Effect of Malt-PEG-Abz@RSL3 micelles on HepG2 cells based on NADPH depletion and GPX4 inhibition in ferroptosis

Article abstract of DOI:10.1080/1061186X.2021.1953511

Ferroptosis is a regulated cell death pathway which depends on iron. Ferroptosis can be induced by limiting intracellular glutathione (GSH) synthesis, or inhibiting the activity of GPX4, or increasing intracellular accumulation of PE-AA-OOH, all of which involve NADPH. Therefore, NADPH depletion, excessive PE-AA-OOH, and GPX4 deficiency are generally considered to be the main characteristics of ferroptosis. In this research, the novel self-assembly nanomicelles modified by maltose ligand (Malt-PEG-Abz@RSL3) with superior nano characteristics were designed and fabricated. Malt-PEG-Abz@RSL3 micelles achieved active targeted drug delivery due to the high expression of glucose transporter (GLUT) and high uptake by HepG2 cells. Maltose-polyethylene glycol broke to release RSL3 for inhibiting GPX4 activity when Malt-PEG-Abz@RSL3 micelles entered the cells. Meanwhile, key coenzyme NADPH that participated in synthesis of GSH and Trx(SH)₂ was depleted by azobenzene moiety, resulting in decreasing GSH and Trx(SH)₂, which dually induced ferroptosis in tumour cells and promoted cell apoptosis.

Full text of DOI:10.1080/1061186X.2021.1953511

Journal of Drug Targeting
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/idrt20
Effect of Malt-PEG-Abz@RSL3 micelles on HepG2
cells based on NADPH depletion and GPX4
inhibition in ferroptosis
Wenhua Li, Xiaoying Liu, Xu Cheng, Wenyuan Zhang, Chen Gong, Chuya Gao,
Haisheng Peng, Bo Yang, Shukun Tang & Haiquan Tao
To cite this article: Wenhua Li, Xiaoying Liu, Xu Cheng, Wenyuan Zhang, Chen Gong, Chuya
Gao, Haisheng Peng, Bo Yang, Shukun Tang & Haiquan Tao (2021): Effect of Malt-PEG-
Abz@RSL3 micelles on HepG2 cells based on NADPH depletion and GPX4 inhibition in
ferroptosis, Journal of Drug Targeting, DOI: 10.1080/1061186X.2021.1953511
To link to this article: https://doi.org/10.1080/1061186X.2021.1953511
Accepted author version posted online: 08
Jul 2021.
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Effect of Malt-PEG-Abz@RSL3 micelles on HepG2 cells based on
NADPH depletion and GPX4 inhibition in ferroptosis
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Wenhua Li , Xiaoying Liu , Xu Cheng , Wenyuan Zhang , Chen Gong , Chuya Gao ,
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Haisheng Peng , Bo Yang , Shukun Tang , and Haiquan Tao
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School of Pharmacy, Harbin University of Commerce, 138, Tong Da Street,
Harbin 150076, China
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Department of Pharmaceutics, Daqing Campus of Harbin Medical University, 1
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Xinyang Rd, Daqing 163319, China
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Cerebrovascular Diseases Department, Zhuhai Hospital affiliated with Jinan
University, Guangdong Province, Zhuhai,79# kangning Road, 519000, China
Abstract
Ferroptosis is a regulated cell death pathway which depends on iron. Ferroptosis
can be induced by limiting intracellular glutathione (GSH) synthesis, or inhibiting the
activity of GPX4, or increasing intracellular accumulation of PE-AA-OOH, all of
which involve NADPH. Therefore, NADPH depletion, excessive PE-AA-OOH, and
GPX4 deficiency are generally considered to be the main characteristics of ferroptosis.
In this research, the novel self-assembly nanomicelles modified by maltose ligand
(Malt-PEG-Abz@RSL3) with superior nano characteristics were designed and
fabricated. Malt-PEG-Abz@RSL3 micelles achieved active targeted drug delivery
due to the high expression of glucose transporter (GLUT) and high uptake by HepG2
cells. Maltose-polyethylene glycol broke to release RSL3 for inhibiting GPX4 activity
when Malt-PEG-Abz@RSL3 micelles entered the cells. Meanwhile, key coenzyme
NADPH that participated in synthesis of GSH and Trx(SH) was depleted by
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azobenzene moiety, resulting in decreasing GSH and Trx(SH) , which dually induced
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ferroptosis in tumor cells and promoted cell apoptosis.
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Keywords: ferroptosis; micelles; NADPH depletion; GPX4 inhibition; HepG2
cells
1. Introduction
Ferroptosis is discovered by Dixon et al. [1] in 2012 and is a regulated cell death
pathway which depends on iron. Different from other known modes of cell death,
such as apoptosis, necrosis and autophagy, ferroptosis is characterized as
morphological, biochemical and genetic features, including smaller mitochondria,
increased membrane density, and excessive accumulation of lipid peroxides and
reactive oxygen species (ROS) [2]. Ferroptosis has been found to inhibit the
proliferation of tumor cells in liver, pancreatic, prostate and breast cancers. Therefore,
induction of ferroptosis in cells is expected to be a prominent potential for tumor
therapy.
Ferroptosis is mainly induced by two different mechanisms. One mechanism is
inhibition of intracellular GSH synthesis or blocking of GSH dependent glutathion
peroxidase 4 (GPX4) activity. GSH can mutually transform between reduced
glutathione (GSH) and oxidized glutathione (GSSG) in cells. Specifically, GSSG is
reduced to GSH by glutathione reductase (GR) with the help of nicotinamide adenine
dinucleotide phosphate (NADPH). GSH is oxidated to GSSG with GPX4, and this
process can result in reduction of lipid peroxides in cells and preventing cells from
oxidative damage. Hence, ferroptosis will be triggered if GSH oxidation process
above is impaired [3]. NADPH is an essential intracellular reducing agent for the
elimination of lipid peroxides, and it is also a major biomarker of ferroptosis
sensitivity [4].
Another mechanism of ferroptosis is arachidonic acid/adrenic acid (AA/AdA).
Accumulation of PE-AA-OOH is a marker of ferroptosis, and the occurrence of
ferroptosis is closely related to the amount of PE-AA-OOH in AA/AdA. The
production of PE-AA-OOH relies on the presence of iron-dependent fenton reaction,
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the action of ferrilipoxygenase, and the presence of oxygen [5,6]. Moreover, the
generation of PE-AA-OOH in cells also dependents on the activity of GPX4, and
PE-AA-OOH can be oxidized to PE-AA-OH in the presence of GPX4 [7-9].
Therefore, NADPH depletion, excessive PE-AA-OOH, and GPX4 deficiency are
generally considered to be the main characteristics of ferroptosis [10].
Direct inhibition of GPX4 activity and indirect regulation of GSH biosynthesis
are two typical ferroptosis induction pathways [11,12]. Both mechanisms suggest that
GPX4 is a central regulator that triggers ferroptosis and is a key target of ferroptosis
[13]. GPX4 is a powerful antioxidant enzyme, and also is the only enzyme that can
use NADPH as an electron donor to reduce the toxic lipid hydroperoxides. GPX4
participates in the production of GSH and is involved in regulating ferroptosis [14,15].
It is found that the decreasing the content and activity of GPX4 can induce ferroptosis
of tumor cells. Meanwhile, researchers found that the content of GPX4 in cancer
tissues is significantly higher than that in adjacent tissues [16]. RSL3, a GPX4
inhibitor, is first discovered to induce ferroptosis in neuronal HT22 cells and mouse
embryonic fibroblasts. In these cells, RSL3 inhibits GPX4 activity and lipid
peroxidation in a concentration-dependent manner, promotes mitochondrial
fragmentation and mitochondrial membrane loss, and accelerates cell apoptosis [17].
In this research, the novel self-assembly nanomicelles modified by maltose ligand
(Malt-PEG-Abz@RSL3) were designed and fabricated based on the above theory.
Malt-PEG-Abz@RSL3 micelles exhibited high cellular uptake by HepG2 due to the
high expression of GLUT on tumor cell membrane. Maltose-polyethylene glycol
broke to release RSL3 for inhibiting GPX4 activity when Malt-PEG-Abz@RSL3
micelles entered the cell. Meanwhile, key coenzyme NADPH that participated in
synthesis of GSH and Trx(SH) was depleted by azobenzene, resulting in decreasing
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GSH and Trx(SH) , which dually induced ferroptosis in tumor cells and promoted cell
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apoptosis, as shown in Figure 1.
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Figure 1. Schematic illustration of Malt-PEG-Abz@RSL3 micelles induced ferroptosis after entering
the HepG2 cells.
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2. Materials and methods
2.1 Materials
HepG2 cells, DiD dye were donated by Peng’s research group; RSL3 (1S, 3R),
3-(4,
5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide
(MTT),
4-(Dimethylamino) benzaldehyde, pyrene, 5,5’-dithiobis (2-nitrobenzoic acid)
(DTNB) were purchased from Innochem; Dulbecco’s modified Eagle’s medium
(DMEM), and fetal bovine serum (FBS) were purchased from Thermo Fisher
Scientific; Anti-GPX4 antibody, β-actin, annexin V-APC/PI apoptosis assay kit,
+
NADP /NADPH assay kit, reduced glutathione (GSH) assay kit were purchased from
Beyotime Biotechnology Co. Ltd. Ultrasonic cell disruption system (Ningbo Xinzhi
Biotechnology Company, China); Flow cytometry (Calibur, USA); Fluorescence
microscope (Leica, Germany); High speed centrifuge (Eppendorf, Germany);
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Microplate reader (BioTek, USA); Malvem Zetasizer Nano ZS 90 (Malvern
Instruments, Southborough, MA). Transmission Electron Microscopy (TEM) (Hitachi,
Japan).
2.2 Methods
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Scheme 1. The synthetic route of Malt-PEG-Abz and mPEG-Abz.
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2.2.1 Synthesis of Malt-PEG-Abz and mPEG-Abz
The synthesis of Maltose-polyethylene glycol-Azobenzene (Malt-PEG-Abz)
used p-toluidine as the starting material, and the amino group was oxidized to nitroso
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group by potassium hydrogen sulfate composite salt (oxone), and then condensed with
p-phenylenediamine to obtain compound 2 [18]. The amino group activity of
compound 2 was relatively low. By reacting with succinic anhydride, the more active
carboxyl group was introduced and condensed with 10 to obtain 4. At last, compound
11 was added and reacted with 4 to obtain 5 (Malt-PEG-Abz) through click reaction
[19].
Because methoxypolyethylene glycols (mPEG) had low activity, if it directly
reacted with compound 3, the carboxyl group and secondary amine of compound 3
would undergo self-cyclization, and most of them were impurities with few products.
Therefore, the hydroxyl group of mPEG needed to be modified to a more active
amino group. The modification of the amino group first activated the hydroxyl group
of mPEG with p-toluenesulfonyl chloride, then replaced it with sodium azide, and
finally reduced it with palladium on carbon (Pa/C) to obtain compound 8. Compound
8 was condensed with compound 3 through an esterification reaction to obtain 9
methoxypolyethylene glycol-azobenzene (mPEG-Abz) with a higher purity and
yield rate.
2.2.1.1 Synthesis of compound 1 and 2
P-benzylamine (3 g, 28 mmol) was dissolved in CH Cl (50 mL) under an argon
2
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atmosphere in a three-neck flask. A solution of oxone (34.4 g, 56 mmol) was
dropwise added into the mixture and stirred at room temperature overnight. The
resulting crude mixture was dissolved in CH Cl and then washed with water. The
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organic layer was dried over sodium sulfate, filtered, and removed excess solvent
under reduced pressure to afford the product 1 (2.62 g, 77.3%). Compound 1 (2.6 g,
21.7 mmol), and P-phenylenediamine (2.6 g, 24 mmol) were dissolved in acetic acid
(80 mL) under an argon atmosphere in a round-bottomed flask protected from light.
The mixture was stirred at room temperature for 3 d during which time the solvent
was removed by vacuum. The resulting crude mixture was washed with saturated
sodium bicarbonate solution. The organic layer was dried over sodium sulfate, filtered,
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and removed excess solvent under reduced pressure. The crude product was purified
by flash column chromatography on silica gel (CH Cl ) to afford the pure product 2
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(0.8 g, 17.5%): HRMS (ESI) Calcd for C H N [M+H] 212.1; found 212.1.
1
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2.2.1.2 Synthesis of compound 3
Compound 2 (630 mg, 3 mmol), succinic anhydride (600 mg, 2 mmol)
4-dimethylaminopyridine (732 mg, 2 mmol) were dissolved in CH Cl (40 mL) under
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an argon atmosphere in a round-bottomed flask protected from light. The mixture was
stirred at room temperature for 2 d. The resulting crude mixture was washed with 1 m
aqueous HCl. The organic layer was dried over sodium sulfate, filtered, and removed
excess solvent under reduced pressure. The crude product was purified by flash
column chromatography on silica gel (MeOH/CH Cl 1:10) to afford the pure product
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3 (700 mg, 75.4%): HRMS (ESI) Calcd for C H N O [M+H] 312.1; found 312.1.
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2.2.1.3 Synthesis of compound 4
Compound 3 (200 mg, 0.64 mmol), N-hydroxysuccinimide (110 mg, 0.96 mmol)
and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (732 mg, 2 mmol)
were dissolved in CH Cl (10 mL) under an argon atmosphere in a round-bottomed
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flask protected from light. The mixture was stirred at room temperature for 30 min.
Compound 10 (400 mg, 0.64 mmol) was added into the mixture and stirred at room
temperature overnight. The resulting crude mixture was washed with 1 m aqueous
HCl. The organic layer was dried over sodium sulfate, filtered, and removal of excess
solvent under reduced pressure. The crude product was purified by flash column
chromatography on silica gel (MeOH/CH Cl 1:10) to afford the pure product 4 (500
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mg, 83.3%): HRMS (ESI) Calcd for C(21+2n)H(25+4n)N O(3+n) [M+H] 820.5, 864.5,
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908.5 et.al;found 820.5, 864.5, 908.5 et.al.
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2.2.1.4 Synthesis of compound 5
Compound 4 (300 mg, 0.34 mmol) was dissolved in tert-butanol (5 mL) in a
round-bottomed flask protected from light. A solution of compound 11 (134 mg, 0.34
mmol) in H O (5 mL), 0.05 m aqueous CuSO (200 μL, 0.1 mmol) and 0.05 m
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aqueous sodium ascorbate (700 μL, 0.34 mmol) were added. The mixture was stirred
at room temperature overnight. The solvent of resulting crude mixture was removed
by vacuum, dissolved in CH Cl and then filtered out the insoluble impurities. The
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crude product was purified by flash column chromatography on silica gel
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(MeOH/CH Cl 1:10) to afford the pure product 5 (240 mg, 55.3%): H NMR (400
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MHz, DMSO-d6) : δ 10.30 (s, 1H), 8.02 - 7.95 (m, 1H), 7.94 - 7.91 (m, 1H), 7.87 -
7.74 (m, 6H), 7.41 - 7.34 (m, 2H), 5.56 - 5.50 (m, 2H), 5.49 - 5.42 (m, 2H), 5.24 -
5.19 (m, 1H), 5.16 - 5.11 (m, 1H), 5.05 - 4.98 (m, 2H), 4.94 - 4.86 (m, 5H), 4.58 -
4.50 (m, 4H), 4.50 - 4.43 (m, 4H), 4.28 - 4.18 (m, 2H), 4.02 - 3.94 (m, 2H), 4.85 -
3.77 (m, 4H), 3.73 - 3.66 (m, 4H), 3.63 - 3.55 (m, 5H), 3.53 - 3.45 (m, 75H), 3.27 -
3.16 (m, 8H), 3.10 - 2.97 (m, 5H), 2.94 - 2.85 (m, 3H), 2.84 - 2.81 (m, 1H), 2.77 -
2.74 (m, 1H), 2.63 - 2.57 (m, 2H), 2.47 - 2.43 (m, 3H), 2.42 - 2.37 (m, 3H), 2.24 (s,
+
1H). HRMS (ESI) Calcd for C(37+2n)H(51+4n)N O(14+n) [M+2H] , 629.8, 673.8, 717.9
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et.al; found 629.8, 673.8, 717.9.
2.2.1.5 Synthesis of compound 6
mPEG1200 (12 g, 10 mmol), p-toluenesulfonyl chloride (3.82 g, 20 mmol) and
triethylamine (2.8 mL, 2 mmol) were dissolved in CH Cl (20 mL) under an argon
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atmosphere in a round-bottomed flask. The mixture was stirred at room temperature
overnight. The resulting crude mixture was washed with 1 m aqueous HCl. The
organic layer was dried over sodium sulfate, filtered, and removed excess solvent
under reduced pressure. The crude product was purified by flash column
chromatography on silica gel (MeOH/CH Cl 1:10) to afford the pure product 6 (8.9 g,
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+
65.7%): HRMS (ESI) Calcd for C(10+2n)H(14+4n)O(4+n)S [M+2H] 644.4, 688.4, 732.4
et.al; found 644.4, 688.4, 732.4 et.al.
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2.2.1.6 Synthesis of compound 7
Compound 6 (8.9 g, 6.6 mmol), and sodium azide (0.86 g, 13.2 mmol) were
dissolved in N, N-Dimethylformamide (16 mL) under an argon atmosphere in a
round-bottomed flask. The mixture was stirred at 80 ℃ for 5 h. The resulting crude
mixture was dissolved in CH Cl and washed with waters. The organic layer was
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dried over sodium sulfate, filtered, and removed excess solvent under reduced
pressure to afford the pure product 7 (8.0 g, 98.8%): HRMS (ESI) Calcd for
+
C(3+2n)H(7+4n)N O
[M+2H] 623.9, 645.9, 667.9 et.al; found 623.9, 645.9, 667.9
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et.al.
2.2.1.7 Synthesis of compound 8
Compound 7 (8.0 g, 6.5 mmol), and 5% Pa/C (0.8 g, 10%) were dissolved in
methanol (20 mL) under a hydrogen atmosphere in a round-bottomed flask. The
mixture was stirred at room temperature overnight. The resulting crude mixture was
filtered and removed excess solvent under reduced pressure to afford the pure product
+
8 (7.7 g, 99.1%): HRMS (ESI) Calcd for C(3+2n)H(9+4n)NO(1+n) [M+2H] 566.9, 588.9,
610.9 et.al; found 566.9, 588.9, 610.9 et.al.
2.2.1.8 Synthesis of compound 9
Compound 3 (100 mg, 0.32 mmol), N-hydroxysuccinimide (55 mg, 0.48 mmol)
and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (92 mg, 0.32
mmol) were dissolved in CH Cl (10 mL) under an argon atmosphere in a
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round-bottomed flask protected from light. The mixture was stirred at room
temperature for 30 min. Compound 8 (400 mg, 0.32 mmol) was added into the
mixture and stirred at room temperature overnight. The resulting crude mixture was
washed with 1 m aqueous HCl. The organic layer was dried over sodium sulfate,
filtered, and removed excess solvent under reduced pressure. The crude product was
purified by flash column chromatography on silica gel (MeOH/CH Cl 1:10) to afford
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the pure product 9 (400 mg, 83.0%): H NMR (400 MHz, DMSO-d6): δ 10.30 (s, 1H),
7.97 - 7.93 (m, 1H), 7.87 - 7.73 (m, 6H), 7.42 - 7.36 (m, 2H), 3.70 - 3.67 (m, 1H),
3.52 - 3.49 (m, 135H), 3.43 - 3.41 (m, 4H), 3.25 - 3.23 (m, 4H), 3.22 - 3.17 (m, 3H),
2.78 - 2.74 (m, 2H), 2.62 - 2.57 (m, 2H), 2.42 - 2.38 (m, 3H), 2.23 (s, 1H). HRMS
+
(ESI) Calcd for C(20+2n)H(24+4n)N O
[M+2H] , 669.4, 713.4, 735.4 et.al; found
4
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669.4, 713.4, 735.4 et.al.
2.2.2 Preparation and characteristics of micelles
Malt-PEG-Abz (20 mg) was dissolved into methanol (20 mL), and RSL3 (2 mg)
was dissolved into CHCl (1 mL). These two liquids were ultrasonically homogenized
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and mixed in the round-bottomed flask. The organic solvents were evaporated to form
a thin film of the polymer and drug. Deionized water (5 mL) was added to redissolve
the film at room temperature to obtain Malt-PEG-Abz@RSL3 micelles. Sephadex
G50 was used to remove the unencapsulated RSL3. mPEG-Abz@RSL3 micelles were
prepared in accordance with the above method of Malt-PEG-Abz@RSL3 micelles.
Moreover, Malt-PEG-Abz@placebo micelles were prepared without RSL3 as control.
The particle size, polydispersity index (PDI), and zeta potential (ZP) were measured
using a Malvem Zetasizer Nano ZS 90. Transmission Electron Microscopy (TEM)
was used to observe the morphology of samples. The surface tension of micelles at
different concentrations were measured with surface tensiometer, and the critical
micelle concentration (CMC) was obtained by drawing the concentrate-surface
tension curve. In addition, the changes of particle size were measured at 0, 2, 4, 6, 8,
12, 24, 48 and 72 h respectively to observe the stability. RSL3 was dissolved in
DMSO to prepare a standard curve after detecting at 350 nm using UV-5100B
spectrometer. Sephadex G50 was used to remove the unencapsulated RSL3.
Chloroform (400 μL) and methanol (500 μL) were added into micelles (100 μL) for
getting the ultrasound 10 min. Encapsulation efficiency (EE) and loading efficiency
(LE) of micelles were measured by the following equation.
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EE (%, w/w) = RSL3encapsulated content /RSL3total content;
LE (%, w/w) = RSL3encapsulated content/RSL3encapsulated content+ micelles content
.
2.2.3 MTT assay
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HepG2 cells were seeded at a density of 1.0×10 cells/mL in 96-well plates and
cultured for about 24 h. Then the culture medium in each well was replaced with 200
µL of DMEM, and micelles with different concentration (2.5, 5, 10 μg/mL) were
added. After 24 h, MTT (5 mg/mL, 10 µL) was added to each well and the cells were
incubated for another 4 h. Then, MTT-containing solution was removed and 100 µL
of DMSO was added to dissolve the MTT formazan crystals. Finally, the absorbance
at 490 nm of each well was recorded by an ELISA microplate reader.
2.2.4 Hemolytic test
Erythrocyte suspension (200 μL, 2%) were incubated with the different
concentration micelles (2.5, 5, 10, 25, 50, 100 μg/mL) (200 μL) at 37 ℃for 1 h. After
centrifugal separation, the supernatant (100 μL) was collected and added to the
96-well plates, then was determined at 540 nm using UV-vis. The same volume of 200
μL distilled water and 200 μL normal saline were mixed with 200 μL 2% erythrocyte
suspension as positive control (100% hemolysis) and negative control (0% hemolysis),
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Hemolytic rate % = (Asample - Anegative control) / (Apositive control - Anegative control) × 100%.
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2.2.5 Cellular uptake by flow cytometry
The cellular uptake efficiency of drugs was studied in HepG2 cells. HepG2 cells
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were seeded at a density of 1.0×10 cells/mL in 12-well plates and cultured for 24 h,
then the Malt-PEG-Abz@RSL3, mPEG-Abz@RSL3, Malt-PEG-Abz@placebo
micelles (1 mM) were added with 20 μL per well. After 4 h incubation, the cells were
washed twice with PBS, and added 0.25% trypsin. After centrifugal separation, the
supernatant was collected and measured by flow cytometry.
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2.2.6 Cellular uptake by confocal microscopy
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The logarithmic growth phase HepG2 cells (1×10 cells/mL) were seeded into
8-well plates and incubated with Malt-PEG-Abz@RSL3, mPEG-Abz@RSL3,
Malt-PEG-Abz (1 mM, 20 μL) for 4 h. Then, cells were washed twice with PBS and
stained with DiD for 5 min. Finally, the stained cells were washed twice with PBS and
analyzed by confocal microscopy.
2.2.7 Calcein AM-PI staining
Calcein AM and propidium iodide (PI) staining (viability and necrosis) was
performed using a calcein AM/PI kit. The logarithmic growth phase HepG2 cells
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(1×10
cells/mL) were seeded in 8-well plates and incubated with
Malt-PEG-Abz@RSL3, mPEG-Abz@RSL3, Malt-PEG-Abz (1 mM, 20 μL) for 12 h.
After being removed the culture medium and gently washed twice with PBS, the cells
were stained with calcein AM-PI assay solution and incubated at 37 ℃ for 30 min.
Digital images of viable (green fluorescence-excitation wavelength: 494 nm, emission
wavelength: 517 nm) and dead (red fluorescence-excitation wavelength: 535 nm,
emission wavelength: 617 nm) cells in selected areas were visualized using
fluorescence microscopy.
2.2.8 Cell apoptosis
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HepG2 cells (1×10 cells/mL) were seeded into 12-well plates and treated with
Malt-PEG-Abz@RSL3，mPEG-Abz@RSL3，Malt-PEG-Abz (1 mM, 20 μL) for 12 h.
Then, cells were centrifuged at 1000 rpm for 5 min to discard the supernatant. The
cells were then transferred to a 1.5 mL tube containing 195 µL of binding buffer.
FITC-conjugated Annexin V (5 µL) and propidium iodide (PI) (10 µL) were added to
the tube. The cells were gently mixed in a vortexer, protected from light, and
incubated for 10-20 min at room temperature. After incubation, stained cells were
measured by flow cytometry and apoptosis was quantitatively confirmed by analyzing
the percentage of early apoptotic cells using Annexin-VFITC/PI double staining.
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2.2.9 Redox biomarker analysis of ferroptosis
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Depleted NADPH detection: HepG2 cells (1×10 cells/mL) were seeded into
6-well plates and treated with Malt-PEG-Abz@RSL3, mPEG-Abz@RSL3,
Malt-PEG-Abz (1 mM, 20 μL) for 24 h. Cells without drugs were considered as
control group. All cells were washed three times with cold PBS and centrifuged at
1000 rpm for 5 min. The depleted NADPH in cells was measured in each group
+
according to the instructions of the NADP /NADPH ratio kit.
5
Detection of GSH content: HepG2 cells (1×10 cells/mL) were seeded into
6-well plates and treated with Malt-PEG-Abz@RSL3, mPEG-Abz@RSL3,
Malt-PEG-Abz (1 mM, 20 μL) for 24 h. Cells in each group were collected, and the
content of GSH in the cells was detected according to the instructions of GSH kit.
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Detection of Trx(SH) content : HepG2 cells (1×10 cells/mL) were seeded into
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6-well plates and treated with Malt-PEG-Abz@RSL3, mPEG-Abz@RSL3,
Malt-PEG-Abz (1 mM, 20 μL) for 24 h. The level of Trx(SH) was quantified using
2
the same DTNB titration approach [20]. The cell lysate (10 μL) was mixed with 90 μL
of DTNB (1 mM), then incubated for 5 min, and detected at 412 nm using microplate
reader.
2.2.10 Western blot
The expression of GPX4 was analyzed by western blot. In brief, the cell lysates
containing identical protein (40 μg) were subjected to standard electrophoresis,
followed by antibody incubation at 4 °C. The dilution ratio for the primary antibody
was 1:2000 (β-actin-specific antibody) and 1:2500 (GPX4-specific antibody). The
dilution ratio was 1:5000 for both GPX4 and β-actin in terms of the secondary
antibody. The protein bands were performed via the western blot detection reagents
and visualized using the Fusion SoloS imaging system (Vilber, France).
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3. Results
3.1 Synthesis of Malt-PEG-Abz and mPEG-Abz
The azobenzene-terminated PEG was used as the initiator for synthesizing
Malt-PEG-Abz, maltose as a ligand for targeting tumor cells. Malt-PEG-Abz was half
1
oil and half solid (240 mg, 55.3%)， H NMR spectrum and HRMS (ESI) of
1
Malt-PEG-Abz were shown in Figure 2A,B. H NMR (400 MHz, DMSO-d6) : δ
10.30 (s, 1H), 8.02 - 7.95 (m, 1H), 7.94 - 7.91 (m, 1H), 7.87 - 7.74 (m, 6H), 7.41 -
7.34 (m, 2H), 5.56 - 5.50 (m, 2H), 5.49 - 5.42 (m, 2H), 5.24 - 5.19 (m, 1H), 5.16 -
5.11 (m, 1H), 5.05 - 4.98 (m, 2H), 4.94 - 4.86 (m, 5H), 4.58 - 4.50 (m, 4H), 4.50 -
4.43 (m, 4H), 4.28 - 4.18 (m, 2H), 4.02 - 3.94 (m, 2H), 4.85 - 3.77 (m, 4H), 3.73 -
3.66 (m, 4H), 3.63 - 3.55 (m, 5H), 3.53 - 3.45 (m, 75H), 3.27 - 3.16 (m, 8H), 3.10 -
2.97 (m, 5H), 2.94 - 2.85 (m, 3H), 2.84 - 2.81 (m, 1H), 2.77 - 2.74 (m, 1H), 2.63 -
2.57 (m, 2H), 2.47 - 2.43 (m, 3H), 2.42 - 2.37 (m, 3H), 2.24 (s, 1H).
mPEG-Abz without maltose ligand was synthesized as the control according to
1
the 4 steps of the reaction. mPEG-Abz was half oil and half solid (400 mg, 83.0%). H
1
NMR spectrum and HRMS (ESI) of mPEG-Abz were shown in Figure 2C,D. H
NMR (400 MHz, DMSO-d6): δ 10.30 (s, 1H), 7.97 - 7.93 (m, 1H), 7.87 - 7.73 (m,
6H), 7.42 - 7.36 (m, 2H), 3.70 - 3.67 (m, 1H), 3.52 - 3.49 (m, 135H), 3.43 - 3.41 (m,
4H), 3.25 - 3.23 (m, 4H), 3.22 - 3.17 (m, 3H), 2.78 - 2.74 (m, 2H), 2.62 - 2.57 (m, 2H),
2.42 - 2.38 (m, 3H), 2.23 (s, 1H).
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Figure 2. Synthesis of Malt-PEG-Abz and mPEG-Abz compounds. A: H NMR spectrum of
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Malt-PEG-Abz; B: HRMS of Malt-PEG-Abz; C: H NMR spectrum of mPEG-Abz; D: HRMS of
mPEG-Abz.
3.2 Characteristics of different micelles
The results of particle size, ZP, PDI, EE and LE of different micelles were
showed in Table 1, and there was no significant statistical difference in these three
groups (Figure 3A,B). Malt-PEG-Abz@RSL3, mPEG-Abz@RSL3, Malt-PEG-Abz
micelles were spherical (Figure 3C). The three micelles had a maximum UV
absorption at 350 nm (Figure 2D). Critical micelle concentration (CMC) of
Malt-PEG-Abz was about 15 μm, but Malt-PEG-Abz@RSL3，mPEG-Abz@RSL3
were about 20 μm (Figure 3E). This might be due to the change of micelles surface
activity caused by the addition of RSL3. The stability experiment found that within 24
h, the particle size in different groups did not change much, but gradually increased
after 24 h (Figure 3F).
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Table 1. Particle size, ZP, PDI, EE and LE of different micelles (n=3).
Samples
Size(nm)
115.1±3.5
109.5±2.4
99.5±2.3
ZP(mV)
PDI
EE(%)
LE(%)
Malt-PEG-Abz@RSL3
mPEG-Abz@RSL3
Malt-PEG-Abz
-23.5±2.6 0.324±0.07 50.36±3.14 4.2±0.41
-24.7±3.1 0.370±0.31 52.36±3.72 4.6±0.36
-25.7±3.3 0.345±0.28
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Figure 3. Characterization of different micelles. A: Size distribution; B ：Zeta potential
distribution; C: The morphology of Malt-PEG-Abz@RSL3 using TEM. Scale bar: 100 nm；D:
Ultraviolet absorption (a.u.); E: Critical micelle concentration; F: Stability of different micelles in
water during 72 h.
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3.3 MTT assay and hemolytic test
Cell viability assay evaluation of Malt-PEG-Abz@RSL3, mPEG-Abz@RSL3,
and Malt-PEG-Abz in HepG2 cells were showed in Figure 4A.
Malt-PEG-Abz@RSL3 had the strongest cell toxicity compared with other drugs at
the concentration of 5 μg/mL and 10 μg/mL. Malt-PEG-Abz also had some
cytotoxicity, because Abz depleted NADPH to disrupt the physiological balance in the
cell and indirectly induced cell death. Hemolysis ratio of Malt-PEG-Abz@RSL3,
mPEG-Abz@RSL3, and Malt-PEG-Abz were all less than 5%, showing no hemolysis
(Figure 4B).
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Figure 4. Cell viability and hemolysis ratio in different groups. A: MTT assay; B: Hemolysis ratio (%).
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3.4 Cellular uptake abilities of different micelles
Malt-PEG-Abz@RSL3 was taken most in by HepG2 cells, followed by
Malt-PEG-Abz. It was because of the high expression of GLUT on the tumor cell
membrane, which took up more of the maltose modified nanoparticles. The cellular
uptake abilities of Malt-PEG-Abz@RSL3 and Malt-PEG-Abz exhibited significant
*
*
differences compared with the mPEG-Abz@RSL3 group, P＜0.01 (Figure 5A, B).
The results of fluorescence confocal detection were consistent with those of flow
cytometry (Figure 5C).
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Figure 5. Cellular uptake abilities of different micelles. A: Statistical analysis of fluorescence
*
*
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intensity, compared with the mPEG-Abz@RSL3 group, P＜0.01; B: Cellular uptake by HepG2 cells
using flow cytometry; C: Confocal fluorescence images in HepG2 cells. Scale bar: 25 μm.
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3.5 Calcein AM-PI staining and cell apoptosis
The evaluation strategy of drug cytotoxicity to tumor cells included the apoptosis
of HepG2 cells, and the judgement of live and dead cells stained by Calcein AM-PI
dyes. Calcein AM-PI staining revealed a large area of red fluorescence of cell death in
Malt-PEG-Abz@RSL3 group. The second was mPEG-Abz@RSL3, and the weakest
was Malt-PEG-Abz (Figure 6A). Cell apoptosis rate of Malt-PEG-Abz@RSL3 was
84.31%, mPEG-Abz@RSL3 was 52.11%, and Malt-PEG-Abz was 12.16% (Figure 6
B).
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Figure 6. Calcein AM-PI staining and cell apoptosis in different micelles. A: Fluorescence images
of HepG2 cells cultured with different micelles. Viable cells were stained with Calcein-AM (green),
and dead/late apoptotic cells were stained with propidium iodide (PI) (red). Scale bar: 100 μm. B: Flow
cytometiric analysis apoptosis of HepG2 cells. Q1-LL: living cells; Q1-UR: late apoptotic cells; Q1-LR:
early apoptotic cells.
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3.6 Redox biomarker analysis of ferroptosis
+
NADP /NADPH ratio was selected as an indicator to evaluate electron binding
+
capacity of micelles in depleting NADPH. It had been found that NADP /NADPH
ratio was high in Malt-PEG-Abz@RSL3 and Malt-PEG-Abz group. This was due to
the high uptake of the maltose modified Malt-PEG-Abz micelles by the HepG2 cells,
and N=N of Abz would deplete NADPH. There were significant differences in
+
NADP /NADPH ratio between the three groups and control group, as shown in
Figure 7A. Content of GSH and Trx(SH) in Malt-PEG-Abz@RSL3 group were
2
lowest, the second was mPEG-Abz@RSL3, and the last one was Malt-PEG-Abz.
Malt-PEG-Abz@RSL3 not only released the GPX4 inhibitor RSL3, resulting in a
decline in the production of GSH synthesis, but also Abz depleted NADPH, a key
coenzyme in GSH synthesis, leading to a further decrease in GSH content. Trx(SH)2
synthesis also required GPX4 and NADPH, so a similar trend appeared like GSH
(Figure 7B). GPX4 protein expression in Malt-PEG-Abz@RSL3 was lowest, and the
second was mPEG-Abz@RSL3. However, Malt-PEG-Abz had no effect on GPX4
protein expression (Figure 7C,D).
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Figure 7. Analysis of redox biomarker NADPH, GSH and Trx(SH) in different micelles. A:
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+
****
***
####
Relative NADP /NADPH ratio,
P＜0.0001, P＜0.001, compared with control;
P＜0.0001
***
**
compared with Malt-PEG-Abz@RSL3; B: Relative content of GSH and Trx(SH)2, P＜0.001, P＜
*
##
#
0.01, P＜0.05 compared with control; P＜0.01, P＜0.05 compared with Malt-PEG-Abz@RSL3; C:
****
Statistical analysis of GPX4 expression; D: Relative GPX4 expression detection by western blot.
P
#
###
##
＜0.0001, compared with control; P＜0.0001, P＜0.01 compared with Malt-PEG-Abz@RSL3.
4. Discussion
As one of the basic substances of life, sugar has been widely applied in the design
of targeted nano-drug. Sugar modified nanoparticles can not only greatly improve
drug solubility, but also achieve the tumor targeting of drugs through GLUT.
Researches have shown that GLUT highly expresses on the tumor cell membrane to
help the cells take in more sugar [21]. The flow cytometry and fluorescence confocal
results showed that the number of Malt-PEG-Abz@RSL3 and Malt-PEG-Abz
micelles entered the cells was significantly higher than that of mPEG-Abz. This sugar
ligand modified nanoparticles can achieve active targeted drug delivery, which is
consistent with literature reports [22-25].
The essence of ferroptosis is the disorder of lipid oxide metabolism in cells. The
accumulation of ROS in lipids will weak the antioxidant capacity and cause the redox
imbalance of cells, which induces the ferroptosis. In this research, RSL3 and Abz
directly and indirectly control the synthesis production of GSH and Trx(SH) , thus
2
induce ferroptosis [26]. After Malt-PEG-Abz@RSL3, mPEG-Abz@RSL3,
Malt-PEG-Abz entering the cells, Abz moiety will break to release RSL3 in anaerobic
tumor cells. Meanwhile, N=N of Abz will deplete NADPH. The both synthesis
processes of GSSH to GSH and Trx(SS) to Trx(SH) require NADPH to provide
2
electrons, but the depletion of NADPH by Abz has hindered the synthesis of GSH and
Trx(SH) and increased the sensitivity for ferroptosis.
2
GPX4 can degrade small molecular peroxides and some lipid peroxides to
restrain lipid peroxidation. It has been found that down-regulation of GPX4
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expression in cells is more sensitive to ferroptosis. In contrast, up-regulation of GPX4
expression results in ferroptosis tolerance. Western blot results showed that the
expression of GPX4 was down-regulated in Malt-PEG-Abz@RSL3,
mPEG-Abz@RSL3, and Malt-PEG-Abz groups, among which the strongest one was
Malt-PEG-Abz@RSL3, and the weakest one was Malt-PEG-Abz without
encapsulating RSL3. However, GPX4 expression was down-regulated in
Malt-PEG-Abz micelles, which might be related to the depletion of NADPH by Abz,
indirectly leading to the low expression of GPX4. GPX4 is a selenoprotein required
for the efficient reduction of peroxidized phospholipids [27-29] and suppresses the
activation of arachidonic acid (AA)-metabolizing enzymes [30], which may
contribute to the process of phospholipid peroxidation. The process of Trx(SH)2
transform into Trx(SS) and GSH transform into GSSG have been hindered after
Malt-PEG-Abz@RSL3 inhibiting the activity of GPX4. Therefore, the process of
PE-AA-OOH transform into PE-AA-OH in AA/ ADA which relies on the two
pathways above is also blocked, resulting in increased level of PE-AA-OOH and
inducing occurrence of ferroptosis.
In a word, Malt-PEG-Abz@RSL3 micelles can reduce the amount of necessary
NADPH and inhibit the activity of GPX4, which will induce ferroptosis and achieve
the purpose of tumor cell apoptosis.
5. Conclusion
Malt-PEG-Abz@RSL3 was designed and modified with sugar ligand, which not
only improved the stability of drugs, but achieved active targeted drug delivery due to
the overexpressed GLUT and high uptake by HepG2 cells. Meanwhile,
Malt-PEG-Abz@RSL3 micelles could release RSL3 to inhibit GPX4 and deplete
NADPH in cells, which dually induced ferroptosis in cells for tumor therapy.
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Acknowledgments
The authors thanks to the National Natural Science Foundation of China
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supported this word (No.81671814), the Heilongjiang Provincial Natural Science
Foundation (No. ZD2016013), the Fundamental Research Funds for the Provincial
Universities (JFWLD202002, 2018XN-22, 2018XN-24), Cultivation Project of
Zhuhai People’s Hospital in 2019 (2019PY-11).
References
[1] Dixon SJ, Lemberg KM, Lamprecht MR, et al. Ferroptosis: an iron-dependent
form of nonapoptotic cell death. Cell. 2012;149(5):1060-1072.
[2] Dixon SJ, Patel DN, Welsch M, et al. Pharmacological inhibition of
cystine-glutamate exchange induces endoplasmic reticulum stress and ferroptosis.
Elife. 2014;3:e2523.
[3] Zhang ZZ, Lee EE, Sudderth J, et al. Glutathione depletion, pentose
phosphate pathway activation, and hemolysis in erythrocytes protecting cancer cells
from vitamin c-induced oxidative stress. J Biol Chem. 2016;291(44):22861-22867.
[4] Shimada K, Hayano M, Pagano NC, et al. Cell-line selectivity improves the
predictive power of pharmacogenomic analyses and helps identify NADPH as
biomarker for ferroptosis sensitivity. Cell Chem Biol. 2016;23(2):225-235.
[5] Conrad M, Pratt DA. The Chemical Basis of Ferroptosis. Nat. Chem. Biol.
2019;15:1137-1147.
[6] Friedmann Angeli JP, Krysko DV, Conrad M. Ferroptosis at the Crossroads of
Cancer-Acquired Drug Resistance and Immune Evasion. Nat. Rev. Cancer.
2019;19:405-414.
[7] Friedmann Angeli JP, Conrad M. Selenium and GPX4, a Vital Symbiosis.
Free Radical Biol. Med. 2018;127:153−159.
[8] Liu H, Schreiber SL, Stockwell BR. Targeting Dependency on the GPX4
Lipid Peroxide Repair Pathway for Cancer Therapy. Biochemistry. 2018;57:
2059-2060.
2
3

5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
[9] Hirschhorn T, Stockwell BR. The Development of the Concept of Ferroptosis.
Free Radical Biol. Med. 2019;133:130-143.
[10] Dixon SJ, Stockwell BR. The Hallmarks of Ferroptosis. Annu. Rev. Cancer
Biol. 2019;3:35-54.
[11] Stockwell BR, Friedmann Angeli JP, Bayir H, et al. Ferroptosis: A Regulated
Cell Death Nexus Linking Metabolism, Redox Biology, and Disease. Cell.
2017;171:273-285.
[12] Meng X, Deng J, Liu F, et al. Triggered All-Active Metal Organic
Framework: Ferroptosis Machinery Contributes to the Apoptotic Photodynamic
Antitumor Therapy. Nano Lett. 2019;19:7866-7876.
[13] Friedmann AJ, Schneider M, Proneth B, et al. Inactivation of the ferroptosis
regulator Gpx4 triggers acute renal failure in mice. Nat Cell Biol. 2014;16(12):
1180-1191.
[14] Ingold I, Berndt C, Schmitt S, et al. Selenium utilization by GPX4 is
required to prevent hydroperoxide-induced ferroptosis. Cell. 2018;172(3):409-422．
[15] Eaton JK, Furst L, Ruberto RA, et al. Selective covalenttargeting of GPX4
using masked nitrile-oxide electrophiles. Nat Chem Biol. 2020; 30: 497-506．
[16] Lin YS, Shen YC, Wu CY, et al. Danshen improves survival ofpatients with
breast cancer and dihydroisotanshinone I induces ferroptosis and apoptosis of breast
cancer cells. Front Pharmacol. 2019;10:1226．
[17] Jelinek A, Heyder L, Daude M, et al. Mitochondrial rescue prevents
glutathione peroxidase-dependent ferroptosis. Free Radic Biol Med. 2018;117:45-57.
[18] Yu B C, Shirai Y, Tour J M. Syntheses of new functionalized azobenzenes for
potential molecular electronic devices. Tetrahedron. 2006;62(44):10303-10310.
[19] Zhang C, Chen Z, Li W, et al. Influences of Different Sugar Ligands on
Targeted Delivery of Liposomes. Journal of Drug Targeting, 2020, 28(35):1-28.
[20] Duan D, Zhang B, Yao J, et al. Shikonin Targets Cytosolic Thioredoxin
Reductase to Induce ROS-Mediated Apoptosis in Human Promyelocytic Leukemia
HL-60 Cells. Free Radical Biol. Med. 2014;70:182-193.
2
4

5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
[21] Chen F, Huang G. Application of glycosylation in targeted drug delivery. Eur
J Med Chem. 2019;182:111612.
[22] Goff RD, Thorson JS. Assessment of chemoselective neoglycosylation
methods using chlorambucil as a model. J Med Chem. 2010;53(22):8129-8139.
[23] Miot-Noirault E, Reux B, Debiton E, et al. Preclinical investigation of
tolerance and antitumour activity of new fluorodeoxyglucose-coupled chlorambucil
alkylating agents. Invest New Drugs. 2011;29(3):424-433.
[24] Hwu J R, Hsu C I, Hsu M H, et al. Glycosylated nordihy-droguaiaretic acids
as anti-cancer agents. Bioorg Med Chem Lett. 2011;21(1):380-382.
[25] Hossain F, Andreana PR. Developments in carbohydrate-based cancer
therapeutics. Pharmaceuticals. 2019;12(2):84.
[26] Guo XL, Liu F, Deng J, et al. Electron-Accepting Micelles Deplete Reduced
Nicotinamide Adenine Dinucleotide Phosphate and Impair Two Antioxidant Cascades
for Ferroptosis-Induced Tumor Eradication. ACS Nano. 2020; 14(11):14715-14730.
[27] Friedmann Angeli JP, Schneider M, Proneth B, et al. Inactivation of the
ferroptosis regulator Gpx4 triggers acute renal failure in mice. Nat. Cell Biol.
2014;16:1180-1191.
[28] Yang, WS, SriRamaratnam R, Welsch ME, et al. Regulation of ferroptotic
cancer cell death by GPX4. Cell. 2014; 156: 317-331.
[29] Ingold I, Berndt C, Schmitt S, et al. Selenium utilization by GPX4 is
required to prevent hydroperoxide- induced ferroptosis. Cell. 2018;172:409-422.
[30] Kagan VE, Mao GW, Qu F, et al. Oxidized arachidonic and adrenic PEs
navigate cells to ferroptosis. Nat. Chem. Biol. 2017;13:81-90.
5
5
97
98
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5