Hyaluronic acid conjugated β-cyclodextrin-oligoethylenimine star polymer for CD44-targeted gene delivery

Abstract

A new CD44-targeted gene delivery system, the star-shaped cationic polymer containing a β-cyclodextrin (β-CD) core and multiple branched oligoethylenimine (OEI) arms with conjugated oligomer of hyaluronic acid (HA), was synthesized by reductive amination between β-CD-OEI star polymer and HA, and was characterized for pDNA condensation and nanoparticle formation, followed by evaluation for targeted gene delivery of luciferase reporter gene and wild type p53 gene in CD44-positive and CD44-negative cell lines. The β-CD-OEI-HA polymer contained 6 arms of OEI (600 Da) and a short HA segment. It could fully condense pDNA to form nanoparticles with sizes ranging from 100 to 200 nm at N/P ratios of 8 or higher. The conjugation of HA reduced cytotoxicity of β-CD-OEI-HA/pDNA polyplexes. It was found that CD44 receptor was highly expressed and localized at the membrane of MDA-MB-231 breast cancer cell line, while no CD44 was found at the membrane of MCF-7 epithelial cell line. Compared with PEI (25 kDa) and β-CD-OEI star polymers, β-CD-OEI-HA demonstrated significant increased gene transfection efficiency in MDA-MB-231 cells, while such effect was absent in MCF-7 cells. The targeted delivery of wild type p53 gene by β-CD-OEI-HA in MDA-MB-231 cells resulted in an increased cell cycle arrest at sub-G1 phase.

Introduction

Cancer is the leading cause of death in the world. Currently, about 25% of deaths in the United States are attributed to cancer (Jemal et al., 2010). In the last two decades, gene therapy has been extensively explored and considered as a promising substitute for conventional chemotherapy (Park et al., 2006). Recently, re-establishing tumor suppressor regulated cellular pathways shows prospective outcome of an effective anti-cancer therapy (Roth et al., 2010). For example, the p53 protein acts as an internal guardian of the genome, and it can induce cell cycle arrest and apoptosis in response to carcinogenic signaling (Junttila and Evan, 2009). Introducing wild-type p53 gene into human lung cancer cells using a retroviral vector successfully induced apoptosis (Fujiwara et al., 1993), indicating a sufficiency of restating single gene function for cancer cell death in spite of multiple genetic disorder. Considering the fact that p53 gene mutations exist in approximately 50% of human cancers (Olivier et al., 2010, Robles and Harris, 2010), it is reasonable to restore the tumor-suppressive function of wild type p53 as an alternative means for cancer therapy (Roth, 2006).

Successful gene therapy depends on an efficient and safe delivery system that delivers the therapeutic genes to a specific target tissue or organ (Schaffer and Lauffenburger, 1998). Non-viral vectors arises as a promising approach for gene therapy due to their markedly safety over viral vectors (Glover et al., 2005). Polyethylenimines (PEIs) with linear or branched structure have been widely used as gene delivery vector, because of its superior transfection efficiency due to the efficient DNA condensation and proton sponge effect (Boussif et al., 1995, Coll et al., 1999, Park et al., 2006). However, their application in vivo has largely been limited by the disadvantages including high cytotoxicity (Clamme et al., 2003), lack of specificity and aggregation in blood (Chollet et al., 2002). To overcome these disadvantages, an alternative strategy is to introduce a less toxic and biocompatible or biodegradable backbone/core to assemble low molecular weight PEIs. In the last decade, several polymers that were characterized as low cytotoxicity and high transfection efficiency have been developed. These include the star-shaped copolymers (Li et al., 2010b, Yang et al., 2007a), comb-shaped copolymers (Chen et al., 2010, Ping et al., 2011, Xu et al., 2009a), and polymers with grafting low molecular weight PEIs (Li et al., 2006, Yang et al., 2007b, Yang et al., 2009).

Cyclodextrins (CDs) are a series of natural cyclic oligosaccharides composed of 6–8 (α-1,4)-linked α-d-glucopyranose units, named α-, β-, and γ-CD, respectively (Li and Loh, 2008). They have low toxicities and do not induce immune responses in mammals (Davis and Brewster, 2004). CDs have been profusely exploited to improve bioavailability of poorly soluble drugs, to prevent side effects or to enhance membrane permeability in pharmaceutical industry. Gonzalez et al. (1999) were the first group to introduce CD-based polymers for gene delivery. Since then, numerous attempts have been reported to utilize CDs to deliver nucleic acid with various structure, such as cross-linking CDs (Huang et al., 2006, Huang et al., 2008, Tang et al., 2006), CD-pendent polymers (Choi et al., 2005, Liu et al., 2008, Zhang et al., 2010), and polyrotaxanes (Li et al., 2006, Yang et al., 2007b, Yang et al., 2009). Among CDs, β-CD has been extensively used because of its availability and suitable cavity size for the widest range of molecules (Challa et al., 2005). In addition, the CD-containing gene delivery system can be further modified by inclusion complex formation (Davis and Brewster, 2004). Recently, a series of CD-centered, star-shaped polymers have been developed and evaluated for in vitro gene delivery (Wen et al., 2014, Xu et al., 2009b, Yang et al., 2007a, Zhao et al., 2014, Zhao et al., 2013).

Hyaluronic acid or hyaluronan (HA), is a natural linear polysaccharide that has pivotal roles in various biological functions, such as stabilizing and organizing the ECM, regulating cell adhesion, motility, mediating cell proliferation and differentiation (Knudson and Knudson, 1993). Due to its excellent biocompatibility and biodegradability, HA has been extensively investigated for biomedical applications such as tissue engineering (Mironov et al., 2005), drug delivery (Lee et al., 2008b), and molecular imaging (Lee et al., 2008a). In addition, HA specifically binds to its cell surface receptors, CD44 and RHAMM (receptor for HA mediated motility) to regulate cell proliferation and movement. CD44 activation enhanced its downstream pathways, e.g., Rac1, leading to tumor growth, progression and metastasis (Bourguignon et al., 2000). More recently, CD44 expressed at cell surface has been considered to be a stem cell marker in breast cancer (Reya et al., 2001). Therefore, we proposed that the specific binding of HA to its receptor CD44 could be utilized for therapeutic application targeting to CD44-positive breast cancer cells.

In the present study, we report a new CD44-targeted gene delivery system for safe and efficient delivery of therapeutic pDNA, which is based on a design of HA-conjugated β-CD star polymer with multiple oligoethylenimine (OEI) arms. The polymer, denoted β-CD-OEI-HA, was synthesized by reductive amination between cationic β-CD-OEI star polymer and low molecular weight HA. Our data demonstrated that the β-CD-OEI-HA polymer exhibited low cytotoxicity, high efficiency to condense pDNA, and high specificity for gene delivery in CD44-positive MDA-MB-231 breast cancer cells. In addition, β-CD-OEI-HA polymer mediated wild type p53 gene delivery sufficiently and effectively induced cell growth inhibition in the CD44-positive MDA-MB-231 cells. Our study provides a potential gene delivery method that specifically targets the aggressive CD44-positive breast cancer cells.