Efficient inhibition of uveal melanoma via ternary siRNA complexes

Abstract

Uveal melanoma (UM) is rare yet the most common and malignant primary intraocular tumor in adults. Due to the lack of effective treatment, the mortality rate of UM has remained high over the past few decades. In the present study, hyaluronic acid (HA) coated chitosan (Chi)/siRNA ternary complexes have been developed and characterized as a novel therapeutic strategy molecularly targeting hypoxia-inducible factor 1α (HIF-1α) pathway for the treatment of UM. The cytotoxicity, cellular uptake, and siRNA silencing effect of the developed siRNA complexes were evaluated. In addition, whether the developed ternary complexes can inhibit UM migration and invasion was investigated. Results showed that the developed ternary siRNA complexes were negatively charged and with a particle size below 190 nm. The ternary siRNA complexes showed excellent cellular uptake and lysosome escape ability with low cytotoxicity. In addition, the ternary complexes were able to downregulate both HIF-1α and VEGF expression in UM cells, and successfully inhibit UM migration and invasion. These results demonstrated that the biocompatible ternary siRNA complexes are promising for local treatment of UM in the posterior segment with future clinical application potential.

Introduction

Uveal melanoma (UM) is rare yet the most common primary intraocular tumor in adult, accounting for 5% of all melanomas and 13% of melanoma deaths (Harbour, 2012, Kaliki and Shields, 2016). At present, the common first-line treatments for this malignancy include eye enucleation, radiation (e.g. brachytherapy), and surgical resection. However, aggressive treatment such as enucleation and irradiation is not an “immediate treatment” option when tumor is below 16 mm in diameter and 9 mm in thickness (Shields et al., 2009). Nearly half of UM patients ultimately develop distant metastases (predominantly to the liver) that are notoriously resistant to systemic chemo- and immunotherapy, with a median survival of five to seven months after the detection of metastasis (Bhatia et al., 2012, Damato, 2006, Diener-West et al., 1992, Moser et al., 2015, Spagnolo et al., 2012). Despite advances in the diagnosis and localized treatment over the past decades, there is currently no effective therapies for the treatment of UM (Munzenrider, 2001).

Posterior UM (e.g. choroidal and ciliary body) accounting for more than 95% of UM cases, exhibit a strong tendency for lethal liver metastasis, thus poor prognosis and high mortality (Carvajal et al., 2017, Woodman, 2012). The uvea is one of the most capillary-rich tissues of the body, yet lack of lymphatic drainage. Therefore, UM metastatic spread occurs strictly via the hematogenous route (Amirouchene-Angelozzi et al., 2015, Onken et al., 2014). Metastasis is a major challenge not only in UM therapy, but also in overall clinical management of many cancers. It is considered that metastasis involves critical interactions between tumor cells and microenvironment. For instance, hypoxia environment promotes metastatic progression of cancer cells. Clinically, overexpression of hypoxia-inducible transcription factors in various cancer types is associated with increased distant metastasis and poor survival (Rankin and Giaccia, 2016). Downregulation of hypoxia-inducible factor (HIF) has been shown to decrease metastatic potential of various tumor cells such as breast cancer, lung cancer, and melanoma (Hanna et al., 2013, Hiraga et al., 2007, Liao et al., 2007, Wong et al., 2011, Zhang et al., 2012). HIF-1α, a transcription factor regulates the expression of secreted factors that mediate angiogenesis and tumor metastasis, is strongly associated with the gene expression profile of Class 2 UM (very likely to metastasize) (Asnaghi et al., 2014, Hu et al., 2016, Martinengo et al., 2014, Mouriaux et al., 2014). Although downregulating HIF-1α via either lentivirus-mediated short hairpin RNA (shRNA) delivery or a small molecule HIF-1α inhibitor digoxin showed inhibition of UM cell invasion in vitro (Asnaghi et al., 2014), these therapeutic strategies are not practical for localized treatment against UM metastasis in the posterior segment due to their intrinsic toxicity and side effects.

In the present study, a novel, biocompatible ternary siRNA complex was developed to deliver molecularly targeted anti-HIF-1α siRNA in the posterior eye segment to inhibit UM progression and invasion via downregulating HIF-1α gene expression in UM cells. Chitosan is one of the most commonly used polycations in gene delivery due to its relatively low toxicity, low immunogenicity, and high biocompatibility (Gao et al., 2005, Ghosh et al., 2007, Mansouri et al., 2004, Mao et al., 2010). However, high positive charge density in high molecular weight (MW) chitosan can lead to slow siRNA release due to strong electrostatic interaction, resulting in delayed or impaired gene silencing effect (MacLaughlin et al., 1998, Mao et al., 2010). Moreover, high MW chitosan is often associated with poor water solubility under physiological environment, which may hamper its complexion ability with siRNA (Dehousse et al., 2010). Accordingly, a water-soluble, low MW chitosan oligosaccharide lactate (Chi) has been selected as a carrier to condense anti-HIF-1α siRNA to form Chi/siRNA nanocomplexes. In order to avoid immobilization of the nanocomplexes in the vitreous humor and facilitate their transport to the tumor site (Martens et al., 2015, Xu et al., 2013). An anionic coating of hyaluronic acid (HA), a non-immunogenic and ubiquitous biopolymer in human organisms including vitreous humor, was used to shield the cationic charge of the Chi/siRNA nanocomplexes and facilitate transport of the nanocomplexes in the vitreous humor (Martens et al., 2015, Xu et al., 2013). The therapeutic strategy via ternary Chi/siRNA nanocomplexes for the treatment of UM was assessed.