Efficient inhibition of uveal melanoma via ternary siRNA complexes
Graphical abstract
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.
Section snippets
1. Materials and cell culture
Chitosan oligosaccharide lactate (Chi, MW: 5 kDa), formaldehyde solution (molecular biology), Transwell® insert (6.5 mm insert, 8.0 μm polycarbonate membrane, Corning®), 0.45 μm Immobilion®-P PVDF transfer membrane, and sodium dodecyl sulfate (SDS) were purchased from Millipore Sigma (St. Louis, MO). Sodium hyaluronate (HA, MW: 20 kDa) was purchased from Lifecore Biomedical (Chaska, MN). UranyLess EM stain was purchased from Electron Microscopy Sciences (Hatfield, PA). Water
Characteristics of siRNA complexes
The positive charge in the Chi backbone makes it easy for Chi to interact with negatively charged siRNA, to form nanosized complexes via electrostatic interaction (Howard et al., 2006, Katas and Alpar, 2006). As shown in Table 1, the hydrodynamic diameter (DH) of all siRNA complexes were below 200 nm. The binary complexes with a N/P molar ratio of 40/1 had smaller particle size (174.3 ± 7.72 nm) and PDI (0.26 ± 0.013) than that of the complexes with a N/P molar ratio of 20/1 (195.6 ± 2.55 nm,
Conclusions
We have developed a novel molecularly targeted therapeutic strategy based on biocompatible ternary siRNA complexes for the treatment of UM, the most common and malignant primary intraocular tumor in adults. The developed ternary siRNA complexes were below 190 nm in size and negatively charged, and had above 80% siRNA loading efficiency. Owing to the combination effect of HA and chitosan, the ternary complexes demonstrated improved cellular uptake and lysosomal escape ability. In addition, we
CRediT authorship contribution statement
Lingxiao Xie: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Data curation, Writing - original draft, Writing - review & editing, Visualization. Yan Yang: Formal analysis, Writing - review & editing. Jie Shen: Conceptualization, Resources, Writing - review & editing, Visualization, Supervision, Project administration, Funding acquisition.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgement
This work was fully supported by the Institutional Development Award (IDeA) Network for Biomedical Research Excellence from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103430.
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