br KRAS is a small GTPase involved in the regulation
KRAS is a small GTPase involved in the regulation of numerous cellular processes, including growth, proliferation, survival and other aspects of cellular biology accordingly to its active/inactive state (Ellis and Clark, 2000). KRAS mutations impair the ability of the KRAS pro-tein to switch between states, hence mutated KRAS acquires oncogenic properties; such mutations are observed in approximately 30% of tu-mors. In particular, pancreatic and colorectal cancers (Cox et al., 2014; Ross et al., 2017; Stephen et al., 2014), are involved in tumor initiation and maintenance (Chin et al., 1999; Collins et al., 2012; Zhang et al., 2006) and are associated with poor prognosis and increased resistance to treatment, including targeted therapies (e.g. EGFR-TKIs (Überall et al., 2008)). Therefore, it is not surprising that KRAS is considered an attractive target, but unfortunately an elusive one, since its intracellular location (Blasco et al., 2011) and very low concentration make its
selective inhibition very challenging. To date, probably the most suc-cessful approach has employed farnesyl transferase inhibitors (KRAS activation requires post-translational farnesylation), and the use of small molecules to block mutant KRAS and RAS family protein and their downstream effectors have been developed or are under clinical trial studies (Appels et al., 2005; Asati et al., 2017; Shin et al., 2018; Wang et al., 2013). Alternatively, small interfering RNA (siRNA) has been employed to target KRAS (Collisson et al., 2012; Hatzivassiliou et al., 2013; Kamerkar et al., 2017; Ross et al., 2017), with some systems translated to up to phase III trials for the intra-tumoral delivery of siRNA to downregulate KRAS, and reduce KRAS activity in pancreatic tumors (Zorde Khvalevsky et al., 2013).
Recently, it has been shown that the regulation of KRAS-mediated signaling in lung adenocarcinoma is strongly linked to CD44 expression (Zhao et al., 2013): interestingly, CD44 is both a diagnostic/prognostic marker  and a targetable internalization receptor , hence this
Corresponding author at: Division of Pharmacy and Optometry, Stopford Building, FBMH, University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom. E-mail address: [email protected] (A. Tirella).
Available online 26 February 2019
association may open the way to more selective KRAS-targeted treat-ments. In both its standard and higher molecular weight variant isoforms (CD44v2-v10), CD44 has a major role as a cell surface receptor for hyaluronic SP-600125 (HA) (Mattheolabakis et al., 2015; Ponta et al., 2003) responsible for both its recognition, binding and internalization (Culty et al., 1992). In tumors, CD44 is often expressed as its higher molecular weight variant isoforms, known to alter cellular behavior and signaling pathways (Culty et al., 1992; Misra et al., 2008). The variant isoform CD44v6 is of particular interest: it is not only expressed when tumor associated fibroblasts are activated, but also triggers receptor kinase activities suggesting again a correlation between CD44 and KRAS in adenocarcinomas (Kim et al., 1994; Misra et al., 2011). Unsurprisingly, HA has been widely employed in the context of CD44-targeting thera-pies, e.g. to improve water solubility or overcome drug resistance (Auzenne et al., 2007; Coradini et al., 1999; Luo et al., 2000, 2002), with some successful cases currently in clinical trials (Bassi et al., 2011; Rios de la Rosa et al., 2018a,b). We are specifically interested in HA-pre-senting colloidal carriers, which include liposomes (Surace et al., 2009), solid nanoparticles (Li et al., 2013; Ma et al., 2012; Yu et al., 2013), or self-assembly nano-systems (Ganesh et al., 2013; Janes et al., 2001; Lallana et al., 2017). These systems in principle, combine CD44 targeting and CD44-mediated internalization with the Enhanced Permeation and Retention (EPR) effect (Stylianopoulos and Jain, 2015), which can fur-ther help the selectivity of a targeted therapy.
Optimal therapeutic strategies should specifically target the mutated KRAS gene and have minimal systemic toxicity. To improve the se-lectivity and delivery of anticancer therapeutics, an effective strategy may require target-ligand interactions and formulation of nanoparticles able to promote internalization and cargo release at the desired in-tracellular site to effectively address the clinical translation aspects (Birzele et al., 2015; Karousou et al., 2017; Rios de la Rosa et al., 2017a). One strategy that our group and others have explored over the past decade is the use of HA-decorated nanoparticles in order to deliver nu-cleic acid via CD44-HA interactions. HA provides stability, low protein adsorption and CD44-targeting to the nanoparticles, which include also a polycation that binds both to HA and the payload (‘glueing’ together the carrier) and would then be responsible for endosomal disruption typi-cally through the ‘proton sponge’ mechanism (Almalik et al., 2013b; Deng et al., 2014; Lallana et al., 2017; Parajó et al., 2010). A critical attribute for such systems is indeed the nature of the polycation, which impacts dramatically on the transfection efficiency. Chitosan; for ex-ample, requires a careful optimization of molecular weight and degree of acetylation to achieve effective siRNA delivery (Lallana et al., 2017). In the context of achieving a CD44-mediated KRAS silencing therapy, we here investigated the influence of two main descriptors of the polycation performance, i.e. size and charge density. To enable this, we prepared a range of nanoparticles employing high molecular weight chitosan and low molecular weight poly(hexamethylene biguanide), commercially known as Nanocin (Chindera et al., 2016). The two respectively act as a very large, poorly charged polycation, and as a very small, densely charged one. Nanoparticles with anti-KRAS siRNA were obtained via combination of chitosan or Nanocin and HA coating and compared in-vestigating nanoparticles: stability/efficacy in the presence of RNases and after storage, ability to deliver siRNA in CD44+ tumor cells (color-ectal cancer cell line HCT-116), and reduction in KRAS expression. We