Date of Award

6-1-2015

Degree Type

Dissertation

Embargo Date

7-6-2017

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

Advisor(s)

Mathew M. Maye

Subject Categories

Physical Sciences and Mathematics

Abstract

Metal and semiconductor nanocrystals (NCs) have unique optical and physical properties that are dependent on size, composition and morphology. When NCs are coupled to biomolecules, their properties are combined to create unique materials with biomimetic capabilities that can function as biosensors, cellular imaging agents or drug delivery vehicles. Most NCs are synthesized in air free, non-polar conditions, so surface chemistries must be tuned to accommodate hydrophilic biomolecules. This can be achieved through ligand exchange or polymer encapsulation procedures. This work takes advantage of both phase transfer routes to functionalize gold nanoparticles (AuNPs), quantum dots (QDs), and quantum rods (QRs) with DNA and proteins for self-assembly, energy transfer and drug delivery applications.

In the first project, we explored the ability to assemble QDs into clusters with a high degree of control through DNA-mediated interactions. The hydrophobic QDs were first transferred to buffers using a polymer encapsulation approach that used an amphiphilic polymer. The polymer encapsulated QDs were successfully functionalized with oligonucleotides through both EDC/NHS coupling and click chemistry. The final QD/DNA conjugates were assembled into multicolor QD clusters through a colloidal stepwise approach. One of the greatest challenges of this project was an inconsistent batch-to-batch QD/DNA coupling efficiency, which was attributed to the presence of excess polymer, QD aggregates and poor stoichiometry. Purifying QDs via ultracentrifugation in a sucrose density gradient removed excess polymer, leading to a decreased optical scattering and increased DNA loading that was beneficial for increasing coupling efficiency. In these clusters, a decrease in the QD donor emission and an increase in the QD acceptor emission indicated that QD-QD FRET occurred. One disadvantage to using QDs as energy acceptors is their broad absorption profile, which causes them to be coexcited with the donor. To overcome this limitation, a bioluminescent protein can be used to generate QD emission through bioluminescence resonance energy transfer (BRET) without external excitation.

In the next project, CdSe/CdS quantum rods (QRs) were functionalized with the bioluminescent firefly protein, Photinus pyralis (Ppy). The aim of this project was to improve the long-term stability of the QR/Ppy conjugates. To make these conjugates, hydrophobic CdSe/CdS QRs are rendered hydrophilic through a ligand exchange with histidine (His) followed by an additional ligand exchange to conjugate hexahistagged Ppy proteins to QRs (QR/His/Ppy). In these conjugates, there was a decrease in the stability of the BRET over time. The retention of the BRET signal was significantly improved by changing the QR capping ligand prior to protein conjugation from His to glutathione (GSH). This is because the GSH ligands that remain on the QR surface after Ppy coupling are more highly charged than His, leading to more efficient electrostatic repulsions between QRs. To incorporate the improved QR/Ppy nanoconjugates into the QD/DNA clusters, the QR emission should be a result of non-radiative energy transfer contributions only to prevent simultaneous excitation of the energy donor and acceptor. To investigate the contribution from radiative energy transfer to the BRET signal, control experiments were performed that indicated that most of the BRET signal arises from non-radiative energy transfer from the Ppy to the QR.

In the last project, DNA functionalized AuNPs were used as drug carriers for idarubicin (IDA), a clinically approved chemotherapeutic agent. To construct these conjugates, AuNPs are synthesized using a citrate reduction method and a ligand exchange is carried out to exchange the citrate capping molecules with thiol modified DNA and thermoresponsive polymers. Drug binding was investigated using DNA denaturation measurements and kinetic studies. An increase in duplex DNA melting temperature with drug loading verified IDA intercalation at the dsDNA. The kinetics of drug release were investigated at physiological temperature, where the presence of drug outside of a dialysis membrane was monitored through IDA fluorescence. The low drug release, small dissociation rate constant of 0.05 min-1 and high equilibrium constant of 3.0 x 108 M-1 demonstrates that these nanoconjugates can act as efficient vehicles for in vivo drug delivery.

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Open Access

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