M.Sc Thesis


M.Sc StudentHagit Shalom Luxenbu
SubjectExploring the Nano-Ghosts Diagnostic Potential:Towards
Theranostic Applications
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Full Professors Machluf Marcelle
Full Thesis textFull thesis text - English Version


Abstract

The goal in cancer nanodrug delivery is producing selective targeted systems, as an effort to improve therapeutic approaches for cancer treatments, while reducing drugs' side effects and improving their pharmacokinetics and efficacy. Besides for therapeutic purposes, nanocarriers have also been more and more used for imaging applications, as well as combining both therapeutic and diagnosis to develop theranostic systems. This emerging field would allow noninvasive assessment of the bio-distribution and the targeted site accumulation of the nano-carriers and a real-time monitoring of the therapeutic responses. Most of the targeted nano-delivery systems are usually engineered by conjugating them with moieties that improve their selectivity. In our lab, we have developed a novel targeted delivery system for cancer therapy, which is based on cell-derived nano-vesicles termed Nano-Ghosts (NGs) produced from the cytoplasmic membrane of human Mesenchymal Stem Cells (hMSCs). These NGs retain the surface molecules that are responsible for MSCs’ cell-cell and cell-matrix interactions, thereby preserve their unique targeting capabilities towards cancer. In contrary to other cell membrane-derived vesicles, the NGs can be produced using a technologically scalable and pharmaceutically applicable process and were shown to efficient target and accumulate in tumor sites while demonstrating therapeutic response. Our goal in this work was to better localize and detect the NGs in vitro and in vivo, without interfering with their natural MSC-borne targeting capabilities. Moreover, we aimed to investigate how exposing the MSCs to inflammatory factors and cancer-derived conditioned media, prior to NGs production, affects the NG's targeting capabilities. For the diagnostic approach, we have labeled the NGs with lipophilic tracers. Labeling the NGs with these fluorescent probes were shown to be preferable relative to NGs' identification by antibodies. Furthermore, DiD lipophilic tracer had better performance than his analogue DiI, both in vitro and in vivo. Since labeling with lipophilic tracers didn’t answer the need for noninvasive imaging in vivo, procedures for loading fluorescent contrast agent into the NGs were successfully conducted. DyLight488? fluorescent dye was loaded into the NGs either passively, during the NGs' preparation process, or by post-perpetration electroporation. Dylight-loaded NGs were uptaken by A549 lung cancer cells and have shown favorable distribution in vivo. Further loading improvements should be implemented to noninvasively assess the bio-distribution and the targeted site accumulation of the NGs. Moreover, we discovered that stimulation of the MSCs with cytokines or cancer-derived conditioned media, prior to NGs production, altered the NGs’ cellular uptake by PC3, U87 and A549 cells. Stimulated NGs’ cellular uptake by PC3 cells in vivo was also investigated and supported the in vitro results.    

In summary, the success in loading the NGs with a contrast agent is promising and emphasizing the NGs' theranostic potential. Moreover, understanding the effect of stimulations prior the NGs production can allow programing of the NGs. Combining both applications will allow a better alternative for systems currently exist.