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Access Type

WSU Access

Date of Award

January 2019

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Biomedical Engineering

First Advisor

Sandro R. da Rocha

Second Advisor

Mahendra Kavdia

Abstract

Diseases of pregnancy are the leading cause of maternal and neonatal morbidity and mortality affecting more than 20% (26 million) of all pregnancies annually. Those diseases include preeclampsia, preterm labor, intrauterine growth restriction and gestational diabetes, many of which are caused by compromised functions of the placenta. Placenta is a specialized organ that is only present during pregnancy where it creates a maternal-fetal interface that is responsible for many functions that contribute to the development of the fetus. Unfortunately, there is currently no treatment for any of those diseases. Our work focuses on the development of a novel nanoplatform that can be used to target the placenta so as to deliver therapeutics that can range from small hydrophilic and hydrophobic molecules or biologics which can be encapsulated or conjugated to the the nanoformulations .

In this work, we have established and characterized an in vitro placental model based on BeWo cell monolayers that mimic the placental barrier, the syncytiotrophoblast (SynT). Next, we synthesized lipid conjugates with gentamicin, as a targeting moiety that is a substrate to megalin receptors, which are expressed on the SynT, on the maternal (apical) side of the placenta. Using those targeting lipids, we successfully prepared and characterized targeting liposomes nanocarriers and showed that they are more significantly taken up by the BeWo cells in polarized monolayers compared to liposomes lacking the targeting lipid. Furthermore, we tested our liposomal formulations under different conditions: 1) saturated megalin receptors 2) a different cell line (HepG2) that does not express megalin, further confirming that liposomal targeting capability is receptor-mediated endocytosis, with results reinforcing the ability of the liposomes to target the in vitro SynT model and that the internalization mechanism is megalin mediated endocytosis. Subsequently, we established an in vivo model of the SynT using timed-pregnant Balb/c mice. On gestational day 18.5, we administered the liposomal formulations and assessed the biodistribution in placentas, kidneys, and fetuses. Placental analysis show significant accumulation of the targeting liposomes compared to the control non-targeting liposomes and the free Cy5.5 (drug model). Moreover, kidneys show significant accumulation, which was expected due to the fact that those organs also express megalin receptors. Fetal tissues showed no significant different between the three groups suggesting that the fetus should experience no toxicity due to limited exposure to the nanocarriers. This conclusion confirms our hypothesis and satisfies our goal of successfully establishing a nanoplatform for the targeted delivery of therapeutic to the placenta.

Once we had demonstrated the targeting ability of the liposome nanocarriers, we set out to develop another formulation based on biodegradable polyester dendrimers. Liposomes have the translational power given their well known toxicity profile, while dendrimers offer certain advantages including their small size and ability to control the release profile and embed trigger release mechanisms to further improve PK. We synthesized dendrimer-Cy5.5-PEG conjugates and are currently working on the conjugation of GM-PEG conjugates to the dendrimers to do the targeting studies. Initial PK work has been in non-pregnant animals for the PEGylated dendrimer, and in vitro and biodistribution studies will be completed once the targeting dendrimers are prepared.

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