Two-methoxyestradiol (2ME or 2ME2) is a highly water insoluble metabolite of estrogen, which possesses antiangiogenic activity and can upregulate death receptor 5 and interferon-signalling pathway, exhibiting anticancer effects. It is practically water insoluble and drug crystals milled to nano-size are evaluated in humans without success to date. While 2ME can inhibit tumor growth, its insolubility (<1ug/ml) may relate to low oral bioavailability. Some studies used vehicles to improve bioavailability. To address insolubility and improve antitumor activity, we evaluated 2ME’s ability to be incorporated into lipid nanoparticles (LNPs) that have been shown to be safe and to provide some tumor cell selectivity. To do so, we investigated different lipid compositions in their abilities to incorporate water-insoluble 2ME. The drug-lipid particles were made with solvent evaporation and hydration method. The free 2ME was removed to determine the degree of drug incorporation into LNP. Drug concentrations were determined with a high-performance chromatography (HPLC) assay. The LNP particle diameter was measured by photon correlation spectroscopy. Among tested lipid compositions, DSPC and DSPE-PEG2000 mixture provided about 80% drug incorporation at drug-to-lipid mole ratio of 1:6. The diameters of the 2ME-LNPs were 47.2±7.2nm. The anti-cancer activity was evaluated in two cell lines, MDA-MB-231 and A549. The 50% effective drug concentration to inhibit cell growth or EC50 of 2ME in MDA-MB-231 breast cancer cells were 0.83µM and 32.4µM for free and LNP respectively. For lung cancer A5492 cells, EC50 was recorded at 2.13 and 3.35 µM respectively for free vs. LNP formulation. These results demonstrated that (1) lipid incorporation overcomes 2ME water insolubility and (2) the drug-lipid particles provide high (~80%) incorporation efficiency thus likely reducing the overall cost, (3) a stable lipid-drug formulation that can be easily scaled and (4) the antitumor cell potency may vary with different cancer cells.

Vitamin D is an essential hormone in the body that helps regulate calcium and phosphate homeostasis. The effects of vitamin D in the body depend on dietary intake, sun exposure, and the ability of the body tissues to convert vitamin D into biologically inactive and active forms. One of the metabolic processes involves the sulfation of 25-hydroxyvitamin D₃ [25(OH)D₃], a form of the hormone that is a major circulating vitamin D metabolite in humans and can be excreted into the bile to affect the function of intestinal epithelial cells. In this research experience, I studied the relative efficiency of the 25(OH)D₃ sulfation reaction by liver sulfotransferase enzyme (SULT2A1) and the effects of the sulfate metabolite on intestinal epithelial cell gene expression. Using purified SULT2A1 and pooled human liver cytosol, I helped to determine the rate of the sulfation reaction over various 25(OH)D₃ concentrations. The Michaelis-Menten equation was used to determine kinetic parameters, which established the efficiency of 25(OH)D₃ sulfation. In order to determine which enzymes are regulated by 25(OH)D₃-sulfate, I tested the effects of 25(OH)D₃-sulfate on the expression of different gene targets in human intestinal cell systems. LS180 cells were used as a human cell model in order to examine how changes in concentration of phosphate solution and 25(OH)D₃-sulfate affect the enzyme gene expression in the cells. The experiments consisted of LS180 in various concentrations of phosphate and 25(OH)D₃-sulfate solutions. The cells were incubated, and lysed to isolate their RNA. Then, the RNA was converted into DNA to be retrieved via a PCR machine, which amplified the difference in relative mRNA expression among different enzymes. This study is expected to provide in vitro evidence that 25(OH)D₃–sulfate plays an important role in regulation of intestinal gene expression, which contributes to optimization of individualized drug therapies using vitamin D.