Anisamide-targeted PEGylated gold nanoparticles designed to target prostate cancer mediate: Enhanced systemic exposure of siRNA, tumour growth suppression and a synergistic therapeutic response in combination with paclitaxel in mice

Abstract

Small interfering RNA (siRNA) has recently illustrated therapeutic potential for malignant disorders. However, the clinical application of siRNA-based therapeutics is significantly retarded by the paucity of successful delivery systems. Recently, multifunctional gold nanoparticles (AuNPs) as non-viral delivery carriers have shown promise for transporting chemotherapeutics, proteins/peptides, and genes. In this study, AuNPs capped with polyethylenimine (PEI) and PEGylated anisamide (a ligand known to target the sigma receptor) have been developed to produce a range of positively charged anisamide-targeted PEGylated AuNPs (namely Au-PEI-PEG-AA). The anisamide-targeted AuNPs effectively complexed siRNA via electrostatic interaction, and the resultant complex (Au₁₁₀-PEI-PEG₅₀₀₀-AA.siRNA) illustrated favourable physicochemical characteristics, including particle size, surface charge, and stability. In vitro, anisamide-targeted AuNPs selectively bound to human prostate cancer PC-3 cells, inducing efficient endosomal escape of siRNA, and effective downregulation of the RelA gene. In vivo, prolonged systemic exposure of siRNA was achieved by anisamide-targeted AuNPs resulting in significant tumour growth suppression in a PC3 xenograft mouse model without an increase in toxicity. In addition, a combination of siRNA-mediated NF-κB knockdown using anisamide-targeted AuNPs with Paclitaxel produced a synergistic therapeutic response, thus providing a promising therapeutic strategy for the treatment of prostate cancer.

Introduction

Prostate cancer is a leading cause of cancer-related fatalities for male population; in 2018 a total of 1,276,106 new cases and 358,989 deaths from this malignant disorder are forecasted to occur worldwide [1]. Recent advances in understanding the molecular pathology underlying prostate carcinogenesis have provided significant opportunities for the application of gene-based therapeutic strategies [2]. Small interfering RNA (siRNA), which results in sequence-specific post-transcriptional gene silencing in mammalian cell lines, has shown impressive anti-cancer potential [3]. However, the paucity of successful delivery systems dramatically retards the clinical translation of siRNA-based therapeutics for cancer [4].

Recently, the development of novel organic and inorganic materials has revolutionised the field of siRNA delivery for treatment of solid tumours [5], [6], [7] and haematopoietic malignancies [8], [9]. As a result, a variety of non-viral siRNA delivery nanoparticle (NP) formulations have been developed for prostate cancer therapy [10]. Despite the promise, significant challenges such as inefficient siRNA encapsulation or complexation, in vitro and in vivo NP instability, non-specific cell binding, poor endosomal or lysosomal escape, and low gene knockdown efficacy, remain to be overcome before siRNA-based therapeutics can be widely accepted for use in patients with prostate cancer [10], [11].

Among the diverse range of nanoparticulate delivery carriers, gold NPs (AuNPs) have been utilised to develop siRNA nanomedicines mainly due to favourable physicochemical properties [12]. Previously, spherical positively charged AuNPs were synthesised using surfactant-free methods in the presence of _L-cysteine methyl ester hydrochloride [HSCH₂CH(NH₂)COOCH₃·HCl] [13] and poly (ethylenimine) (PEI) [14] as the capping agents, respectively. Although these positively charged AuNPs (Au-_L-cysteine and Au-PEI) demonstrated effective gene silencing in vitro, activity was curtailed due to non-specific binding to serum proteins [13], [14]. When Au-_L-cysteine was further modified with polyethylene glycol (PEG), stability in serum-containing medium was improved [15]; however, these PEGylated AuNPs displayed poor cellular uptake due to the “PEG dilemma” phenomenon [16]. Bi-functional PEG moieties on AuNPs have therefore been exploited to facilitate conjugation of targeting ligands, resulting in cell-specific internalisation [17].

Recently, sigma receptors have been found to overexpress in a variety of human cancer cell lines and patient tumour tissues (e.g. prostate carcinoma) [18], [19]. Anisamide (AA) is known as a ligand to target the sigma receptors overexpressed on the prostate cancer cell membrane [20], [21]. Informed by our previous studies, a range of novel spherical AuNPs were developed in this study for targeted delivery of siRNA in the treatment of prostate cancer. The spherical Au core was initially coated with PEI to achieve a cationic surface (Au-PEI) capable of complexing siRNA and inducing endosomal escape. To enhance stability in physiological environments and mediate selective uptake in prostate cancer cells, Au-PEI was further modified by PEGylated anisamide to generate a PEGylated Au-AA targeted construct, Au-PEI-PEG-AA. The resultant complex of Au-PEI-PEG-AA with siRNA against RelA gene (a gene product from the NF-κB transcription factor complex [22]) was investigated alone and in combination with paclitaxel for therapeutic efficacy in a prostate carcinoma xenograft mouse model.