(16b) Triple Delivery Nanoscale Device for siRNA, Vismodegib and Gemcitabine Co-Delivery to Treat Pancreatic Cancer

Pancreatic cancer (PC) has an extremely poor prognosis with a five-year survival rate of less than 7%.¹ PC is characterized by extensive desmoplasia, which limits the delivery and efficacy of chemotherapy. Sonic hedgehog (Shh)-signaling plays an important role in the tumor growth through desmoplasia, metastasis and angiogenesis.² Shh also regulates epithelial to mesenchymal transition (EMT), is overexpressed in PC stem cells and contributes to resistance and PC progression. ³ In addition, previous studies from our lab have shown that MUC4 is aberrantly overexpressed in human PC and has a role in the progression and metastasis of PC cells.⁴ Currently applied standard therapy for PC, involving surgical cytoreduction followed by systemic gemcitabine (GEM) based chemotherapy, has not provided desired outcome yet. As an alternative, small interfering RNA (siRNA) based therapy has been proposed for the PC treatment. However, targeted co-delivery of siRNA along with GEM and Vismodegib (VIS), an SHH antagonist, is challenging due to varying physicochemical properties, drug release rates, and pharmacokinetics. There is currently no ideal delivery system available to provide transport across vascular endothelial barriers, diffusion through extracellular matrix, and that can facilitate cellular entry and endosomal escape, while maintaining siRNA stability and providing site-specific release and activity.

In this study, we proposed the use of a temperature and pH responsive, cationic, amphiphilic and biocompatible pentablock (PB) copolymer based triple delivery nanoscale device (TDND) for in vitro and in vivo combined delivery of siRNA, VIS and GEM for the treatment of PC. This novel TDND facilitated cellular uptake due to amphiphilic character, endosomal escape due to protonatable tertiary amine groups in the cationic end blocks, and provided efficient simultaneous siRNA, VIS and GEM release in the cytoplasm through temperature responsive micellization. We hypothesized that targeting MUC4 expression through siRNA treatment and Shh signaling through VIS treatment via the TDNDs results in suppression of MUC4 expression, down regulation of SHH signaling pathway, inhibition of desmoplasia, pancreatic stellate cells and cancer stem cells. This may lead to an improved therapeutic outcome of GEM in PC through improving its perfusion in the tumor.

The PB copolymer consisting of temperature responsive PluronicF127 (poly(ethyleneoxide)-block-poly(propyleneoxide)-block poly(ethyleneoxide) (PEO-PPO-PEO)) middle block and pH responsive cationic (poly(2-diethylaminoethyl methacrylate)) (PDEAEM) end blocks was synthesized through atom transfer radical polymerization (ATRP). The TDND was prepared through the electrostatic complexation of PB copolymers with siRNA and subsequent self-assembly with VIS and GEM encapsulated PluronicF127 layers at different ratios. The TDND was characterized to evaluate size, zeta potential, serum and RNase stability. The simultaneous siRNA, VIS and GEM release test was conducted. In vitro toxicity, transfection efficiency, cellular uptake and endosomal escape capability of TDNDs were evaluated against Capan-1 PC line.

Our results indicated that at N/P ratios (N/P: molar ratios of nitrogens (N) in the pentablock copolymer to phosphates (P) in siRNA) above 7.5, complete complexation occurs which provides siRNA protection and stability while preventing the premature release. The stability of the TDND system was also tested in the presence of serum and RNase enzymes for 72h. It was observed that the TDNDs with N/P ratio of 12.5, 25 and 50 showed good siRNA protection and stability against serum and RNase enzyme. The designed TDND system with N/P ratio of 50 also provided efficient transfection and endosomal escape in Capan-1 human pancreatic adenocarcinoma cell line. This TDND system showed significantly higher transfection (~2.5 fold higher) than commercially available agent, Lipofectamine. The tumor cell killing efficiency of the TDND in Capan-1 pancreatic carcinoma cell line was also tested. TDND with N/P ratio of 50 (siRNA, GEM and VIS co-loaded) showed a synergistic effect in killing the cancer cells (80% of the cells were killed) compared to the control cases. We also investigated the simultaneous release of siRNA, GEM and VIS. It was observed that the thermoresponsive gelling TDND systems acted like a drug depot and were able to provide simultaneous release of siRNA, VIS and GEM and reached a plateau within 72h. Almost 90% of the siRNA and GEM were released within 5 days, while ~33% of VIS was released. The in vivo therapeutic efficacy of the TDNDs were also tested and showed significant reduction in tumor growth.

Overall, the TDND developed in this study was capable of overcoming the current hurdles of combination siRNA/drug delivery and showed significant activity in PC cell lines in vitro and in vivo for translational medicine and future clinical applications. This strategy provided an enhanced understanding of the mechanism of action underlying the synergistic activity of combined delivery.

 

1. Siegel, R. L.; Miller, K. D.; Jemal, A., Cancer statistics, 2016. CA: a cancer journal for clinicians 2016, 66 (1), 7-30,

2. Bailey, J. M.; Swanson, B. J.; Hamada, T.; Eggers, J. P.; Singh, P. K.; Caffery, T.; Ouellette, M. M.; Hollingsworth, M. A., Sonic hedgehog promotes desmoplasia in pancreatic cancer. Clinical cancer research : an official journal of the American Association for Cancer Research 2008, 14 (19), 5995-6004, PMID:PMC2782957.

3. Lee, C. J.; Li, C.; Simeone, D. M., Human Pancreatic Cancer Stem Cells: Implications for How We Treat Pancreatic Cancer. Translational Oncology 2008, 1 (1), 14-18,

4. Rachagani, S.; Torres, M. P.; Kumar, S.; Haridas, D.; Baine, M.; Macha, M. A.; Kaur, S.; Ponnusamy, M. P.; Dey, P.; Seshacharyulu, P.; Johansson, S. L.; Jain, M.; Wagner, K. U.; Batra, S. K., Mucin (Muc) expression during pancreatic cancer progression in spontaneous mouse model: potential implications for diagnosis and therapy. Journal of hematology & oncology 2012, 5, 68, PMID:PMC3511181.