ROS-responsive thioether-based nanocarriers for efficient pro-oxidant cancer therapy

https://doi.org/10.1016/j.jiec.2019.03.030Get rights and content

Abstract

A high level of intracellular reactive oxygen species (ROS) is one of the remarkable intrinsic features of cancer cells. Therefore, ROS-responsive drug carriers have received great attention for cancer-selective drug delivery. In this study, ROS-responsive thioether-bearing polymers (TEP) were synthesized for effective intracellular delivery of piperlongumine (PL) into cancer cells. PL is a pro-oxidant drug that induces cytotoxic oxidative stress in cancer cells. PL-loaded TEP nanoparticles (PL-TEP NPs) were successfully formulated by a nanoemulsion method. PL-TEP NPs showed ROS-sensitive disassembly, which leads to ROS-sensitive drug release. In addition, PL-TEP NPs showed higher cytotoxicity in human breast cancer cells (MCF-7) than in normal human dermal fibroblast cells (hDFB), demonstrating their cancer cell-specific pro-oxidant therapy. This study demonstrates that ROS-responsive TEP NPs are effective drug carriers for efficient intracellular delivery of hydrophobic drug, PL.

Graphical abstract

ROS-responsive thioether-bearing polymers (TEP) were synthesized and used as drug carriers for intracellular delivery of piperlongumine (PL) in cancer cells. The PL-loaded TEP nanoparticles exhibited ROS-triggered disassembly in cancer cells, leading to efficient cancer cell-specific apoptosis.

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Introduction

Reactive oxygen species (ROS) including hydrogen peroxide, hydroxyl radicals, and superoxide anions are highly reactive chemical by-products generated mainly from cell mitochondria [1]. Recent studies have reported that cancer cells express high levels of intracellular ROS due to their genetic and metabolic alterations [2], [3], [4]. Therefore, intracellular ROS in cancer cells has been utilized as a unique cancer-related stimulus to mediate intracellular delivery of therapeutic agents. Various types of ROS-responsive drug delivery systems have been developed because of their promising potential for cancer-targeted drug delivery [5], [6].

Pro-oxidant cancer therapeutics that can selectively induce cytotoxic oxidative stress in cancer cells have gained much attention recently [4], [7]. Piperlongumine (PL), derived from Piper longum Linn, has been known as an effective pro-oxidant anticancer drug because they can selectively induce apoptosis in cancer cells through excessive production of intracellular ROS and inhibition of PI3K/Akt/mTOR signaling pathway [8], [9]. Despite the therapeutic potency of PL, its utility is hampered by its poor water-solubility due to its hydrophobic nature [10]. Therefore, development of drug carriers that can effectively incorporate hydrophobic PL is indispensable to enhance its bioavailability and anticancer efficacy [10], [11].

Nanocarriers have been extensively investigated as efficient platforms for anticancer drug delivery [12], [13]. Drug-loaded nanocarriers offer many advantages including enhanced bioavailability, increased cellular uptake, and cancer-targeted delivery via the enhanced permeability and retention (EPR) effect [13], [14], [15]. Among the various methods to fabricate nanoparticles, nanoemulsion, a heterogeneous system of two immiscible liquids stabilized by emulsifiers, has gained wide interest due to its distinct capacity to solubilize hydrophobic drugs [16].

In this study, an ROS-responsive thioether-bearing polymer (TEP) was synthesized and utilized as ROS-sensitive nanocarrier for efficient intracellular delivery of hydrophobic PL in cancer cells. Under ROS conditions, hydrophobic thioether-containing polymers undergo phase transition via oxidation of the hydrophobic sulfide groups to hydrophilic sulfoxide or sulfone moieties. Thioether groups are sensitive to low concentrations of ROS such as 100 μM H2O2, a biologically relevant ROS concentration [17]. Therefore, thioether group-containing polymers have been exploited as promising ROS-responsive nanocarriers for the treatment of inflammation and cancer that are associated with high levels of ROS [17], [18]. As shown in Fig. 1, PL can be stably encapsulated into hydrophobic TEP nanoparticles (TEP NPs) using the conventional water-in-oil-in-water (w/o/w) double nanoemulsion method. After PL-loaded TEP NPs (PL-TEP NPs) are internalized by the cancer cells, they undergo hydrophobic-to-hydrophilic transition in response to high levels of intracellular ROS in cancer cells, which accelerates the disassembly of the NPs (Fig. 1). Then, the encapsulated PL can be rapidly released into the cytoplasm, thus leading to apoptosis in the cells through excessive production of intracellular ROS. The physical properties and drug encapsulating efficiency of PL—TEP NPs were investigated. ROS-responsive disassembly of PL—TEP NPs and their drug release behaviors were evaluated. In addition, cancer cell-specific cytotoxicity of PL—TEP NPs was investigated in vitro.

Section snippets

Materials

Piperlongumine (PL) was purchased from Cayman Chemical (Ann Arbor, MI, USA). hDFB human dermal fibroblast cells and MCF-7 human breast cancer cells were purchased from ATCC (Manassas, VA, USA). 2′,7′-dichlorfluorescein diacetate (DCF-DA), Rhodamine B isothiocyanate (RhB), MTT powder, and doxorubicin (DOX) were supplied from Sigma-Aldrich (St. Louis, MO, USA). Diethylene glycol diacrylate, 2,2′-thiodiethanethiol, and dimethylphenylphosphine, poly(vinyl alcohol) (PVA) were purchased from Alfa

Synthesis of TEP and characterizations of PL-TEP NPs

We designed TEP as a drug carrier with low cytotoxicity and ROS-responsive thioether groups. Thioether groups can be oxidized to hydrophilic sulfoxide or sulfone [20], [21]. Highly reactive thiol-ene addition reaction was utilized to synthesize TEP [22]. Chemical structures and molecular weights of TEP were confirmed by 1H NMR and GPC. As shown in (Fig. 2B), the 1H NMR spectrum clearly indicates the disappearance of acrylate groups of DEGDA, indicating the successful conjugation of

Conclusion

ROS-responsive thioether-bearing polymer was synthesized and used for the intracellular delivery of PL. PL-loaded TEP NPs were fabricated by w/o/w double nanoemulsion method. PL-TEP NPs showed a ROS-sensitive drug release profile due to ROS-triggered oxidation of thioether groups of the TEP. In vitro study demonstrated that TEP has low cytotoxicity and PL-TEP NPs exhibit more efficient cellular uptake and anticancer activity in MCF-7 cancer cells compared to free PL. Importantly, PL-TEP NPs

Conflicts of interests

All authors declare that they have no conflicts of interests.

Acknowledgements

This work was supported by the Incheon National University Research Grant in 2017. This work was also supported by the National Research Foundation of Korea (NRF) grants funded by the Korea government (MSIT) (Basic Research Laboratory Program, NRF-2017R1A4A1015036).

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