Author ORCID Identifier

https://orcid.org/0000-0001-7267-4424

Date of Award

Fall 12-12-2022

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Sciences

First Advisor

Didier Merlin

Second Advisor

Andrew Gewirtz

Third Advisor

Chunhua Yang

Abstract

Inflammatory bowel disease (IBD), comprised of Crohn’s disease (CD) and ulcerative colitis (UC), is chronic inflammation affecting more than 1.5 million people in the United States and 2.2 million people in Europe. CD can affect any portion of the gastrointestinal (GI) tract and is commonly patchy with a transmural inflammation pattern. UC is limited to the large intestine and has a continuous inflammatory pattern that involves only the mucosa. A commonality is a dysregulated immune response in the intestine. IBD develops in genetically susceptible individuals due to complex interactions between environmental stimuli, genome, microbiome, and an inappropriate mucosa immune response. However, the current treatment is not effective enough to show significant improvement in many patients. Here, we developed two therapeutic approaches to treat intestinal mucosal wounds found in ulcerative colitis in vitro and in vivo.

In the first section, I studied fecal metabolites derived from genetic knockout (KO) of peptide transporter-1 (PepT1) mice which are known to show resistance to acute colitis and colitis-associated cancer (CAC). The previous publication provided evidence that the PepT1 KO microbiota is sufficient to protect against colitis and CAC. Given that PepT1 KO alters the gut microbiome and thereby changes the intestinal metabolites that are ultimately reflected in the fecal samples. Therefore, we investigated the fecal metabolites of PepT1 KO mice using a liquid chromatography-mass spectrometry (LC-MS)-based untargeted metabolomics technique. We found that the specific fecal metabolite, tuberonic acid (TA), increased seven times higher in KO mouse fecal samples when compared to wild-type (WT) mouse feces. Accordingly, we performed research on whether the increased TA could have an anti-inflammatory effect. In vitro study discovered that TA not only prevented lipopolysaccharides (LPS)-induced inflammation in macrophages but also accelerated the epithelial cell healing processes.

In the next section, we studied mRNA-based therapeutics using lipid nanoparticle encapsulation. Inspired by the colon-targeting ability of ginger-derived nanoparticles (GDNPs) from our laboratory, we reversely engineered lipid nanoparticles (LNPs) that comprise the most abundant lipids in GDNPs, including phosphatidic acid (PA), monogalactosyldiacylglycerol (MGDG), and digalactosyldiacylglycerol (DGDG) at 5:2:3 ratio. We encapsulated IL-22 mRNA within the newly generated lipid nanoparticles (nLNPs) as enhanced IL-22 expression in the colon is known to have a potent anti-inflammatory effect against ulcerative colitis (UC). The IL-22 mRNA-loaded nLNPs were shown to be approximately 200 nm in diameter and have a zeta potential of -18 mV. Oral administration of the IL-22/nLNPs complex increased the protein expression level in the colonic mucosa. During acute colitis, mice fed with the IL-22/nLNPs experienced an accelerated healing process, as indicated by the recovery of higher body weight and longer colon length as well as reduction of colonic MPO activity, fecal lipocalin concentration, histological index, and mRNA expression level of pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β). These results indicate that the reversely engineered nLNPs are an effective mRNA delivery system to treat ulcerative colitis.

The last two sections elaborate on two different protocols to generate and characterize novel, natural, non-toxic ginger-derived nanoparticles (GDNPs) and ginger-derived lipid vesicles (GDLVs) that can be utilized as well-defined drug vehicles for treating inflamed intestinal mucosa. The first method uses sucrose gradient ultracentrifugation with extracted ginger juice to purify GDNPs. The concentration of obtained GDNPs was measured by a protein quantification assay kit and microplate reader at an absorbance of 750 nm. The second method uses liquid-liquid extraction to isolate lipid vesicles from GDNPs from the first protocol. Ginger-derived lipid vesicles (GDLVs) were generated by thin-film hydration and dissolved therapeutic agent, siRNA, by brief suspension by sonication. This GDLVs/siRNA complex was measured for its size and zeta potential, then its TEM and AFM images were acquired. In addition, in vivo test was performed to ensure its delivery functionality, bio-distribution, and efficiency. These two protocols provide a novel and crucial method to develop an efficient and safe drug delivery system that causes no immunotoxicity when orally administered. Furthermore, the nanostructure of GDNPs is the primary source of reversely engineered lipid nanoparticles for the mRNA-loaded delivery system (Page 41).

In conclusion, these therapeutic studies prove that the two different approaches of treating fecal metabolite and IL-22-loaded lipid nanoparticles to accelerate the healing process of intestinal inflammation are effective both in vitro and in vivo. Metabolites have emerged as essential effectors in mediating commensal microbiomes in human physiology. Positive results to show the anti-inflammatory effect of the specific metabolite derived from genetic knockout of PepT1 could be the groundwork of novel metabolites-based therapies. Safe and efficient RNA-based gene therapy using lipid nanoparticles was propelled into the spotlight due to the pandemic outbreak. The short-term oral administration of IL-22 mRNA protected by nLNPs successfully demonstrated a powerful mucosal healing effect. This may provide a potent gene therapy to treat ulcerative colitis.

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