Doctor of Philosophy
Date of Defense
Prof. Keith J. Stine
Prof. Alexi V. Demchenko
Prof. Michael R. Nichols
Prof. Chung F. Wong
Glycoalkaloids (GAs) are secondary metabolites found mostly in higher plant species and some marine invertebrates. They are known to form complexes with 3β-hydroxy sterols such as cholesterol causing membrane disruption. So far the visual evidence showcasing the complexes formed between glycoalkaloids and sterols has been mainly restricted to some earlier studies using Brewster angle microscopy. This study aimed to develop a method for topographic and morphological analysis of sterol-glycoalkaloid complexes. Langmuir-Blodgett (LB) transfer of monolayers comprising of glycoalkaloid tomatine, sterols, and lipids in varying molar ratios onto mica followed by AFM examination was performed. The AFM method used required minimal sample preparation and allowed visualization of sterol-glycoalkaloid aggregation at nanometer resolution. While aggregation was observed in a mixed monolayer of tomatine with cholesterol and mixed monolayers with coprostanol, no sign of complexation was observed for the mixed monolayers of epicholesterol and tomatine. Similar aggregates were observed in ternary mixtures of tomatine with cholesterol and phospholipids 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or egg sphingomyelin (egg SM). Further, the effect of complexation on membrane integrity was studied using a supported bilayer composed of DMPC and cholesterol in a 7:3 molar ratio. Disruption of the bilayer was observed within five minutes of tomatine introduction into the aqueous medium above the supported bilayer.
The effectiveness of GAs against various cancer cells are reported in the literature. One major problem that prevents their practical application is the hemolytic nature of GAs. The possibility of employing mesoporous silica nanoparticles (MSNs) to circumvent this problem is explored. MSNs due to their high pore volume and tunable pore size enable loading of a large volume of molecules. Further surface modification of MSNs provides an opportunity to design a carrier system compatible with the host body and specific to target cells. Amino functionalized MSNs (MSN-NH2) coated with polydopamine (PD) and polyethylene glycol (PEG) were synthesized. The hemolytic activity of the nanoparticles (NPs) was tested. PD and PEG-coated MSNs showed no sign of hemolysis. Further, the PEGylation of MSNs resulted in reduced phagocytosis. GA α-tomatine was loaded inside the pores of MSN-NH2. Thermogravimetry analysis (TGA) indicated a loading of 4.7 %. The developed drug delivery system (DDS) showed a significant decrease in the hemolytic activity of α-tomatine. The effectiveness of the DDS was tested against human liver cancer (HepG2) cells. Cell viability assay suggested the effectiveness of the developed DDS was comparable to free tomatine. The cells showed signs of late apoptosis. An increase in cell viability upon the use of, QVD-OPh, a poly caspase inhibitor suggested caspase-mediated apoptosis might be a plausible cause of cell death.
AFM is a useful tool that finds its application across different branches of science. In material sciences, AFM is used to characterize the surface properties of materials such as roughness, and surface coverage. AFM was used for the characterization of nano porous gold (NPG) the observed structure was comparable to the structure observed under SEM. Further, the ability of AFM to record three-dimensional data was utilized to confirm the presence of the anti-cancer drug doxorubicin (DOX) on the surface of NPG. Further, AFM was utilized to capture images of RBCs and HepG2 cells. The effect of α-Tomatine on HepG2 cells was also visualized using AFM.
Nepal, Bishal, "Complexation of Glycoalkaloid α- Tomatine with Sterols and Its Potential Application as an Anti-Cancer Drug" (2021). Dissertations. 1086.
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