Mantle cell lymphoma (MCL) is an extremely difficult to treat subtype of non-Hodgkin lymphoma (NHL) with a low patient survival rate compared to most common cancers. Recently, nanoparticle delivery systems have received a great deal of interest for treating NHL. One of the more promising cargo options for these systems is short interfering RNA (siRNA). siRNA is a 18-23 nucleotide long double stranded RNA that is used to inhibit the protein expression of target mRNAs in a sequence specific manner. MCLs have several commonly overexpressed genes compared to normal cells making it an ideal candidate for siRNA therapies. For RNA interference to occur, A delivery vehicle is needed for the siRNA to reach the cytoplasm of the cell. In this thesis, ionizable lipid-like materials termed lipidoids are formulated into lipid nanoparticles (LNPs) to deliver siRNA. A new library of lipidoids is constructed to gain a better understanding of how the lipidoid tail-structure affects the silencing ability of LNPs. A novel tail precursor is identified as conferring potency to LNPs. Then, LNPs are used to silence genes within difficult to transfect MCL cells. LNPs targeting the anti-apoptotic protein Mcl-1 exhibit potent gene silencing and cause an increase in the fraction of cells undergoing apoptosis. This is important because there is no therapeutic that is FDA approved that targets this commonly overexpressed protein. Because of this LNP’s potency, siRNAs targeting multiple genes can be encapsulated into LNPs without causing unwanted toxicity. LNPs targeting several genes in multiple pathways cause a larger fraction of MCL cells to undergo apoptosis compared to cells treated with LNPs targeting only one gene. A major issue in cancer therapeutics is that the majority of nanoparticles accumulate in the liver. In an effort to improve the delivery of LNPs to target cells, changes to their formulations and administration methods are investigated as a means to improve LNP circulation time, biodistribution, and silencing ability. Overall, this work identifies lipidoid nanoparticles as potent siRNA delivery systems to treat MCL and investigates key properties for further improvement in LNP siRNA delivery to target cells.

Intestinal diseases affect millions of people worldwide. Recently, a number of proteins have been shown to be upregulated in the intestinal cells of patients that contribute to disease progression. Therefore, these diseases could be amenable to RNA interference technology (RNAi). Utilizing RNAi to deliver short interfering ribonucleic acid (siRNA) to intestinal cells shows promise for the treatment of diseases by specifically suppressing the expression of disease relevant proteins. A class of lipid nanoparticles termed lipidoid nanoparticles (LNPs) have been shown previously to potently deliver siRNA to several cell types in vitro and in vivo. Here, we seek to establish the utility of lipidoid nanoparticles (LNPs) in the context of oral siRNA delivery to intestinal cells for the treatment of intestinal diseases. Initial in vitro studies demonstrated that the siRNA-loaded LNPs mediated potent, dose dependent, and durable gene silencing in Caco-2 intestinal cells without inducing significant cytotoxicity or altering intestinal barrier function. LNP stability studies revealed that LNPs in an aqueous buffer remained stable for long periods of time when stored in the refrigerator (2 °C) compared to the freezer (-20 °C) or at room temperature. In addition, LNPs remained stable upon lyophilization with the addition of trehalose or sucrose to the LNP solution before freeze-drying. To determine potential for oral LNP delivery, we studied LNP stability under gastrointestinal (GI) tract conditions. LNPs remained potent and stable following exposure to solutions of varied pH, including pH values as low as 1.2. However, efficacy decreased following exposure to increasing concentrations of pepsin and bile salts. Mouse oral biodistribution studies indicated that siRNA-loaded lipid nanoparticles were retained in the GI tract for at least 8 hours. Confocal microscopy confirmed that nanoparticles entered the epithelial cells of the mouse small intestine and colon. Oral LNP therapeutic efficacy was measured in an inflammatory bowel disease (IBD) mouse model by targeting the upregulated genes myosin light chain kinase (MLCK) and Interleukin 18 receptor (IL18R) and were found to prevent some IBD disease progression. Lastly, a formulation for the co-delivery of siRNA and messenger RNA (mRNA) was developed and it was discovered that a negatively charged polymer can be used to improve LNP efficacy. Together, these studies have advanced our knowledge of lipid nanoparticle stability, and potential as an orally delivered intestinal therapeutic.

Delivery vehicles are necessary for many therapeutics to overcome the various challenges in their path. It is clear, however, that the relationship between delivery vehicles and the immune system is a complex one. One such delivery vehicle is the lipidoid nanoparticle, which has been shown to be potent in several cell types. This thesis details the first time lipidoids have been used for wound delivery, and demonstrates the successful silencing of an inflammatory protein, TNFα, in the context of diabetic ulcers. Knockdown is seen in an in vitro macrophage-fibroblast coculture model, as well as in nondiabetic and diabetic mice wound models. Lipidoids silence roughly half of the TNFα gene expression in the diabetic wound and have been shown to help the wound close faster than untreated controls. Of course, immune activation can decrease therapeutic efficacy or trigger dangerous reactions in the patient. Learning more about what chemical moieties cause an immune response would allow for the design of a particle that could better resist immune clearance and avoid the creation of a secondary response. This thesis investigated the effect of a lipidoid library on the immune system using a two pronged approach. The lipidoids were first tested against human peripheral blood mononuclear cells and then were injected into mice to probe the in situ immune response. Several types of B cells were examined in this latter case, namely germinal center B cells, plasma cells, and memory B cells. A T cell dependent response occurred, favoring memory B cells for most of the lipidoids tested. There was an increase in free antibody in the blood that reflected this increase in antibody producing cells. Nitrogen rings and carbon tail lengths of eleven and twelve carbons were particularly reactive, though it appears that the amine head group determines immune response more than the tail. Further work will analyze whether these increases in immune cells reflect a loss of therapeutic efficacy, as current ramifications are unclear. An in-depth T cell subset analysis with flow cytometry would also help complete the picture.