Effective drug delivery is severely restricted by the presence of complex pathophysiological barriers in solid tumors. In human pancreatic adenocarcinoma, mature and hypopermeable tumor blood vessels limit the permeation and penetration of chemo or nanotherapeutics to cancer cells and substantially reduce the treatment efficacy. New, clinically-viable strategies are therefore sought to breach the neoplastic barriers that prevent optimal tumor-specific drug delivery. Here, we present an original idea to boost targeted drug delivery by selectively knocking down the tumor vascular barrier in a poorly permeable human pancreatic cancer model. For the first time, we demonstrate that clinical irradiation (10 Gy, 6 MV) can induce tumor vascular modulation when combined with tumor endothelial-targeting gold nanoparticles. Active disruption of tumor blood vessels by nanoparticle-combined radiotherapy led to increased vessel permeability and improved tumor uptake of two prototypical model nanodrugs: i) a shortcirculating nanocarrier with MR-sensitive gadolinium (Gad-NC; 8 kDa; t1/2=1.5 h) and ii) a longcirculating nanocarrier with fluorescence-sensitive NIR dye (FL-NC; 30 kDa; t1/2=25 h).Functional changes in the altered tumor vessel dynamics, measured by relative changes in permeability (Ktrans), flux rate (Kep) and extracellular interstitial volume (Ve) were consistent with the concomitant increase in nanodrug delivery. This combination of radiation-induced antivascular and nanodrug-mediated anti-tumor treatment offers high therapeutic benefit for tumors with pathophysiology that restricts efficient drug delivery.

Introduction: The objective of this project is to study the biological pathways activated in irradiated pancreatic cancer cells pre-treated with gold nanoparticles. Metallic nanoparticles emit Auger electrons and photoelectrons upon exposure to X-rays. When selectively delivered to tumors, these nanoparticles can locally enhance<?__anchored_object__ "ro_u170cins1e758"?><?__anchored_object__ "ro_u170cins1e759"?> the effects of radiation therapy. Previous in vitro work has primarily studied the effectiveness of nanoparticle-enhanced therapy, without elucidating the underlying biological mechanisms. Understanding the biological mechanisms (such as changes in gene expression) of how nanoparticles enhance radiation therapy can help in the further design of more effective nanoparticles. Methods: Gold nanoparticle sensitization was tested using a human based pancreatic cancer cell line, Capan-1. Nanoparticle toxicity was tested using a combination of MTS and Alamar Blue assays after 24 hours of treatment. Gene expression was tested using immunofluorescence and western blot techniques. Cells were pretreated with 0.8 mg gold nanoparticles 24 hours prior to radiation. Cells were then harvested at various time points to determine how gene expression changes over time. Radiosensitization was carried out using a clonogic cell survival assay where cells were pretreated with 0.8 mg gold nanoparticles 24 hours prior to radiation and then re-plated at various densities 4 hours post radiation, and allowed to incubate for 14 days. Statistical differences were determined using ANOVA followed by student t-tests. Results: The MTS and Alamar blue assays showed low gold nanoparticle toxicity towards cells up to a dose of 1mg for Capan-1 cells. Capan-1 cells pretreated with 0.8 mg gold nanoparticles for 24 hours prior to radiation showed lower cell survival than cells only treated with radiation. Western blots and immunofluorescence data showed that certain genes, such as γ-H2AX, were upregulated when pretreated with gold nanoparticles and irradiated compared to cells treated with radiation alone. It was also shown that γ-H2AX expression peaked around 30 minutes post radiation and then returned to basal levels after 24 hours. Conclusions: Capan-1 cells experience more DNA damage when pretreated with gold nanoparticles than when only treated with radiation. γ-H2AX, which is a DNA damage-related protein, was upregulated in the combined dose showing that there was more DNA damage in the combined treatment. It was also shown that protein and gene expression analysis needs to be carried out at various time points post radiation to obtain a better understanding of the mechanisms involved in radiation induced damage to pancreatic cancer cells. Supported by NSF-DGE- 0965843 and CA188833-02. Citation Format: Autumn D. Paro, Ilan Shanmugam, Anne van de Ven, Rajiv Kumar, Thomas J. Webster, Srinivas Sridhar. Biological mechanisms involved in nanoparticle-enhanced radiation therapy for pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr B44.