ enzyme-linked immunosorbent spot assays. The AuNV design has shown successful peptide conjugation with approximately 90% yield while remaining smaller than 80 nm in diameter. DCs took up AuNVs with minimal toxicity and were able to process the vaccine peptides on the particles to stimulate cytotoxic T lymphocytes (CTLs). These high-peptide density AuNVs stimulated CTLs better than free peptides and have great potential as carriers for various vaccine types. For vaccine adjuvant delivery, we conjugated a modified CpG oligodeoxynucleotide immune stimulant to gold nanoparticles using a simple and scalable self-assembled monolayer scheme that enhanced the functionality of CpG in vitro and in vivo. The use of a triethylene glycol (TEG) spacer on top of the traditional poly-thymidine spacer increased CpG macrophage stimulatory effects without sacrificing DNA content on the nanoparticle, which directly correlates to particle uptake. These TEG modified CpG-AuNP complexes induced macrophage and dendritic cell tumor infiltration, significantly inhibited tumor growth, and promoted survival in mice when compared to treatments with free CpG. Apart from using AuNPs to deliver vaccines, we conjugated dendrimers onto small gold nanoparticles to condense DNA for plasmid delivery. Although EDC/sulfo-NHS coupling methods have been previously used in several AuNP conjugates, in depth analysis of the conjugation scheme proved to be critical for maintaining particle stability and functionality of the dendrimer particularly for lower generations. Reaction time of the conjugation and carboxyl spacing were found to be major factors for synthesizing dendrimer-AuNP complexes. Other than immunotherapy agents or plasmids, AuNPs can also be used to deliver chemotherapy agents. Our design uses molecular beacons (MBs) that target mutated or over expressed mRNAs and use them as a molecular trigger for chemotherapy or drug release. This strategy is referred to as Molecular Activated Stealth Chemotherapy (MASC). Understanding the proper synthesis of MB-AuNPs is critical for the successful fabrication of the final MASC particles. We developed a model that successfully predicted the spacing and elevation effects on MB and its function while conjugated to AuNPs. In conclusion, we constructed multimodal theranostic magnetic hollow gold nanoshells for MRI contrast and PTT. Furthermore, we designed gold nanoparticles to deliver vaccines, plasmids, and chemotherapy, which were released by mRNA. The various designs can serve as platforms for cancer diagnostics, circulating tumor cells collection immunotherapy, viral or bacteria vaccination, gene therapy, and personalized medicine. Future work focuses on in depth analysis of these designs and combining them for more efficient nanocarriers designs.