Novel combination therapy using photodynamic therapy and targeted VEGF-A siRNA nanoparticle delivery in head and neck cancer models

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Photodynamic therapy (PDT) promotes hypoxic condition to tumor cells leading to overexpression of angiogenic markers such as vascular endothelial growth factor-A (VEGF-A). Combination with antiangiogenesis therapy is expected to enhance the efficacy of PDT. In this study, PDT was combined with lipid-calcium-phosphate nanoparticles (LCP NPs) to encapsulate VEGF-A siRNA (siVEGF-A) and enhanced with anisamide to target sigma receptor overexpressing human head-and-neck squamous cell carcinoma (HNSCC) The measured LCP NPs size was 37.9 ± 2.4 nm and the zeta potential of 46.2 ± 0.6 mV attributing to the addition of anisamide. VEGF-A was up-regulated 24 hours post-PDT and sigma receptor were found to be expressed in HNSCC cells. In vitro SCC4 and SAS cellular uptake study using haloperidol to compete with the targeted LCP NPs containing Texas-Red Oligo DNA revealed that the nanoparticles were delivered through the sigma receptor. Cell viability was conducted at 25 nM of transfected siVEGF-A for combination therapy. In vitro combined therapy of PDT and siVEGF-A showed higher cell killing effect compared with PDT or gene therapy alone. In vivo xenograft animal model results show tumor growth inhibition for both SCC4 and SAS of decreased tumor volume to ~30% in groups treated with PDT and siVEGF-A compared to PBS group. In vivo VEGF-A expression were significantly reduced for groups treated with siVEGF-A compared with PBS and PDT and siScrambled. H&;E staining of tumor showed less mitotic figures for groups treated with PDT and siVEGF-A. Immunostaining of CD31, α-SMA and VEGF-A were significantly decreased for groups treated with siVEGF-A. Cleaved caspase-3 and in situ TUNEL assay show more apoptotic cells and reduced Ki-67 expression for treated groups compared to PBS group. In vivo toxicity assay suggests that there is no animal toxicity of the delivered dose. In summary, results suggest that PDT combined with targeted gene therapy targeting angiogenesis could be a potential therapeutic mean to achieve better therapeutic outcome for head-and-neck cancer.