Type of Document Dissertation Author Kogot, Joshua M Author's Email Address firstname.lastname@example.org URN etd-11052008-165903 Title Surface Assembly and Intracellular Delivery of Biomolecule Conjugated Nanomaterials Degree Doctor of Philosophy Department Chemistry and Biochemistry, Department of Advisory Committee
Advisor Name Title Geoffrey Strouse Committee Chair Brian Miller Committee Member Gregory Dudley Committee Member Timothy Logan Committee Member P. Bryant Chase Outside Committee Member Keywords
- Bimodal MR Contrast
- Gene Delivery
- Gold Nanoparticle
Date of Defense 2008-09-22 Availability unrestricted AbstractThe scope of this dissertation is two-fold: assembly of histidine-tagged peptides and proteins directly to the surface of a 1.5 nm AuNP and the assembly and delivery of nano-medical materials for MR imaging and gene therapy. Chapter 1 is a general introduction and describes the reason I chose to pursue my graduate career at the interface of biochemistry and nanomaterials. The introduction chapter provides a historical background for both biochemistry and nanomaterials, and shows the parallels of these two fields. Although biochemistry has been more extensively studied in the past century, the opportunities and current applications make nanotechnology a very attractive field. The merger of biochemistry and nanomaterials in the last decade has opened new doors to medical imaging, medical devices, drug delivery, and has expanded our ability to probe fundamental biochemistry and biophysics questions once out of our reach.
Chapters 2 and 3 describe an attempt to elucidate and confirm the ability to bind hexahistidine-tagged (His6) peptides and proteins directly to the surface of a AuNP. Until now, only Cys amino acids could be used to bind directly to a AuNP surface, otherwise elaborate surface modifications such as antibody-antigen interactions and AuNP surfaces modified with metal chelators such as NTA were necessary for controlled peptide / protein surface interactions. Using FT-IR, NMR, CD, and fluorescence spectroscopy, the direct, covalent attachment of His6 peptides is confirmed and the directed attachment of a model protein (FGF1) is described. The His6 – AuNP interaction is shown to occur through the N of the His side chain and is able to displace a Cys peptide for biding, suggesting that the binding interaction is tight due to the multi-chelator effect of the six His residues. Furthermore, studies with the FGF1 protein confirm that the AuNP binding does not perturb the structure / function of this protein and suggests that the His6-tag can be a viable alternative to engineered Cys residues for future bio-nano applications with proteins.
In Chapter 4 I present my research with developing new, high field and bimodal contrast agents for MR and optical microscopy imaging. MRI has become a valuable, non-invasive clinical diagnostic tool, and with the development of new contrast agents (CA), it is slowly becoming a technique with sensitivities rivaling radionuclei PET scans. The goal of this research was to develop a bimodal contrast agent using an InP/ZnS semiconductor quantum dot appended with a peptide and macrocyclic lanthanide chelator (DOTA). Besides developing a bimodal contrast agent, the goal was to use this material as high field (21.1T), which required the use of Dy(III) as the paramagnetic ion in the DOTA chelator. Bimodal contrast was achieved by transfecting CHO cells with the InP/ZnS-peptide-DOTA-Dy(III) and observing quantum dots optically via live cell imaging or by MR microscopy by suspending the CHO cells in an agarose tissue mimic. The bimodal contrast succeeded in both the fluorescence optical detection and the negative contrast detection by high field MR imaging.
Chapter 5 describes the ability to co-label a 5.7 nm AuNP with siRNA and linearized DNA for simultaneous dual gene regulation. The study was performed using two fluorescent proteins as optical signatures, eGFP (siRNA knockdown) and DsRed-express (delivered linear DNA). Using cationic liposome transfecting agents, the bimodal gene regulator is delivered to Lec-1 cells and CHO cells and shows efficient gene knockdown for eGFP up to 42 h after transfection but shows less efficiency for transient expression of the linearized gene, which was not detected until 36 h after transfection. An NSET molecular beacon study with dye-labeled linearized DNA and short dsDNA revealed that the short strand was able to release from the AuNP surface more efficiently and had more rapid and efficient endosomal escapes, which is known to be a rate limiting step in transient gene expression. The linearized DNA showed slower AuNP surface release and substantially less endosomal escape. The molecular beacon results confirm that the shorter siRNA strand can interfere with eGFP much more rapidly and efficiently given the rate of endosomal escape, whereas the low transient protein expression is not surprising given the low endosomal escape rate for the larger 2 kilobase, double-strand linearized plasmid DNA regulator.
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