Date of Award
Doctor of Philosophy (PhD)
Chemistry and Biochemistry
Xiao-Hong Nancy Xu
Multidrug resistance (MDR) exists in both prokaryotic and eukaryotic cells. MDR is responsible for ineffective treatment of a wide range of diseases, such as infections and cancer. The ATP-binding cassette (ABC) membrane transporters (efflux pumps) are one of the largest and most diverse super-families of membrane proteins found in all living organisms, ranging from bacteria to humans. All ABC transporters share a common structure of four core domains; two transmembrane domains (TMD) with variable sequence and topology and two nucleotide-binding domains (NBD) with conserved sequences. Conventional methods for the study of the efflux functions are radioactively labeled substrates and fluorescent dyes, which provide the sum of accumulation kinetics of a population of cells. Notably, individual cells act independently of efflux and accumulation kinetics and therefore, the accumulation kinetics of single cells is lost in such bulk measurements. Furthermore, single fluorophores or radioisotopes themselves do not possess distinctive size-dependent physicochemical properties that would allow their sizes to be measured in situ in real time. Therefore, they cannot serve as various sized pump substrates for the study of size-dependent efflux function of single membrane transporters in single live cells. This dissertation focuses on development of single nanoparticle (NP) plasmonic spectroscopy and design of three different sized drug nano-carriers to study the size-dependent efflux function of ABC membrane transporters (e.g., MsbA and BmrA) in single live cells, two model organisms such as E.coli (gram-negative) and B.subtilis (gram-positive) bacterial cells., aiming to understand and overcome MDR. We designed, synthesized, and purified silver (Ag) nanoparticles (NPs) with diameters of 2.4 ± 0.7 nm, 13.0 ± 3.1, and 92.6 ± 4.4 nm, functionalized them with monolayer of 11-amino-1-undecanethiol hydrochloride (MUNH2), and linked them with antibiotics ofloxacin (Oflx) via two-step reaction process. We determined the conjugation ratio (the number of Oflx molecules attached to a single NP) of 2.4 ± 0.7, 13.0 ± 3.1, and 92.6 ± 4.4 nm as 8.6x102, 9.4x103, and 6.5x105 respectively. We characterized single nano-carriers using dark-field optical microscopy and spectroscopy (DFOMS). These drug nano-carriers exhibit photostability, which enabled us to study the size-dependent efflux functions of single membrane transporters in single live cells in real-time for a desired period of time. Using these three different sized nano-carriers, we found the size dependent inhibitory effects of the drug nano-carriers in single live cells, two strains of B.subtilis, WT (normal expression of BmrA) and ∆BmrA (deletion of BmrA). We have demonstrated that single plasmonic NPs can serve as unique size-dependent molecular imaging probes to study efflux functions of single membrane transporters in single live cells in real-time. Furthermore, we found that the smaller drug nano-carriers are much more biocompatible than the larger drug nano-carriers. In other words, the larger drug nano-carriers show higher inhibitory effects against the bacterial cells. Thus, the smallest nano-carriers (2.4 ± 0.7 nm) could be used to study the efflux function of ABC (BmrA) membrane transporters in single live cells, while the large nano-carriers could be used to effectively deliver antibiotics that could overcome MDR and enhance antibiotics efficacy.
Cherukuri, Pavan K.. "Design of Drug Nano-Carriers for Study of Multidrug Resistance in Single Live Cells" (2016). Doctor of Philosophy (PhD), dissertation, Chemistry and Biochemistry, Old Dominion University, https://digitalcommons.odu.edu/chemistry_etds/12