1. MODULATION OF INTRACELLULAR CERAMIDE USING POLYMERIC NANOPARTICLES TO OVERCOME MULTIDRUG RESISTANCE IN CANCER CELLS Lilian E. van Vlerken 1, Dinesh Shenoy 1, Zhenfeng Duan 2, Michael Seiden 2, Shashi Mehta 3, and Mansoor M. Amiji 1 1 Department of Pharmaceutical Sciences, School of Pharmacy, Northeastern University, Boston MA 02115 2 Department of Hematology and Oncology, Massachusetts General Hospital, Harvard Medical School, Boston MA 02114 3 Department of Pathology, Roger Williams Medical Center, Providence, RI 02908 Abstract Introduction Materials and Methods ResultsConclusion The development of multidrug resistance (MDR) in many tumor types is a major barrier to successful anti-cancer therapy. One of the mechanisms that leads to such chemoresistance is inhibition of apoptotic signaling in MDR cancer cells through glycosylation of the apoptotic mediator ceramide. The purpose of this study was to investigate whether MDR could be reverted by co- administering exogenous Ceramide with a chemotherapeutic (Paclitaxel), co-encapsulated in polymeric nanoparticles to produce a multifunctional anticancer therapy. For starters, accumulation and localization of the nanoparticles intracellularly was observed by fluorescent microscopy. Next, to determine efficacy of this novel therapeutic strategy, the percent cell death of drug sensitive vs. multidrug resistant cancer cells in response to the nanoparticle treatment was quantified. Lastly, apoptotic activity in response to the paclitaxel/ceramide co-therapy was measured. Results indicate that nanoparticle delivery of the co- therapy reduces chemoresistance of the MDR cells to paclitaxel 100-fold, to produce a chemosensitivity profile in the MDR cells that is similar to their drug-sensitive counterpart cell-line. Co- therapy of ceramide with paclitaxel appeared to increase apoptotic activity 2 fold in the MDR cells, suggesting that delivery of exogenous ceramide reinstates the apoptotic signal to resensitize MDR cells to chemotherapy. In conclusion, a combination therapy of paclitaxel with ceramide, delivered in polymeric nanoparticles, appears to greatly re-sensitize drug resistant ovarian tumor cells to chemotherapy. The results demonstrate the promising potential for clinical use of this therapeutic strategy to overcome MDR. › PEO-PCL nanoparticles were prepared by the controlled solvent displacement and loaded with 10% w/w paclitaxel (PTX) or 20% w/w C6-ceramide (CER). › Intracellular nanoparticle trafficking was performed by loading the PEO-PCL nanoparticles with rhodamine-PTX (0.1% w/w) or NBD-C6-CER (0.1% w/w), and incubating the particles with the DS and MDR cells for 0.5, 1, 2, or 6 hours. Cells were fixed, and imaged by fluorescence microscopy. › The wildtype (drug sensitive (DS)) human ovarian cancer cell line SKOV3 was maintained in culture alongside an SKOV3TR subculture selected for MDR in the presence of PTX. › For cytotoxicity studies, the DS and MDR cells were subjected to dose-response studies against PTX, CER and PTX combined with CER, delivered as free drugs in solution or delivered in PEO-PCL nanoparticles. The P- glycoprotein inhibitor Verapimil (VPM) was used as a positive control for MDR modulation. Resulting cell death/viability after 6 days of treatment was measured by the MTS (formazan) assay. › Apoptotic activity in response to PTX/CER co-treatment was measured by Yo-Pro-1 and propidium iodide staining (Vybrant #7, Invitrogen), and quantitated by Laser Scanning Cytometry and fluorescence microscopy. A therapeutic strategy that co-administers PTX with CER, delivered in polymeric nanoparticles significantly re-sensitizes drug resistant tumor cells to chemotherapy, while also increasing chemosensitivity of DS cancer cells. The results demonstrate the promising potential for clinical use of this therapeutic strategy as an anticancer treatment for MDR as well as non-MDR cancer types. › Multidrug Resistance (MDR) refers to the development of a cross-resistance to a multitude of structurally and functionally unrelated drugs. › The development of MDR poses a great problem in cancer therapy, where tumors survive despite invasive chemotherapy. › Among the many mechanisms responsible for development of MDR in the cancer cell, alterations in apoptotic signaling appears to greatly contribute to the phenomenon › Many MDR cancer cells overexpress Glucosylceramide Synthase (GCS), which converts the apoptotic signaling mediator ceramide to an inactive form (glucosylceramide), rendering the apoptotic signal incomplete. › Exogenously administering ceramide to MDR cells should overcome this blockade and reinstate apoptotic signaling initiated by chemotherapeutic stress. › Nanoparticles are useful drug delivery vehicles for cancer therapy, since they 1) protect the drug from inactivation and metabolism inside the body, 2) preferentially accumulate at the tumor site by enhanced permeability and retention, and 3) deposit the drug, or a combination of drugs, intracellularly. Objective The purpose of this study was develop a novel therapeutic approach using polymeric nanoparticles for co-administration of ceramide with the chemotherapeutic paclitaxel, to overcome MDR in ovarian cancer. Acknowledgements L.E. van Vlerken is a recipient of an NSF IGERT fellowship in nanomedical science and technology. This study was supported by NIH grants CA-095522 and CA-119617. SKOV3TR Figure 2 -. Dose response relationship of SKOV3 vs. SKOV3TR cells to PTX. 0 20 40 60 80 100 120 0.00010.0010.010.1110100 [PTX] (  M) % cell viability SKOV3 › PEO-PCL nanoparticles are spherical in shape with a mean diameter of 211.6 ±1.8 nm ( figure 1a ) › PTX and CER nanoparticles are readily endocytosed by SKOV3 and SKOV3TR cells to release the drug load intracellularly by 6 hours ( figure 1b, 1c ) › CER co-treatment not only greatly re-sensitizes the MDR cells to PTX chemotherapy, but also increases cell-kill efficacy in DS cells. Delivery of the drugs encapsulated in PEO-PCL nanoparticles enhances chemosensitization of both DS and MDR cells (figure 3a,3b ). › PTX/CER co-treatment increases apoptotic activity 2-fold in both the MDR and DS cells ( figure 4a,4b ), suggesting that exogenous CER reinstates apoptotic signaling, triggered by PTX, in the MDR cells. Figure 1 – Imaging of PEO-PCL nanoparticles by Scanning Electron Microscopy (A), and i ntracellular accumulation of the PEO-PCL nanoparticles loaded with rhodamine-PTX (red) and NBD- CER (green) into SKOV3 (B) and SKOV3TR (C) cells after 6 hours incubation. A. B.C. Figure 3 - Therapeutic efficacy of PTX/CER nanoparticle treatment. Percent cell viability of SKOV3 (A) and SKOV3TR (B) after 6 days of PTX, CER, or PTX/CER treatment delivered as free drug (top panel) or within PEO-PCL nanoparticles (bottom panel). PTX/VPM treatment (SKOV3TR) is a positive control for MDR modulation. (* indicates statistical significance of p<0.001 between PTX and PTX/VPM treatment; n=8 samples/group) % apoptotic cells 0 20 40 60 controlCER PTXPTX + CER p<0.05 controlCER PTXPTX + CER p<0.05 A. B. Figure 4 - Percent apoptotic SKOV3 (A) and SKOV3TR (B) cells in response to the CER, PTX or the PTX/CER treatment after 24 hours. SKOV3 cells (A) received 10 nM PTX and/or 10  M CER, while SKOV3TR cells (B) received 1  M PTX and/or 10  M CER. (n=6 samples/group)