Date of Award

January 2018

Document Type

Thesis

Degree Name

Medical Doctor (MD)

Department

Medicine

First Advisor

David H. Stitelman

Abstract

Congenital disorders represent a large unmet clinical need with significant morbidity and premature mortality resulting from mutations and anomalies that can be diagnosed, and eventually treated, in utero. Nanoparticles (NP) have been used as versatile protective vessels for the delivery of a variety of therapeutic agents. Importantly, the potency of NPs can be amplified by modifications to the molecular composition as well as to the delivery to allow targeting to specific tissues. We hypothesize that optimization of NP targeting both by nanoparticle modification and the route of administration can increase overall uptake in specific cells and tissues of interest. To this end, we sought to 1) determine the fetal biodistribution of NPs based on gestational timing and route of administration; 2) optimize antibody target selection on tissues of interest; and 3) demonstrate conjugation-dependent uptake of targeting NPs in target cells. Fluorescent-dye loaded NPs were injected intra-amniotically (IA) or intravenously (IV) in mouse fetuses at 15-18 days gestation. Accumulation of NPs in fetal organs was determined by imaging and microscopy. Antibody target selection was performed by immunohistochemistry to assess accessibility as well as antibody binding curves to assess receptor density and binding affinity. Selected antibodies were conjugated onto fluorescent NPs. Epithelial cells were treated with these targeting NPs and selective uptake was determined over time by flow cytometry. Fetal mice start breathing and swallowing amniotic fluid at E16 gestation, allowing for delivery of IA injected NPs into the lungs and gut. IV injections at E15 and E16 result in greatest accumulation in fetal liver. Characterization of epithelial cell surface markers was able to predict targeting results. Conjugation of anti-mucin1 (MUC1) and anti-intracellular adhesion molecule (ICAM) antibodies resulted in conjugation-dependent increased uptake in epithelial cells. Targeting of NPs to tissues can be directed by timing and route of injection. Internalization of NPs can be significantly enhanced by conjugation to antibodies specific to target cells and tissues. Application of targeting technology in the scope of fetal therapy is novel and will contribute significantly towards advanced treatments and cures for congenital disorders.

Comments

This thesis is restricted to Yale network users only. It will be made publicly available on 06/27/2020

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