(T1230-03-13) Effect of α-tocopherol Conjugation on Polycation-Mediated siRNA Delivery to Orthotopic Pancreatic Tumors

Purpose: Pancreatic Cancer is one of the deadliest cancers with low 5-year survival rate of around 10% [1]. Pancreatic ductal adenocarcinoma (PDAC) being the most common and aggressive forms of Pancreatic cancer represents 90% of all cases [2]. Despite several diagnostic and therapeutic advances in the last decade, the death rate has enhanced by around 1% per year [3, 4]. The high mortality rate is due to the asymptomatic nature of the early stage of the PDAC [5, 6]. Since it is already known that CXCR4 and STAT3 display crucial effects on proliferation [7], metastasis [8], angiogenesis [9, 10], and formation of immunosuppressive microenvironment in pancreatic tumors. Here, we hypothesized that conjugation of α-tocopherol (TOC), a naturally occurring hydrophobic molecule, to a polycation (PAMD), synthesized from CXCR4-antagonist AMD3100, will improve delivery of therapeutic siRNA to silence STAT3 in PDAC tumors [11, 12].

Methods: PAMD was synthesized using previously standardized method through Michael Addition [38]. Tocopherol was then conjugated to PAMD using 1,1’- carbonyl di-imidazole (CDI) coupling to make PAMD-TOC. The content of Tocopherol was determined from ¹H-NMR and the molecular weight for PAMD was analyzed using GPC. The nanoparticles were prepared using siRNA at different w/w ratio and characterized using gel retardation assay, DLS, and TEM imaging. Zeta potential was also analyzed. Further ahead, cellular uptake was done for the nanoparticles in two different cells lines- KPC8060 (murine) and Panc-1 (human). Anti-cancerous and anti-migration effect of the nanoparticles was also analyzed in these 2 cell lines. Biodistribution of nanoparticles was done in PDAC models. Anti-tumor efficacy of the nanoparticles was also done in syngeneic PDAC and orthotopic human xenograft mouse model along with the study of the effect of nanoparticles on survival of KBP8060 orthotopic PDAC model.

Results: PAMD-Toc was synthesized with 8wt% Toc content. On CXCR4 redistribution assay it was found that CXCR4 internalization was inhibited with the AMD3100, PAMD and PAMD-Toc. On cytotoxicity test, PAMD-Toc showed lower toxicity as compared to PAMD in both KPC-8060 and Panc-1 cells. On gel retardation assay, PAMD-Toc condensed siRNA at w/w ratio of more than 2 with smaller size of 100nm and 14mV zeta potential. Confocal microscopy showed improved cellular uptake for the PAMD-Toc/siRNA in both the cell lines. These nanoparticles showed deeper penetration in the spheroids of both the cell lines. Cells treated with PAMD-Toc/siSTAT3 showed strong effect on inhibition of the colony formation. Similar reduction in the migration of the cells was seen when they were treated with the PAMD-Toc/siSTAT3. CXCL12 was used as a chemoattractant. Upon biodistribution, the PAMD-Toc/siSTAT3 showed significantly higher tumor accumulation as compared to other nanoparticle formulations. Therapeutic efficacy of the PAMD-Toc./siSTAT3 treated mice, both KPC8060 orthotopic PDAC model and orthotopic human xenograft PDAC model showed considerable reduction of tumor volume and metastasis. In fact, KPC8060 orthotopic human xenograft PDAC model treated with PAMD-Toc/siSTAT3 showed 100% survival.

Conclusion: PAMD-Toc enhanced the cellular uptake and penetration ability of nanoparticles in both the KPC 8060 and Panc-1 cell lines. It also contributed to the outstanding transfection, anti-cancer and anti-migration efficacy in vitro as compared to the parent PAMD/siSTAT3 nanoparticles. Tocopherol conjugation also enhanced tumor accumulation and penetration of nanoparticles in both KPC8060 orthotopic PDAC mice and Panc-1 human xenograft orthotopic PDAC mice. It dramatically suppressed the tumor growth and migration in both the animal models by remodeling the immunosuppressive tumor microenvironment. It also improved survival ability in the KPC8060 orthotopic model.

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Acknowledgments: This work was supported by start-up funds from the University Of Nebraska Medical Center and by NIH grant R01 CA235863.

Synthesis of PAMD (A), TOC-CDI (B), and PAMD-TOC (C).



Bio distribution of nanoparticles in KPC8060 orthotropic PDAC mice and PANC-1 human xenograft orthotropic PDAC mice through IP administration (siRNA 2 mg/kg, polymers 6 mg/kg).



Therapeutic efficacy of nanoparticles in A) orthotropic KPC8060 PDAC mice and B) PANC-1 human xenograft orthotropic PDAC mice through IP administration. Primary tumor volume during the treatment was monitored by ultrasound.