Exploitation Of P-Glycoprotein At The Blood Brain Barrier For Targeted Drug Delivery
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Drug development for the central nervous system (CNS) has struggled to reach clinical approval. One reason many drugs do not advance into clinical applications is their low uptake in the CNS due to the blood brain barrier (BBB). Targeted drug delivery to the CNS has been well-studied for over 30 years, and recently has been focused on methods of BBB disruption (e.g., focused ultrasound), circumvention (e.g., convection enhanced delivery) or exploitation (e.g., receptor mediated targeting). Receptor mediated targeting is a method of active transport across the BBB by exploiting endogenous receptorligand interactions. The work outlined in this dissertation has studied a novel drug delivery method for receptor mediated targeting through the exploitation of P-glycoprotein (P-gp). P-gp is naturally overexpressed at the BBB and therefore makes an attractive target for CNS drug delivery. It was hypothesized that this new approach to CNS delivery could be accomplished by creating a polymeric nanoparticle delivery system with a P-gp substrate as a targeting moiety. The work focused on the development of a polylactide (PLA) nanoparticle containing a surfacetethered polyethylene glycol (PEG) linker terminated with rhodamine as a P-gp targeting moiety. Rhodamine dyes are a well-known class of P-gp substrates and the two used in this study, rhodamine 6G (Rho6G) and rhodamine 123 (Rho123) show high and moderate affinity to P-gp, respectively. Due to the novelty of this system, the PEG-Rho linker was first assessed in vitro to determine if it was still capable of interacting with P-gp as a substrate. It was evident that the conjugates of PEG-Rho still remained P-gp substrates; therefore, the PLA-PEG nanoparticle was developed to assess targeting of the drug delivery system in vivo. Before targeting efficiency could be measured in vivo a nanoparticle detection method was needed. The autofluorescence of various tissues poses a problem when considering nanoparticle detection by fluorescence in vivo. Therefore, the time resolved fluorescent properties of europium chelates were utilized to overcome autofluorescence challenges. Europium chelates continue to emit photons microseconds after excitation, whereas the autofluorescent molecules in tissues emit photons for only nanoseconds. By measuring photon emittance at microsecond timescales following excitation, the autofluorescent background was eliminated allowing sensitive detection of the nanoparticles in vivo. Once the Rho-PEG-PLA nanoparticle was synthesized and a detection method to track and quantify the particles in vivo was developed, the targeting efficiency of the systems was assessed. In a mouse model, Rho6G-PEG-PLA nanoparticles accumulated 2.6 times greater in the brain than untargeted control mPEG-PLA nanoparticles. Using a P-gp knockout mouse, the accumulation of Rho6G-PEG-PLA nanoparticles was shown to significantly decrease in the brain compared to the wild type mouse. Thus the conclusion was made that Rho6G-PEG-PLA nanoparticles can actively target P-gp at the BBB and can enhance the accumulation of drug delivery nanoparticles in the CNS.
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