Research Paper
siRNA-loaded biodegradable nanocarriers for therapeutic MAPK1 silencing against cisplatin-induced ototoxicity

https://doi.org/10.1016/j.ijpharm.2017.06.035Get rights and content

Abstract

Ototoxicity represents a major adverse side-effect of cis-diamminedichloroplatinum-II (cisplatin, CDDP). The mitogen-activated protein kinase (MAPK) pathway is thought to play a central role in potentiating the apoptotic effect of CDDP within the cochlea. We hypothesized that prophylactic inhibition of MAPK signaling, using small interfering RNA (siRNA), might confer a protective effect against CDDP-induced apoptosis within the auditory sensory epithelia. To enhance the therapeutic utility of this approach, we synthesized biocompatible siMAPK1-loaded nanoparticles (NPs) and performed physicochemical characterizations for size, morphology, drug loading and release kinetics, using dynamic light scattering, electron microscopy and spectrophotometric analyses, respectively. Our findings show 183.88 ± 6.26 nm-sized spherical siMAPK1-loaded NPs with −27.12 ± 6.65 mV zeta potential and 112.78 ± 0.24 pmol/mg of siMAPK1 loading that exhibit a sustained release profile for prolonged therapeutic efficacy. Synthesized NPs were validated for biocompatibility and prophylactically protected against CDDP-induced cytotoxicity in HEI-OC1 cells and hair cell loss in murine organotypic cochlear explants. Our study confirms a pivotal role for MAPK1 signaling as a potentiating factor for CDDP-induced apoptosis and cochlear hair cell loss, and highlights siMAPK1 NP treatment as a therapeutic strategy for limiting the ototoxic side-effects associated with systemic CDDP administration.

Introduction

Cisplatin (cis-dichlorodiamminoplatinum (II); CDDP) is one of the most potent and efficacious anti-neoplastic agents employed for the treatment of a variety of cancers, including testicular, germ cell, head and neck, bladder, cervical, non-small cell lung, breast, esophageal, cervical, stomach and prostate cancers (Florea and Büsselberg, 2011). However, CDDP treatment and therapeutic dosing are limited by detrimental side effects, such as neurotoxicity, ototoxicity, gastrointestinal disturbance, and acute nephrotoxicity (Arany and Safirstein, 2003, Travis et al., 2014, Karasawa and Steyger, 2015). The major dose-related adverse side effect of cisplatin treatment is irreversible sensorineural hearing loss (Sun et al., 2015). Ototoxicity is one of the frequent side effects of CDDP therapy in patients treated with higher dose regimens, with 75–100% of patients receiving 150–225 mg/m2 CDDP showing some degree of hearing loss (Kopelman et al., 1988, McKeage, 1995). Ototoxicity caused by CDDP can occur within hours to days after drug administration (Rybak et al., 2009). Outer hair cells (OHCs) have been found to be the most vulnerable to CDDP damage (Usami et al., 1996). Experimental and clinical studies have revealed that, OHCs in the basal turn are particularly susceptible to CDDP-induced cytotoxicity (Rybak et al., 2007, Ding et al., 2011). Indeed, histological evaluations of cochlear tissues in animal models of clinical CDDP administration have revealed that ototoxic destruction is primarily focused among the OHCs, particularly within the basal turn (high frequency region) of the cochlea (García-Berrocal et al., 2007; Hellberg et al., 2013; Kuduban et al., 2013).

Over the last 30 years, considerable effort has been devoted to determine the underlying pathophysiology that governs CDDP-induced ototoxicity (Callejo et al., 2015, Karasawa and Steyger, 2015). At cellular level, acute CDDP exposure results in a progressive wave of programmed cell death (apoptosis) within the OHC population that is, at least in part, initiated and perpetuated by DNA damage and oxidative stress (Kopke et al., 1999, Dehne et al., 2001, Karasawa and Steyger, 2015). These intrinsic stressors induce extrinsic signaling through activation of tumor necrosis factor α (TNFα)-like death receptors that further drive the apoptotic response (So et al., 2007). Intrinsic and extrinsic pathways of CDDP-induced OHC pro-apoptotic signaling mediate their destructive influence through a family of cysteine proteases, called caspases, whose cumulative proteolytic activities culminate in OHC destruction.

While many signaling pathways have been implicated in governing and perpetuating this CDDP-induced pathologic response, the mitogen-activated protein kinase (MAPK) family of signaling factors has been shown to play a central role in tipping the balance towards an apoptotic fate. At the nexus of these pro-apoptotic signaling cascades for CDDP ototoxicity, are two isoforms of an extracellular signal-regulated kinase named MAPK1 and MAPK3 (also known as ERK 2 and 1, respectively). Upon activation, MAPK1/MAPK3 phosphorylate a number of key cytoplasmic and nuclear factors that, in this cytotoxic context, promote both intrinsic and extrinsic caspase-mediated cell death (Cagnol and Chambard, 2010). While other members of the MAPK family, such as p38 MAPK and c-Jun-NH2-terminal kinase (JNK), have been shown to play roles in CDDP-induced apoptosis, in vitro studies have revealed that MAPK1/MAPK3 activation (i.e. phosphorylation) and subsequent signaling represent key, rate-limiting events for driving OHC death in this context (Wang et al., 2004, So et al., 2007, Lee et al., 2010). Moreover, the specificity and preeminent status of this signaling pathway is conserved in in vitro models of CDDP-induced nephrotoxicity (Arany et al., 2004, Kim et al., 2005), suggesting that MAPK1/3 signaling may represent a highly attractive and specific target for controlled therapeutic inhibition to mitigate the dose-limiting side-effects of CDDP chemotherapy.

Small interfering RNA-mediated silencing of mRNA transcripts is a canonical approach for gene-specific inhibition (Carthew and Sontheimer, 2009, Ghildiyal and Zamore, 2009). However, several factors limit the clinical application of siRNA. Those factors include poor cellular internalization, physical and chemical instability within bodily fluids (half-life ∼10 min in plasma), and rapid degradation in the lysosome (Grimm, 2009, Takahashi et al., 2009, Whitehead et al., 2009). Encapsulation of siRNA within biocompatible poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) represents a reliable approach to overcome the above disadvantages, including physical protection of siRNA against RNase activity, enhanced cellular uptake, and prolonged in vivo half-life (Woodrow et al., 2009, Wang et al., 2012). Moreover, encapsulation within PLGA NPs allows increased surface exchange, nuclease resistance, controlled drug release and extending the therapeutic window of siRNA-mediated knockdown (Cun et al., 2011). In the present study, we evaluated the efficacy of siRNA-loaded PLGA NPs as delivery vehicles for mediating transcriptional silencing of MAPK1 (ERK2) as a means of mitigating CDDP-induced ototoxicity and provide a proof-of-concept support for their potential therapeutic utility against this chemotherapeutic adverse effect.

Section snippets

Materials

Poly D,L-lactic-co-glycolic acid (PLGA, L/G ratio of 50:50 and molecular weight ranging between: 7 to 17 kDa) was purchased from Boehringer Ingelheim (Ridgefield, CT). Dichloromethane (DCM) was obtained from Burdick and Jackson (Muskegon, MI). Polyvinyl alcohol (PVA, MW: 30–70 kDa) was provided by Sigma Aldrich (Saint-Louis, MO). TE Buffer Solution OmniPur® Grade, pH 8.0 was purchased from EMD Millipore (Billerica, MA). Fluorescein-conjugated non-targeting (scrambled) siRNA (Ambion Silencer

MAPK1 as a therapeutic target for protective inhibition against CDDP-induced ototoxicity

Mitogen-activated protein kinase (MAPK) signaling pathways are known to modulate a variety of cellular processes (Zhuang and Schnellmann, 2006). Specifically, the MAPK3/1 (ERK1/2) signaling nexus has been shown to play key roles in both mediating and ameliorating cellular responses to stress, such as CDDP-induced cytotoxicity (So et al., 2007). To examine CDDP-induced activation of MAPK3/1 in the context of ototoxicity, the canonical murine inner ear cell line, HEI-OC1, was employed. HEI-OC1

Conclusions and future perspectives

Our work has led us to conclude that spherical siMAPK1-loaded PLGA NPs with smooth surfaces, in the nanoscale range were successfully prepared and faithfully recapitulated the therapeutic benefits against CDDP-induced ototoxicity achieved by siMAPK1 silencing using commercial transfection agents, which are generally more cytotoxic at high doses (Kamaci et al., 2011). Our data suggest that siMAPK1-loaded NPs are well-tolerated by inner ear cells and are internalized in a dose-dependent manner

Conflict of interest

The authors declare that the research was conducted in the absence of any current commercial or financial or other relationships that could be construed as a potential conflict of interest.

Funding

The project was co-supported by the Oklahoma Center for the Advancement of Science and Technology (OCAST, award number: AR16-010), and Otologic Pharmaceutics Inc. (OPI).

Author contributions

IY executed all formulation experiments, performed physicochemical characterizations, and contributed to the writing of the manuscript. MBW performed western blotting, RT-qPCR analyses, organotypic culture experiments, and contributed to the writing of the manuscript. WL and XD conducted explant dissections and assisted with microscopy. DE and RK supervised experimentation and data analyses.

Acknowledgements

The authors gratefully acknowledge Dr. Christenson (Department of Molecular & Integrative Physiology, University of Kansas Medical Center, Kansas City, KS) and Dr. Agrahari (Laboratory of Future Nanomedicines & Theoretical Chronopharmaceutics, Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, Kansas City, MO) for their assistance with the nanoparticle tracking analysis (NTA). We are also thankful to Dr. Andria F. Hedrick (School of Pharmacy at the

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