Date of Award
Doctor of Philosophy (PhD)
Chemistry and Biochemistry
Xiao-Hong Nancy Xu
The multidrug ATP-binding cassette (ABC) membrane transporters (efflux pumps) are found in both prokaryotes and eukaryotes and they can extrude diverse structurally unrelated substrates, such as antibiotics and chemotherapeutic agents out of the cells. The efflux pumps are responsible for multidrug resistance (MDR) and the failure of numerous treatments in infections and cancers. All ABC membrane transporters share a common modular topology containing two transmembrane domains (TMDs) and two nucleotide binding domains (NBDs). The underlying molecular mechanisms regarding how the similar structural ABC membrane transporters could selectively extrude a wide variety of substrates and cause MDR, are not yet fully understood. Radioisotopes and fluorophores have been widely used as probes to study efflux kinetics of multidrug membrane transporters in bulk cells which could have masked interesting rare events from individual cells. Moreover, radioisotopes and fluorophores do not process size-dependent physicochemical properties, making them unsuitable to serve as various sized substrates for the study of efflux function of the ABC transporter. In this dissertation, we focus on the development of three different sized single silver nanoparticles (Ag NPs) to serve as both drug nanocarriers and imaging probes to study size-dependent efflux function of ABC membrane transporters in single live cells (e.g., Escherichia coli) in situ in real time. We synthesized and characterized Ag NPs with diameters of 2.4 ± 0.7, 13.0 ± 3.1, and 92.6 ± 4.4 nm, functionalized them with a monolayer of 11-amino-1-undecanethiol (AUT) to prepare AgMUNH2 NPs (control nanocarriers). We then covalently linked the AgMUNH2 NPs with ofloxacin (Oflx) to prepare AgMUNH-Oflx NPs (antibiotic drug nanocarriers) with conjugation ratios of 8.6x102, 9.4x103, and 6.5x105 Oflx molecules per NP, respectively. We studied inhibitory effects of these antibiotic drug nanocarriers against E. coli and found size-dependent inhibitory effects in which the same amount of Oflx carried by the largest nanocarriers exhibited the highest inhibitory effects, and the smallest nanocarriers exhibited the lowest inhibitory effects. The AgMUNH2 NPs did not show significant inhibitory effects on cell growth. Furthermore, we used Ag NP-based nanocarriers as imaging probes to study efflux function of multidrug ABC membrane transporters in single live E. coli cells, because Ag NPs process distinctive size-dependent photostable plasmonic optical properties. We found that the accumulation rates of nanocarriers highly depended on the NP concentration, the presence of ATPase pump inhibitor, and the types and sizes of nanocarriers. Interestingly, the ABC membrane transporters extrude AgMUNH-Oflx NPs more effectively and rapidly than AgMUNH2 NPs indicating that efflux pumps could be equipped with a sensing machinery to detect, recognize and extrude toxic substrates (e.g., antibiotics). Notably, the cells could extrude the smaller nanocarriers more effectively, leading to the least inhibitory effects. Therefore, the smaller drug nanocarriers could serve as excellent imaging probes to study the efflux function while the larger nanocarriers serve as powerful drug delivery vehicles. This study demonstrates the possibility of developing optimal-sized nanocarriers to achieve the maximum drug efficacy and potentially avoiding MDR.
"Study of ABC Membrane Transporters in Single Live Cells"
(2018). Doctor of Philosophy (PhD), Dissertation, Chemistry and Biochemistry, Old Dominion University, DOI: 10.25777/h7yf-dc17