Introduction

As gene-silencing agents, siRNA have significantly expanded the specificity and range of ‘druggable’ targets making them promising agents for precision medicine in cancer¹. Effective delivery and cellular uptake, the major challenges in siRNA nanoparticle (NP) therapeutics, become even more challenging in cancer because of the complexities of the tumor microenvironment (TME) such as the abnormal vasculature. Studies directly relating NP delivery to function are necessary to understand how these NPs navigate through the TME, and to understand the impact of the TME on siRNA delivery and function². Here we used triple negative MDA-MB-231 human breast cancer xenografts derived from cells engineered to overexpress VEGF to understand the role of VEGF and vascularization in NP delivery and function. VEGF overexpression increases vascularization in these tumors³. We used polyethylenimine (PEI) NPs to deliver siRNA to downregulate choline kinase-alpha (Chk-a), an enzyme that is associated with malignant transformation and tumor progression⁴. Chk-α converts choline to phosphocholine (PC), a key precursor of membrane phospholipids.

Methods

Tumors were obtained by inoculating 1×10⁶ MDA-MB-231 wild type cells, or MDA-MB-231 cells engineered to stably express VEGF, orthotopically in female SCID mice. PEI NPs were labeled with Cy5.5 dye for optical imaging with a Pearl® Trilogy Small Animal Imaging System (LI-COR, Lincoln, NE). PEI NPs delivering Chk-a siRNA were administered intravenously with two doses of 3 nmol Chk-a siRNA given 24h apart, once tumors were ~ 300-400 mm³. In vivo and ex vivo optical imaging was used to track NP delivery and distribution in 6 wild type and 6 VEGF overexpressing tumors at 24h following injection of the second dose of siRNA NPs. RT-PCR and immunoblotting were used to evaluate Chk-α levels in tumors excised at 24h after the injection of second dose. High-resolution ¹H MRS of tumor extracts at 11.7 T was used to evaluate changes in choline, phosphocholine, and glycerophosphocholine (GPC).

Results and Discussion

Significantly higher Cy5.5 fluorescence intensity was observed in VEGF overexpressing MDA-MB-231 tumors compared to wild type MDA-MB-231 tumors as shown in the representative in vivo (Fig. 1A) and ex vivo (Fig. 1B) images. Analysis of the tumor/muscle ratio demonstrated a significant increase of the intensity ratio in VEGF overexpressing tumors (Fig. 1C). Representative ex vivo images of Cy5.5 intensities in organs, summarized in Fig. 2, demonstrated that VEGF overexpression in tumors did not alter PEI NP biodistribution in the other organs including the liver, kidney, lung, heart, and spleen. RT-PCR (Fig. 3A) and immunoblots (Fig. 3B) analysis of tumors confirmed that the improved NP delivery in VEGF overexpressing tumors resulted in a significantly improved downregulation of Chk-a mRNA and protein. High-resolution 1H MRS of tumor extracts showed siRNA NP treatment did not significantly reduce phosphocholine in MDA-MB-231 tumors (Fig. 4A, B), whereas a significant decrease of phosphocholine was observed in MDA-MB-231-VEGF tumors (Fig. 4C, D). These data demonstrate the importance of vascular delivery in achieving successful target downregulation and highlight the role of imaging in evaluating NP delivery. Our data indicate that strategies to improve vascular delivery of NPs in tumors are important in the use of siRNA to achieve gene-specific downregulation.

Acknowledgements

Funding support: NIH R01 CA253617 and R35 CA209960