Targeting SOD1 via RNAi with PEGylated graphene oxide nanoparticles in platinum-resistant ovarian cancer

Abstract

Acquired platinum resistance poses a significant therapeutic impediment to ovarian cancer patient care, accounting for more than 200,000 deaths annually worldwide. We previously identified that overexpression of the antioxidant superoxide dismutase 1 (SOD1) in ovarian cancer is associated with a platinum-resistant phenotype via conferring oxidative stress resistance against platinum compounds. We further demonstrated that enzymatic inhibition using small-molecule inhibitors or silencing of SOD1 via RNA interference (RNAi) increased cisplatin sensitivity and potency in vitro. We launched this study to explore the potential therapeutic applications of SOD1 silencing in vivo in order to reverse cisplatin resistance using a graphene-based siRNA delivery platform. PEGylated graphene oxide (GO) polyethyleneimine (GO^PEI-mPEG) nanoparticle was complexed with SOD1 siRNA. GO^PEI-mPEG-siSOD1 exhibited high biocompatibility, siRNA loading capacity, and serum stability, and showed potent downregulation of SOD1 mRNA and protein levels. We further observed that cisplatin and PEI elicited mitochondrial dysfunction and transcriptionally activated the mitochondrial unfolded protein response (UPR^mt) used as a reporter for their respective cytotoxicities. SOD1 silencing was found to augment cisplatin-induced cytotoxicity resulting in considerable tumour growth inhibition in cisplatin-sensitive A2780 and cisplatin-resistant A2780^DDP subcutaneous mouse xenografts. Our study highlights the potential therapeutic applicability of RNAi-mediated targeting of SOD1 as a chemosensitizer for platinum-resistant ovarian cancers.

Introduction

Ovarian cancer, with an annual worldwide incidence of 313,000 cases and mortality of 207,000, is considered the second most lethal gynaecological malignancy [1]. Despite the favourable overall survival when detected in early stage 1—attributed principally to its asymptomatic disease progression—most cases are diagnosed in advanced stages 2 and 3 with a 5-year survival of 31%. The overall global survival has only increased modestly in previous decades due to the constrained availability of treatment options and the clinically acquired chemotherapy resistance [2]. Despite most patients responding to debulking surgery and combinational treatment with platinum and taxols, almost half the patients eventually develop recurrence and become resistant or refractory to additional platinum-based therapeutic interventions [2].

Despite their adverse side effect profile, platinum compounds are still considered potent as the first-line drug of choice as mono- or synergistic therapy for many solid tumours [3,4,5,6]. Besides nuclear DNA adduct formation and subsequent induction of apoptotic signalling, platinum drugs also elicit mitochondrial dysfunction through oxidative stress and mitochondrial DNA (mtDNA) damage [7, 8]. Contingent on the intrinsic platinum sensitivity of the respective malignancy, platinum drugs initially exhibit robust therapeutic efficiency. However, the development of acquired platinum resistance of recurrent tumours renders their subsequent clinical applicability ineffective. Lung, prostate, and colorectal cancers are intrinsically resistant to platinum; acquired resistance is also more common in epithelial ovarian cancer [9]. Implicitly, preventing or reversing this well-documented clinical phenomenon could have profound and widespread clinical therapeutic benefits.

Intrinsic and acquired platinum resistance have been linked to reduced drug uptake, increased efflux, enhanced detoxification, elevated scavenger levels, increased oxidative stress tolerance, upregulated DNA repair mechanisms, and the reprogramming of cellular metabolism to evade cisplatin-induced death [10, 11]. Cancer cells may exhibit one, or more of the aforementioned peculiar mechanisms that ultimately determine their net sensitivity to platinum [12, 13]. However, to date, the targeting of drug-transporting pathways of MDR-1, ATP-7A/7B, CTR1, MRP, and DNA repair pathways, including BRCA1/2, ERCC1, and MMR, provided only modest improvement in survival clinically [14]. In addition, the utilization of MDR1 inhibitors, including but not limited to Zosuquidar, MK-571, and PSC-833, showed moderate efficacy in terms of platinum chemosensitization in ovarian cancer [15]. Thus far, the exact mechanisms and determinant factors driving platinum resistance have not been fully elucidated. Due to genetic and patient sample heterogeneity, patient-specific expression levels of potentially robust platinum resistance biomarkers make the discovery process burdensome.

In the quest for a novel therapeutic target for cisplatin resistance, we previously identified the ROS-neutralizing SOD1 to be overexpressed in cisplatin-resistant ovarian cancer cell lines using quantitative label-free comparative proteomics analysis [16]. The 32 kDa Cu²⁺/Zn²⁺ SOD1 is an abundantly expressed intracellular homodimeric metalloenzyme that converts superoxide anions to hydrogen peroxide that is subsequently transformed by catalase to oxygen and water [17]. Superoxide neutralization by SOD1 is a crucial mechanism in counteracting oxidative damage, as SOD1 knockout Drosophila exhibits reduced lifespan, infertility, and hypersensitivity to oxidative stress [18]. Other studies concluded that RNAi-mediated SOD1 silencing provokes senescence in human fibroblasts and induces apoptosis in HeLa cells via ROS-mediated induction of TP53 [19]. Further, SOD1 plays an essential role in cytoplasmic NRF2-mediated adaptation to oxidative stress and in the mitochondria via UPR^mt [20]. Thus, our previous results suggest the role of SOD1 overexpression as a defense mechanism of ovarian cancer cells to counteract platinum-induced oxidative stress and modulation of ROS-mediated redox signalling [16]. We further concluded that enzymatic inhibition of SOD1 using copper/zinc chelating agents of TETA and ATN-224 reversed platinum resistance [21]. Subsequently, to overcome the off-target side effects caused by non-specific small molecule metal chelators, we further corroborated the chemosensitizing effect of SOD1 downregulation via RNAi in vitro [22].

RNAi is a validated gene therapeutic method to control post-transcriptional gene regulation in various disease states [23]. However, the exogenous introduction of therapeutic siRNA for in vivo applications faces numerous obstacles, including easy degradation, short half-life, filtration by the kidneys, poor cellular uptake due to inherent negative charge, structural instability, and degradation by RNases [24]. Therefore, a meticulous design of any siRNA delivery system is fundamental to fully capture the potential of siRNA therapeutics. Non-viral gene delivery vectors such as cationic lipids, cationic polymers, and polysaccharides, in particular, have gained momentum due to their efficacy and biosafety compared to viral vectors for delivering DNA or RNA cargo into the cells [25].

The two-dimensional graphene oxide (GO), due to its convenient applicability for non-covalent or covalent functionalization via abundant epoxy, carboxyl, and hydroxyl surface groups, has been widely used in combination with various cationic polymers for siRNA and gene delivery [26, 27]. GO is also highly biocompatible in most in vitro and in vivo test systems due to its high colloidal stability, water dispersibility, and surface-to-volume area ratio [28]. We previously synthesized graphene-based siRNA, small molecule, and combined drug delivery systems for various applications [29]. Consequently, in this study, a novel graphene-based nanoparticle platform (GO^PEI-mPEG) was prepared by sequential coupling of nano-graphene oxide with cationic polymer polyethyleneimine (PEI) to achieve SOD1 siRNA delivery and further with polyethylene glycol (PEG) to increase biocompatibility and control surface charge. In addition, the incorporation of GO, due to the fixed lateral dimensions of the 2D sheets, can allow for improved control of a more uniform and reproducible final hydrodynamic nanoparticle size, compared to using PEI-PEG polyplexes alone [30, 31]. Our study aimed to evaluate the in vivo chemosensitizing efficacy of SOD1 siRNA by GO^PEI-mPEG delivery system in cisplatin-resistant mouse xenograft models.

Materials and methods

Nanoparticle preparation

Graphene oxide (GO) synthesis

GO was prepared with a modified Hummer’s method [32]. The mixture of NaCl (35 g) and native graphite flakes (1 g, Alfa Aesar, Haverhill, MA, USA) was ground with a mortar until the colour of the mixture turned grey, then dissolved in deionized water (DIW). NaCl was extracted by multiple washing steps and ultracentrifugation at 8000 rpm for 5 min. The product was dried at 90 °C for 12 h and stirred in a three-necked bottle with H₂SO₄ (23 mL) for 8 h. In an ice bath, KMnO₄ (3 g) was added to the mixture keeping the temperature below 20 °C, until the colour turned dark green. The solution was further stirred in a dimethyl-silicone oil bath at 38 °C for 30 min and at 70 °C for an additional 45 min until the colour of the solution turned dark brown. The mixture was diluted with DIW (5 mL) and stirred for 10 min. Next, the solution was diluted with DIW (40 mL) and stirred for 15 min at 100 °C. Subsequently, the mixture of DIW (10 mL) and H₂O₂ (10 mL) was added and stirred for 10 min, and the final product was washed twice with 5% HCl (50 mL) and numerous times with DIW via ultracentrifugation. The prepared GO batch was dialyzed using 100 kDa dialysis bags. Next, GO (8 mL) was washed twice with DIW, and the –COOH groups were activated by adding NaOH (1.8 g). Finally, the suspension was diluted with DIW up to 15 mL and stirred at 55 °C for 4 h. Next, the solution was neutralized by adding HCl (5 mL), and the mixture was washed multiple times until the pH became neutral. Following a 30-min bath sonication and centrifugation at 13,000 rpm for 5 min, the concentration of the supernatant was measured with an Evolution 201 UV–Vis spectrophotometer (Thermo-Fisher Scientific, Carlsbad, CA, USA).

GO^PEI synthesis

The conjugation of COOH groups of GO and primary amines of PEI was achieved with zero-length carboxyl to amine carbodiimide (EDC) cross-linking. Carboxyl groups were activated with EDC forming an active but unstable O-acylisourea intermediate displaced by nucleophilic attack from the primary amines of PEI. These primary amines formed an amide bond with the activated carboxylic groups. In brief, GO (5 mg) was diluted up to 15 mL in phosphate-buffered saline (PBS) and was bath sonicated for 30 min in ice before adding 30, 60, or 90 mg of PEI^{25 kDa} (Sigma-Aldrich, St. Louis, MO, USA) dissolved in 200 µL of PBS (HyClone Laboratories Inc, Logan, UT, USA). Following 5 min of sonication, NaOH (20 µL) and 5 mg of Pierce™ Premium Grade EDC [1-Ethyl-3-(3-dimethyl aminopropyl) carbodiimide] (Thermo-Fisher Scientific) were added, respectively, then sonicated for 5 min and stirred for 30 min. GO^PEI was prepared using three different PEI concentrations; 30, 60, and 90 mg/mL corresponding with GO^PEI1X, GO^PEI2X, GO^PEI3X, respectively. Next, 10 mg of EDC in 400 µL PBS was added under continuous stirring, sonicated for 30 min, and then stirred overnight. The final EDC concentration was 1 mg/mL in a 10-fold molar or weight excess to GO. The prepared GO^PEI nanoparticles were purified with ultrafiltration using a 100 kDa Amicon Ultra-15 centrifugal filter (Merck Millipore Ltd., Darmstadt, Germany).

GO^PEI-mPEG synthesis

Methyl-PEG (2000 kDa, 5 mg) (Xi’an Ruixi Biological Technology Co., Ltd, Shaanxi, China) in PBS was diluted to 15 mL and sonicated for 5 min. NaOH (20 µL) and EDC (5 mg) were added and sonicated for 30 min. Next, GO^PEI (30 mg) was added, and the solution was sonicated for 5 min before adding EDC (10 mg), then stirred overnight. The nanoparticle was purified with ultrafiltration using a 100 kDa filter (Millipore Sigma, Burlington, MA, USA).

Nanoparticle characterisation

Atomic force microscopy (AFM)

The size and surface morphology of GO and GO^PEI were characterized with AFM. The nanoparticle samples (200 μL) were transferred onto a 1.5 cm × 1.5 cm muscovite mica sheet and imaged with Veeco Dimension 3100 atomic force microscope (Veeco Instruments, Bruker, MA, USA). The images were analyzed using V700 (Veeco) and Nanoscope v.7.00b19 (Veeco) software….