Interleukin-22 (IL-22) has been demonstrated as a critical regulator of epithelial homeostasis and repair; it showed an anti-inflammatory effect against ulcerative colitis. Local microinjection of IL-22 cDNA vector has been shown to be effective in treating ulcerative colitis in mouse models. However, microinjection comes with multiple technical challenges for routine colon-targeted drug delivery. In contrast, oral administration can get around these challenges and provide comparable efficacy. We showed in previous studies that oral administration of new lipid nanoparticles (nLNP)-encapsulated IL-22 mRNA targets the colon region and efficiently ameliorates colitis. This protocol describes the details of preparing and characterizing the nLNP-encapsulated IL-22 mRNA using three major lipids that mimic the natural ginger-derived nanoparticles. It provides an nLNP platform that can be used to orally deliver other types of nucleic acids to the colon.

Oral administration of colon-targeting nanoformulations holds many advantages over the systemic delivery of free drugs, or traditional nontargeting formulations in the treatment of ulcerative colitis (UC). Currently, the most conventional method for constructing colon-targeting drug delivery systems (DDS) is by integrating the biocompatible materials poly(lactic-co-glycolic acid) (PLGA) and polylactic acid (PLA) into a copolymer. This PLGA/PLA-polyethylene glycol-folic acid (PEG-FA) copolymeric nanoformulation effectively delivers the drugs for uptake by various human colon cancer cells (e.g., HT-29 and HCT-116) and mouse colon cancer cells (CT-26). There is, however, a distinct lack of comprehensive protocols for the construction of such copolymer. This protocol details an easy-to-follow single-step method for the construction of a colon-targeting PLGA/PLA-PEG-FA nanoformulation, which encapsulates a fluorescent dye and demonstrates the visualization of its cell uptake in vitro.

We have demonstrated that a specific population of ginger-derived nanoparticles (GDNP-2) could effectively target the colon, reduce colitis, and alleviate colitis-associated colon cancer. Naturally occurring GDNP-2 contains complex bioactive components, including lipids, proteins, miRNAs, and ginger secondary metabolites (gingerols and shogaols). To construct a nanocarrier that is more clearly defined than GDNP-2, we isolated lipids from GDNP-2 and demonstrated that they could self-assemble into ginger lipid-derived nanoparticles (GLDNP) in an aqueous solution. GLDNP can be used as a nanocarrier to deliver drug candidates such as 6-shogaol or its metabolites (M2 and M13) to the colon. To characterize the nanostructure of GLDNP, our lab extensively used atomic force microscopy (AFM) technique as a tool for visualizing the morphology of the drug-loaded GLDNP. Herein, we provide a detailed protocol for demonstrating such a process.

Synthetic nanoparticle-based drug delivery system is widely known for its ability to increase the efficacy and specificity of loaded drugs, but it often suffers from relatively higher immunotoxicity and higher costs as compared to traditional drug formulations. Contrarily, plant-derived nanoparticles appear to be free from these limitations of synthetic nanoparticles; they are naturally occurring biocompatible vesicles that do not generate immunotoxicity and are easy to obtain. Additionally, lipids isolated from plant-derived nanoparticles have shown the capability of assembling themselves to spherical nano-sized liposomal particles. Herein, we employ lipids extracted from ginger-derived nanoparticles and load them with therapeutic siRNA (CD98-siRNA) to create CD98-siRNA/ginger-lipid nanoparticles. Characterization of the CD98-siRNA/ginger-lipid nanoparticles showed that they present a spherical shape, with a diameter of around 189.5 nm. The surface zeta potential of the nanoparticles varies from -18.1 to -18.4 mV. Furthermore, in recent research, the CD98-siRNA/ginger-lipid nanoparticles have shown specific colon targeting capability and excellent anti-inflammatory efficacy in a Dextran Sodium Sulfate (DSS) induced mouse model of colitis.

Exosomes secreted by colonic epithelial cells are present in feces and contain valuable epigenetic information, such as miRNAs, proteins, and metabolites. An in-depth study of this information is conducive to the diagnosis or treatment of relevant diseases. A crucial prerequisite of such a study is to establish an efficient isolation method, through which we can obtain a relatively more significant amount of exosomes from feces. This protocol is designed to effectively isolate a large number of exosomes from contaminants and other particles in feces by a combined method with fast filtration and sucrose density gradient ultracentrifugation. Exosomes generated by this method are suitable for further RNA, protein, and lipid analysis.

Factors implicated in the pathophysiology of intestinal inflammation include defects in intestinal epithelial barrier function, abnormal immune responses, and activities of the gut microbiota. Current agents used to treat human Inflammatory Bowels Disease (IBD), chronic inflammation of digestive tract, have serious side effects. In addition, most of these treatments target the damaging factors while not providing pro-healing factors that repair the damaged intestine. Here we provide a method to isolate, purify and characterize a specific population from ginger (ginger-derived nanoparticles: GDNPs 2) with anti-inflammatory activities. GDNPs 2 as a drug vehicle are a novel natural, nontoxic delivery system, which target the inflamed intestinal mucosa, blocks damaging factors while promoting pro-healing factors and could easily be developed for large-scale production aimed at the treatment of IBD.

Here, we describe an in vitro epithelial wound-healing assay using Electric Cell-Substrate Impedance Sensing (ECIS) technology. The ECIS technology is a real time cell growth assay based on a small (250 μm diameter) active gold electrode which resistance is measured continuously. When intestinal epithelial cells reach confluency on the gold electrode, resistances reach a plateau. For the wound-healing assays, confluent intestinal epithelial monolayers are subjected to a current of 40 kHz frequency, 1,400 μA amplitude, and 30-second duration. This kills the cells around the small active gold electrode, causing detachment and generating a wound that is healed by surrounding cells that have not been submitted to the current pulse. Wound healing is then assessed by continuous resistance measurements for approximately 30 h after wound. Both cell wounding and measurements of the subsequent healing process are carried out under computer control that takes online measurements each 30 s and stores the data. ECIS technology can be used to study the underlying causes for impaired mucosal healing and to test the efficacy of drugs in mucosal healing.