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Biological and Medical Applications of Materials and Interfaces
Tumor Specific Expansion of Oxidative Stress by Glutathione Depletion and Use of a Fenton Nanoagent for Enhanced Chemodynamic Therapy Qiufang Chen, Jun Zhou, Zhe Chen, Qing Luo, Jian Xu, and Guanbin Song ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b09323 • Publication Date (Web): 09 Aug 2019 Downloaded from pubs.acs.org on August 10, 2019
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ACS Applied Materials & Interfaces
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Tumor Specific Expansion of Oxidative Stress by
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Glutathione Depletion and Use of a Fenton
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Nanoagent for Enhanced Chemodynamic Therapy
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Qiufang Chen a, Jun Zhou b, Zhe Chen a, Qing Luo a, Jian Xu c, d and Guanbin Song *a
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aCollege
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Ministry of Education, Chongqing University, Chongqing 400030, P.R. China.
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bSchool
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cChongqing
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Individualized Treatment, Chongqing University Cancer Hospital, Chongqing 400030，
of Bioengineering, Key Laboratory of Biorheological Science and Technology,
of Life Science, Chongqing University, Chongqing 400030, P. R. China. Key Laboratory of Translational Research for Cancer Metastasis and
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China.
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dDepartment
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400030, China.
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*Corresponding author:
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Guanbin Song, e-mail:
[email protected] of Thoracic Surgery, Chongqing University Cancer Hospital, Chongqing
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KEYWORDS: destruction of redox homeostasis, β-Lapachone, Iron oxide, Fenton’s reaction,
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GSH depletion.
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ABSTRACT: Amplifying intracellular oxidative stress effectively destroys cancer cells. In
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addition, iron-mediated Fenton reaction converts endogenous H2O2 to produce hypertoxic
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hydroxyl radical (·OH), resulting in irreversible oxidative damage to combat tumor cells. This
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method is known as chemodynamic therapy (CDT). Overexpressed glutathione (GSH) in tumor
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cells efficiently scavenges ·OH, significantly reducing the curative effects of CDT. To
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overcome this challenge and enhance intracellular oxidative stress, iron oxide nanocarriers
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loaded with β-Lapachone (Lapa) drugs (Fe3O4-HSA@Lapa) were constructed and had both
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Fenton-like agents and GSH depletion properties to amplify intracellular oxidative stress.
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Release of Lapa selectively increases tumor site-specific generation of H2O2 via NAD(P)H:
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quinone oxidoreductase 1 (NQO1) catalysis. Subsequently, the iron ions released from the
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ionization of Fe3O4 in the acidic environment selectively convert H2O2 into highly toxic ·OH
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by Fenton reaction, dramatically improving CDT with minimal systemic toxicity due to low
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NQO1 expression in normal tissues. Meanwhile, released Lapa consumes GSH in the tumor,
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amplifying oxidative stress and enhancing the efficacy of CDT. Designed Fe3O4-HSA@Lapa
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nanoparticles (NPs) exhibit perfect targeting capability, prolonged blood circulation and
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increased tumor accumulation. Furthermore, Fe3O4-HSA@Lapa NPs effectively enhance the
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inhibition of tumor growth and reduce the side effects of anticancer drugs. This work
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establishes a remarkably enhanced tumor-selective CDT against NQO1-overexpressing tumors
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by significantly inducing intratumoral oxidative stress with minimal side effects.
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INTRODUTION
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Increasing evidence has demonstrated that cancer cells are immersed in oxidative stress,
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which causes increased generation of reactive oxygen species (ROS) due to perturbed ROS
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homeostasis, and cancer cells typically exhibit upregulated glutathione (GSH) by activating the
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antioxidant system to adapt to the oxidative stress.1-3 ROS, such as the superoxide radical (O2-)
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and hydrogen peroxide (H2O2), play vital roles in cancer cell homeostasis and signaling
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throughout the life cycle of cancer cells.4-7 However, breaching intracellular thresholds of ROS
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causes irreversible oxidative damage to cellular components, including DNA strand breakage8
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and subsequent cancer cell apoptosis and necrosis.9 Therefore, accumulation of H2O2 in cancer
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cells is used as a trigger for various types of chemotherapies.10-12 ROS production occurs
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through a variety of mechanisms, such as photodynamic therapy, drug action mechanisms,
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biochemical reactions, etc. and has been applied to cancer treatment.13-15 However,
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supernormal glutathione (GSH) levels (concentrations range from 1 to 15 mM)16, 17 in tumor
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cells have increased scavenging capacity for ROS and attempt to maintain redox homeostasis,
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representing one of the greatest barriers to chemo-, radio-, and photodynamic therapies.3, 16, 18
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Therefore, disrupting redox homeostasis via enhancing ROS generation while simultaneously
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inhibiting ROS clearance in cancer cells should amplify oxidative stress to treat cancer more
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effectively.19, 20
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Recently, Fenton reactions, which use ferrous ions (Fe2+) to catalyze the conversion of
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H2O2 into the ·OH with the strongest oxidative capacity and toxicity among ROS species, have
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attracted increasing attention and have been used to destroy cancer cells.21-23 Several iron-
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carrying nanoparticles24, 25 or other metal ion NPs26 that exhibit Fenton-like activity have been
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developed for use as catalysts for Fenton reactions to catalyze in situ H2O2 within solid tumors
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into ·OH to induce cancer cell death. This process is referred to as CDT. It has been shown that
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Fe3O4 NPs have no cytotoxicity in the neutral pH environment of normal tissues, but can self3 ACS Paragon Plus Environment
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sacrifice to produce Fe2+ under acidic conditions of tumor and catalyze H2O2 to generate ·OH
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through Fenton reaction, thus killing cancer cells.27,
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cancer cells is insufficient for Fe3O4 to produce large enough quantities of ·OH for the desired
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curative effect.23 In addition, overproduced GSH in cancer cells mediates potent resistance to
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intracellular oxidative stress by effectively scavenging ·OH, thus weakening the curative effect
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of CDT.26, 29 Therefore, to disrupt redox homeostasis and achieve optimal therapeutic results,
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an innovative formulation that could amplify intracellular H2O2 levels for Fenton reaction
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producing excess ·OH and simultaneously consume GSH levels in cancer cells has therapeutic
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value.
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However, concentration of H2O2 in
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β-Lapachone (Lapa) is a novel anticancer agent that is catalyzed by the nicotinamide
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adenine dinucleotide phosphate (NADPH): quinone oxidoreductase-1 (NQO1) enzyme
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through a futile cycle to generate H2O2. Due to NQO1 being overexpressed 2-100-fold in
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different cancers cells,30 including non-small-cell lung cancer (NSCLC),31 Lapa exhibits
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tremendous selectivity for tumor cells over normal cells. Therefore, adopting Lapa to amplify
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H2O2 levels in cancer cells could significantly improve the efficiency of multifunctional drug
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delivery systems while reducing side effects to normal tissue.32 In addition, NQO1 uses
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NADPH as an electron donor to induce a futile cycle between the quinone and hydroquinone
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forms of Lapa33 that can consume 60 mole of NADPH in 5 minutes per mole of Lapa,33-35
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resulting in severe intracellular NADPH depletion. On the other hand, NADPH is used to
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maintain the reduced state of glutathione (GSH) as a coenzyme of GSH reductase.36-38 Due to
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the depletion of NADPH, the reduction of GSSG to GSH catalyzed by glutathione reductase is
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inhibited. In other words, the Lapa-induced futile cycle depletes NADPH, which inhibits
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glutathione disulphide (GSSG) conversion into GSH, synergistically enhances oxidative stress
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in the tumors.31, 33 Therefore, Lapa not only supplies sufficient H2O2 for Fenton reaction but
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also inhibits GSH levels in cancer cells with negligible damage to normal tissues. 4 ACS Paragon Plus Environment
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In summary, we constructed an innovative biodegradable formulation by loading Lapa
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into Fe3O4 NPs to efficiently suppress tumor growth by expanding oxidative stress and
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disputing redox balance to enhance CDT with desired tumor specificity and curative effects
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(Figure 1). As shown in Figure 1A, protocatechuic acid (PA) and human serum albumin (HSA)
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were sequentially bonded with Fe3O4 NPs. Then, Lapa was encapsulated in HSA, which
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formed vesicles in the water phase. Finally, Lapa drug-loaded self-sacrificing iron oxide NPs
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(Fe3O4-HSA@Lapa NPs) were assembled. Fe3O4-HSA@Lapa NPs were designed based on
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the following considerations (Figure 1B). (i) Magnetic field-guided delivery of Fe3O4-
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HSA@Lapa NPs causes increased Lapa and iron accumulation in tumor tissues. (ii) HSA in
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Fe3O4-HSA@Lapa NPs serves as the drug carrier, which improves the solubility of
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hydrophobic drugs and the biocompatibility of nanoparticles while also to prolong circulation
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of nanoparticles to increase accumulation of nanoparticles in the tumor by the EPR effect.39
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These features maximize the localization of Fe3O4-HSA@Lapa NPs into tumor tissues and
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facilitate internalization of Lapa and iron into cancer cells. (iii) Loaded Lapa selectively
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generates high levels of H2O2 in the reaction catalyzed by NQO1, which is overexpressed in
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A549 cells.40 Moreover, Fe3O4 NPs self-sacrifices by dissolution in an acidic environment
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releasing high levels of iron ions within the cancer cells. An increase in H2O2 subsequently
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reacts with iron ions to produce high levels of highly toxic ·OH via Fenton-like reactions,
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resulting in apoptosis of the cancer cells. (iv) Lapa depletes the elevated GSH levels in cancer
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cells, synergistically elevating oxidative stress.33 (v) In addition, Lapa exhibits relatively low
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cytotoxicity and induces insignificant H2O2 production in normal tissues, producing
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insufficient ·OH via cascading Fenton reaction and causing minimal cytotoxicity in normal
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tissues that have low expression of NQO1.32 These multiple mechanisms reduce side effects,
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increase tumor selectivity and targeting, and enhance therapeutic effects of Fe3O4-HSA@Lapa
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NPs. This elaborate biocompatible multifunctional drug delivery system directly introduces a 5 ACS Paragon Plus Environment
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non-toxic nanoagent into the tumor, which embodies proof of concept in nanomedicine for
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effective and selective tumor therapy.
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Preparation and characterization of Fe3O4-HSA@Lapa NPs
RESULTS AND DISCUSSION
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The design and fabrication of Fe3O4-HSA@Lapa NPs were illustrated in Figure 1A. The
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monodispersed larger Fe3O4 NPs were synthesized using seed-mediated methods.41 The
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resultant Fe3O4 NPs were connected to protocatechuic acid (PA) through ligand-exchange
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reaction, yielding PA-coated Fe3O4 NPs (Fe3O4-PA NPs) with carboxyl groups on their surface.
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Then, HSA protein were covalently bonded with Fe3O4-PA NPs via forming amide bonds
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between carboxylic groups in the PA layer and amine groups of HSA with the assistance of
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EDC. Utilizing the hydrophobic interaction between Lapa and the hydrophobic region of the
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HSA protein, Lapa was loaded into Fe3O4-PA-HSA NPs (Fe3O4-HSA NPs), obtaining the final
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Fe3O4-PA-HSA@Lapa NPs (Fe3O4-HSA@Lapa NPs).
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As demonstrated by TEM results (Figure 2A), prepared Fe3O4 NPs, Fe3O4-HSA NPs and
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Fe3O4-HSA@Lapa NPs were highly uniform, spherical in shape, and monodisperse. The
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average diameter of Fe3O4 NPs was analyzed by a particle size analyzer and was approximately
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10 nm. Then, Fourier transform infrared spectroscopy (FTIR) was performed to monitor
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modification of Fe3O4-HSA@Lapa NPs to verify the successful preparation of Fe3O4-
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HSA@Lapa NPs (Figure 2B). The Fe3O4 NPs (b) demonstrated a strong peak at 589 cm-1,
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which was attributed to the Fe-O bond.42,
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absorbance peaks at 1295 cm-1 and 1118 cm-1 appeared in the resultant nanoparticles, which
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was credited to the carboxyl and phenolic hydroxyl groups of PA, confirming the successful
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modification of PA onto Fe3O4 NPs.44 Moreover, after covalent binding of HSA (d), two
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characteristic peaks at 1655 and 1529 cm-1 were detected, resulting from the stretching
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After surface decoration with PA (c), new
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vibration of -CO-NH- in HSA.45 The FT-IR spectrum of Fe3O4-HSA@Lapa NPs (e) was very
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similar to the spectra of Lapa and unloaded nanoparticles. The pure Lapa spectrum (a) exhibited
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a group of scattered low-intensity absorption peaks between 500 and 1740 cm-1. The
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characteristic peaks of pure Lapa were seen at 1113, 1313, 1568, 1599 and 1635 cm-1.46 In
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comparison, these spectra were also present in the spectrum of Fe3O4-HSA@Lapa NPs (e) and
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absent in unloaded nanoparticles (b, c, d). The characteristic peaks at 1313 and 1113 cm-1 were
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caused by the C-O-C group of Lapa.47 The peak at 1568 cm-1 could be attributed to the bending
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motion of the CH3 groups in the Lapa molecule, while the absorption peak at 1599 cm-1 could
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be attributed to the bending of two CH bonds in the benzene region of Lapa.46, 47 The peak at
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1635 cm-1 represented the combination of CH3 shearing motions and CH benzene bending with
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minor C-C bond stretching.46 FT-IR characterization further verified successful surface
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modification of Fe3O4 NPs and successful preparation of Fe3O4-HSA@Lapa NPs. According
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to dynamic light scattering (DLS) results (Figure 2C), the average diameter of Fe3O4 NPs was
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approximately 10 nm, consistent with TEM results. Following HSA conjugation and drug
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loading, the average diameter of Fe3O4-HSA@Lapa NPs increased to 28 nm. As shown in
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Figure 2D, the zeta potential of Fe3O4 NPs was 1.9 mV, and the zeta potential of Fe3O4-PA
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NPs was -38.4 mV due to the carboxyl groups in PA, verifying successful coating of PA onto
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Fe3O4 NPs. Additionally, the zeta potential of Fe3O4-HSA NPs increased to -22.4 mV due to
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the reaction between PA carboxyl groups and HSA amines, further indicating that HSA had
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bonded with PA in Fe3O4-PA NPs to create Fe3O4-HSA NPs. Taken together, these results
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indicated successful surface modification of Fe3O4 NPs.
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The ligand desorption assay indicates that the desorption rate of HSA on the Fe3O4-
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HSA@Lapa NPs after 4 days was only approximately 5.1% (Figure S1). Furthermore, the
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results of the long-term DLS detection (Figure S2) showed that after incubation in the bionic
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buffer for 4 days, the average hydrodynamic diameter of Fe3O4-HSA@Lapa NPs underwent 7 ACS Paragon Plus Environment
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little changes, and the PDI value was less than 0.2. These results confirmed that Fe3O4-
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HSA@Lapa NPs had good stability in physiological environment. However, in an acidic
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environment (pH 5.4), the particle size of Fe3O4-HSA@Lapa NPs gradually decreased (Figure
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S3), indicating that Fe3O4-HSA@Lapa NPs have undergone acid degradation, which was
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further validated by the DLS results (Figure S4).
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The loading capacity of Lapa was further evaluated using thermogravimetric analysis
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(TGA). The results of TGA curves for Fe3O4 NPs, Fe3O4-PA NPs, Fe3O4-HSA NPs and Fe3O4-
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HSA@Lapa NPs were shown in Figure 2E, indicating the mass fraction of the chemical
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composition of Fe3O4-HSA@Lapa NPs. The difference of the weight loss ratio between curves
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Fe3O4-HSA NPs and Fe3O4-HSA@Lapa NPs were attributed to Lapa loading, and the loading
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efficiency of Lapa was determined to be approximately 4.2 wt%. The release of Lapa from the
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Fe3O4-HSA@Lapa NPs was measured by a UV-vis spectrophotometer, and the results showed
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that, after 96 h incubation, the cumulative release rate of Lapa from the nanoagents reached 75%
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(Figure S5). XRD results demonstrated that Fe3O4-HSA@Lapa NPs retained the crystalline
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structure characteristics of magnetite Fe3O4 NPs (Figure S6). Vibrating sample magnetometer
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(VSM) data shown in Figure S7 verified that Fe3O4-PA-HSA@Lapa NPs were
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superparamagnetic without apparent residual magnetization or coercive force and that the
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saturation magnetization (Ms) value of Fe3O4-HSA@Lapa NPs was 68.2 emu g-1.
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pH-dependent ionization of Fe3O4-HSA@Lapa NPs and ·OH production
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Only iron ions are catalysts of Fenton reactions, so we first investigated the release of iron
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ions from Fe3O4-HSA@Lapa NPs under various pH conditions using ICP-MS. There were no
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iron ions released from Fe3O4-HSA@Lapa NPs at neutral pH, and iron ion release rate
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increased to 1.5% at pH=6.5, reaching 3.2% at pH=5.4 after 100 h, indicating clear pH-
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dependent ionization of Fe3O4-HSA@Lapa NPs (Figure 3B). The release rate of iron ions is
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relatively low, but it is sufficient to catalyze the Fenton reaction. Once the nanoparticles enter 8 ACS Paragon Plus Environment
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the intracellular environment, Fe3+ produced by the degradation of Fe3O4-HSA@Lapa NPs and
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by the Fenton reaction will be reduced to Fe2+ by iron reductase and reductive molecules, thus
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providing a continuous supply of Fe2+ for the Fenton reaction.48-50 Production of ferrous ions
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was further verified using a potassium ferricyanide dispersed agarose gel (Figure S8). pH-
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dependent ferrous ion production from Fe3O4-HSA NPs was observed at pH=6.5 and 5.4. Of
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note, Fe3O4-HSA NPs ionized faster at pH=5.4. The above results indicated that Fe3O4-
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HSA@Lapa NPs were excellent the donors of ferric ions as they maintained a unionized status
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in neutral normal tissues (pH≈7.4), while quickly dissolving into ferrous ions upon entering the
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slightly acidic extracellular microenvironment of tumor (pH≈6.5). Ferrous ion release would
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be particularly intensified in the acidic environment of endosomes or lysosomes (pH≈5.4) after
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endocytosis.
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Next, we further affirmed the ionization properties of Fe3O4-HSA@Lapa NPs after
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endocytosis on a cellular level using ICP-MS (Figure S9). Ionized iron concentrations in A549
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cells incubated with Fe3O4-HSA@Lapa NPs was significantly increased in a time-dependent
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manner, and the addition of a magnet prominently heightened intracellular iron concentrations
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compared to controls, indicating that Fe3O4-HSA@Lapa NPs effectively dissolved into iron
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ions in tumor cells, and magnet enhanced ionization by increasing endocytosis of Fe3O4-
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HSA@Lapa NPs (Figure S10).
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Endocytosis of Fe3O4-HSA@Lapa NPs was further evaluated using ICP-MS (Figure S10).
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A549 cells treated with Fe3O4-HSA@Lapa NPs with or without magnets exhibited ever-
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increasing time-dependent cellular uptake of nanoparticles (p
