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dc.contributor.advisorDahlman, James E.
dc.contributor.authorLokugamage, Melissa Prasangi
dc.date.accessioned2021-06-10T16:52:23Z
dc.date.available2021-06-10T16:52:23Z
dc.date.created2021-05
dc.date.issued2021-04-07
dc.date.submittedMay 2021
dc.identifier.urihttp://hdl.handle.net/1853/64722
dc.description.abstractNanoparticles improve drug efficacy by delivering drugs to sites of disease. To effectively deliver a drug in vivo, a nanoparticle must overcome physical and physiological hurdles that are not present in cell culture, yet in vitro screens are used to predict nanoparticle delivery in vivo. An ideal nanoparticle discovery pipeline would enable scientists to study thousands of nanoparticles in vivo. Here, we discuss technologies, such as DNA barcoding, to enable the efficient delivery of nucleic acid therapies and study the delivery of lipid-based nanoparticles. This work will specifically describe the improvement and development of nucleic acid delivery vehicles. In Chapter 2, we report new DNA barcodes designed with minimal secondary structure, dispersed semi-randomized sequences, and a digital droplet PCR (ddPCR) site can be quantified at low doses in vitro. We demonstrate that in vivo nanoparticle biodistribution measured with fluorescence underestimates the number of targeted cell types in vivo compared to ddPCR DNA barcodes. These study shows that rationally designed DNA barcodes can quantify delivery with higher sensitivity than traditional fluorescence-based assays. In Chapter 3, we quantified how over 100 nanoparticles delivered siRNA to 9 cell types in vivo using a novel siGFP-based barcoding system and bioinformatics. We found nanomaterials containing conformationally constrained lipids formed stable LNPs, herein named constrained lipid nanoparticles (cLNPs). cLNPs delivered siRNA and sgRNA to T cells at doses as low as 0.5 mg / kg, and unlike previously reported LNPs, did not preferentially target hepatocytes. The data suggest natural lipid trafficking pathways can promote T cell delivery, offering an alternative to active targeting approaches. In Chapter 4, we explored the relationship between inflammation and drug delivery. Specifically, we found that TLR4 activation could override LNP-mediated mRNA delivery. We determined that TLR4 activation blocked mRNA translation in several cell types, without significantly reducing LNP uptake Notably, the data suggest that the mechanism could be driven, in part, by TLR4-mediated reductions in endosomal escape and was likely driven by a TLR4-mediated reduction in mRNA translation. More generally, this suggests a LNP which delivers mRNA to one inflammatory disease may not deliver mRNA to another. In Chapter 5, we identify design rules for nebulized LNP delivery of mRNA using an in vivo, iterative, cluster-based experiments. We found that PEG-lipids are crucial for LNP formulation, low PEG molar percentages improve LNPs with neutral helper lipids, and high PEG molar percentages improve LNPs with cationic helper lipids. We also identified an LNP that delivers mRNA to the lungs at low doses and used this LNP to protect adult mice from a lethal H1N1 flu challenge. In Chapter 6, we studied whether a single LNP trait can reduce liver delivery and increase non-liver delivery. Using DNA barcoding, we identified three novel LNPs with distinct in vivo mRNA delivery profiles at clinically relevant doses, enabling us to edit genes in the lung by co-delivering Cas9 mRNA and sgRNA. Finally, in Chapter 7, we proposed new ideas within the field of nucleic acid drug delivery. We discuss ways to continue improving nucleic acid drug delivery. Specifically, we emphasize the need for new screening approaches, ways to understand the genes involved in drug delivery, and the importance of studying drug delivery across larger animal models.
dc.format.mimetypeapplication/pdf
dc.language.isoen_US
dc.publisherGeorgia Institute of Technology
dc.subjectNanoparticles
dc.subjectLipid nanoparticles
dc.subjectRNA
dc.subjectNanomedicines
dc.subjectDrug delivery
dc.titleUTILIZING HIGH-THROUGHPUT IN VIVO NANOPARTICLE SCREENS TO IMPROVE DRUG DELIVERY
dc.typeDissertation
dc.description.degreePh.D.
dc.contributor.departmentBiomedical Engineering (Joint GT/Emory Department)
thesis.degree.levelDoctoral
dc.contributor.committeeMemberSantangelo, Philip J.
dc.contributor.committeeMemberSorscher, Eric
dc.contributor.committeeMemberLam, Wilbur A.
dc.contributor.committeeMemberBotchwey, Edward
dc.date.updated2021-06-10T16:52:23Z


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