1. DESIGN AND DEVELOPMENT OF A POLYMER-BLEND NANOPARTICLE DRUG DELIVERY SYSTEM TO OVERCOME MULTIDRUG RESISTANCE IN CANCER Lilian E. van Vlerken 1, Steven R. Little 2, Zhenfeng Duan 3, Michael V. Seiden 3, Robert Langer 4, and Mansoor M. Amiji 1 1 Department of Pharmaceutical Sciences, Northeastern University, Boston, MA 02115; vanvlerken.l@neu.eduvanvlerken.l@neu.edu 2 Department of Hematology and Oncology, Massachusetts General Hospital, Boston, MA 02114 3 Department of Chemical Engineering, University of Pittsburgh, Pittsburgh, PA 15261 4 Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139 Abstract Introduction Materials and Methods Results Conclusion The development of multi-drug resistance (MDR) is a major barrier to anti-cancer therapy, since this phenotype renders the tumor unresponsive to a multitude of chemotherapeutic options. Alterations in apoptotic signaling have emerged as a common strategy for MDR development, whereby glucosylceramide synthase (GCS) causes bioactivation of the pro-apoptotic mediator ceramide to a non-functional moiety. The objective of this work is to overcome MDR through a nanoparticle-based therapy that administers ceramide (CER) in combination with the chemotherapeutic drug paclitaxel (PTX), to restore apoptotic signaling. For optimal therapeutic efficacy, we have engineered long-circulating polymeric nanoparticles composed of pH responsive poly(beta-amino ester) (PBAE) blended into a hydrophobic matrix consisting of poly(epsilon-caprolactone) (PCL) or poly(lactic-co- glycolic acid) (PLGA). By regulating the blending ratio of the two polymers, we could tune release of the combination therapy, specifically tailored to the tumor environment. Efficacy of the formulation was tested on a breast (MCF7) model of MDR cancer. Optimal size and stability of the nanoparticle formulation was found at a blend of 70%:30% and 80%:20% PCL/PLGA:PBAE, and a drug load of 2.5% PTX and 10% CER. Release studies revealed that the blend composition is pH responsive, where a surge in release occurred when spiked to pH 6.5. Moreover, release of PTX vs. CER could be tuned, where, compared to the 70/30 composition, CER release from the 80/20 PCL/PBAE particle was delayed while PTX release was accelerated. Unlike the other three formulations, the 70/30 PLGA/PBAE formulation released PTX rapidly, upon a drop in pH to 6.5, with a slow sustained release of CER. Efficacy studies then revealed the ability of this tuned therapeutic strategy to greatly chemo-sensitize the MDR cancer type, shown by an increase in cell death up to 79% following treatment with 1  M PTX and 8.6  M CER, compared to treatment with PTX alone at the same dose (27% cell death, p<0.001). Remarkebly, the novel therapeutic approach showed and equally successful chemosensitation profile with the drug-sensitive MCF7 cells. The results demonstrate a promising potential for use of these polymer- blend nanoparticles to fine-tune release drug profiles, where the application can chemosensitize not only MDR but also drug-sensitive cancer phenotypes. › Polymer-blend nanoparticles containing various ratios of PCL/PLGA to PBAE blending were fabricated by controlled solvent displacement › Nanoparticles were characterized for size and shape by SEM and TEM › pH-sensitive pocket formation and drug compartmentalization in the nanoparticle interior was modeled by casting the polymer-blends as films for imaging under light microscopy. Fluorescent derivatives of paclitaxel (rhodamine) and ceramide (NBD) were loaded into the polymer-blends to simulate drug compartmentalization within the polymer-blend nanoparticles › Drug release was performed into PBS containing 1% Tween-80 at pH 7.4 for the first 6 hours, then replaced by release medium at pH 6.5 for the duration of the study. Paclitaxel release was monitored by RP-HPLC, while ceramide release was monitored by fluorescence intensity of NBD-ceramide. › Human breast adenocarcinoma cells (MCF7) were cultured alongside their respective MDR subculture (MCF7TR) that had been selected for resistance in the presence of increasing concentrations of PTX. › Efficacy studies were perfomed on MCF7 and MCF7TR cells by treating the cells with the nanoparticle formulations alongside adequate controls for 6 days, after which remaining cell viability was quantitated using the MTT assay. Polymer-blend nanoparticles can be developed to tune release of combination therapies from a single nanoparticle drug delivery system. In this case, these blend nanoparticles can be used to compartmentalize the drugs inside the nanoparticle to successfully tune release of a paclitaxel and ceramide combination therapy. The overall result is a significant chemosensitization of MDR as well as regular drug sensitive MCF7 breast cancer cells. These results show promising use of this therapeutic strategy to overcome MDR and enhance anti-cancer therapy. Objective The purpose of this study was develop a novel therapeutic approach using polymer-blend nanoparticles for temporally-controlled co-administration of ceramide with the chemotherapeutic drug, paclitaxel, to overcome MDR in cancer. Acknowledgements L.E. van Vlerken is a recipient of an NSF IGERT fellowship in Nanomedical Science and Technology co-sponsored by the NSF and NCI. This study was further supported by the Nanotechnology Platform grant from the NCI (R01-CA119617). Special thanks to Dr. Lara Gamble for the University of Washington NESAC/BIO center for her help with XPS studies, supported by NIBIB grant EB-002027 › Drug sensitive (wild-type) cells show the same amount of cell death at a 10-fold lower concentration of paclitaxel than the MDR cells, verifying the MDR phenotype of the model. A combination therapeutic approach using ceramide then as a co-therapy aside the chemotherapeutic paclitaxel results in significantly increased cell death in not only the MDR, but also the drug sensitive MCF7 breast cancer population (Figure 3) › Polymer-blend nanoparticles using a blend of either 70% PCL:30% PbAE or 70% PLGA:30% PbAE display a spherical appearance and nanometer size-range as indicated by SEM and TEM (Figure 4) › PLGA:PbAE and PCL:PbAE polymer blends cast into films reveal that the two types of polymer-blends display strikingly different morphologies. Moreover, while the PCL:PbAE blend appears to etch away evenly when exposed to acidic solution, only parts of the PLGA:PbAE blend etch away, while leaving the remainder of the film untouched (Figure 5) › Drug compartmentalization into the PCL:PbAE and PLGA:PbAE films is revealed through the use of fluorescent-derivatives of the drugs. While in the PCL:PbAE blend the green and red fluorescence merge completely to suggest that the two drugs are evenly distributed thoughout the blend, in the PLGA:PbAE blend, the green and red fluorescence does not merge, suggesting that the drugs compartmentalize within PLGA:PbAE polymer blend nanoparticle. Moreover, when comparing to Figure 4, the red fluorescent paclitaxel appears to localize itself into the pH sensitive region that dissolves out when the pH changes to 6.5. This supports the hypothesis that paclitaxel localizes into the pH-sensitive pockets of the nanoparticle to allow for temporal release prior to release of ceramide. (Figure 6) › Drug release behavior shows that the PLGA:PbAE blend nanoparticle releases paclitaxel and ceramide with temporal control, where paclitaxel is rapidly released first, followed by a slow sustained release of ceramide. Efficacy studies show that this nanoparticle is then able to significantly enhance chemosensitization of not only MDR, but also drug sensitive cancer cells (Figure 7). › Multidrug Resistance (MDR) refers to the development of a cross-resistance to a multitude of structurally and functionally unrelated drugs. › Among the many mechanisms responsible for development of MDR in the cancer cell, alterations in apoptotic signaling appears to greatly contribute to the phenomenon, whereby the overexpressed enzyme glucosylceramide synthase (GCS) converts the apoptotic signaling mediator ceramide to an inactive form (glucosylceramide), › Previous work demonstrated that administering ceramide as a combination therapy with a chemotherapeutic (paclitaxel) to MDR breast and ovarian human cancer cells could overcome this blockade and reinstate apoptotic signaling initiated by chemotherapeutic stress. › Additionally, the work revealed that the ceramide/paclitaxel combination was optimally effective when ceramide was administered several hours following paclitaxel administration (Figure 1), › For optimal therapeutic efficacy, we developed a polymer blend nanoparticle allowing for simultaneous delivery of the combination therapy, but with controlled release of the two therapeutics. The chemotherapeutic paclitaxel was placed within pH-responsive PBAE pockets that released their load immediately upon internalization of the nanoparticle into the acidic tumor environment, while ceramide was placed within the hydrophobic matrix, composed of PCL or PLGA, which degraded slowly to release their load in a much delayed manner (Figure 2). 0 20 40 60 80 100 PTXPTX + CER sol'n PTX + CER @ t = 6hrs CER + PTX @ t = 6 hrs % cell survival p<0.001 p<0.05 (PCL) or (PLGA) Hydrophobic polymer Location of ceramide (PBAE) pH responsive polymer Location of paclitaxel (PEO) surface modification Figure 2 – illustration of the polymer blend nanoparticle design Figure 1 – Temporal relationship between paclitaxel (PTX) and ceramide (CER) administration to chemosensitize MDR cancer cells 500 nm Figure 4 – scanning electron microscopy (SEM) and transition electron microscopy (TEM – inset) images of a) 70% PCL:30% PbAE and b) 70% PLGA: 30% PbAE nanoparticles a. b. c. d. Figure 5 – Microscopy images of polymer films of a) 70% PLGA:30% PbAE and b) 70% PCL:30% PbAE, and degradation c) 70% PLGA:30% PbAE and d) 70% PCL:30% PbAE after exposure to pH 6.5 conditions (200x magnification) Figure 6 – Drug compartmentalization in the 70% PCL:30% PbAE polymer blend (top row) and the 70% PLGA:30% PbAE polymer blend (bottom row). Brightfield microscopy image with corresponding fluorescence images of red-fluorescent rhodamine- paclitaxel and green-fluorescent NBD-ceramide (200x magnification) brightfieldpaclitaxelceramidemerged PCL: PbAE PLGA: PbAE 0 20 40 60 80 100 % cell death MCF7 TR MCF7 1  M PTX 0.1  M PTX 1  M PTX + CER 0.1  M PTX + CER ** Figure 3 – Cell kill efficacy of the paclitaxel (PTX) and ceramide (CER) combination therapy on MDR breast cancer (MCF7 TR ) as well as wild- type breast cancer (MCF7). ** indicates a statistically significant difference (p<0.001) between PTX + CER and PTX alone (n=8/group) Figure 7 – a) cumulative drug release from the 70% PLGA: 30% PbAE nanoparticles exhibiting temporal control. b) Increase in cell kill efficacy of the 70% PLGA: 30% PbAE nanoparticles (NP) delivering a dose of 1  M paclitaxel (PTX) and 8.6  M ceramide over treatment with 1  M PTX alone. ** indicates a statistically significant difference (p<0.001) between NP and PTX treatment, and $$ indicates a statistically significant difference (p<0.001) between MCF7 TR and MCF7 cells in response to PTX treatment (n=8/group) time (hrs) 20 40 60 80 100 120 051015202530 % drug load released (cumulative) paclitaxel ceramide Drop in pH to 6.5 0 20 40 60 80 100 MCF7 TR MCF7 1  M PTX NP % cell death ** a.b. $$