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Article
Iron oxide nanoparticles induce autophagosome accumulation through multiple mechanism: lysosome impairment, mitochondrial damage and ER stress Xudong Zhang, Hongqiu Zhang, Xin Liang, Jinxie Zhang, Wei Tao, Xianbing Zhu, Danfeng Chang, Xiaowei Zeng, Gan Liu, and Lin Mei Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00405 • Publication Date (Web): 10 Jun 2016 Downloaded from http://pubs.acs.org on June 13, 2016
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Molecular Pharmaceutics
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Iron oxide nanoparticles induce autophagosome accumulation
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through multiple mechanism: lysosome impairment, mitochondrial
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damage and ER stress
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Xudong Zhang1, 2, 3, a, Hongqiu Zhang1, 2, a, Xin Liang2,4, Jinxie Zhang1, 2,, Wei Tao1,2,
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Xianbing Zhu1,2 , Danfeng Chang1, 2, Xiaowei Zeng1, 2, Gan Liu1, 2 , & Lin Mei1, 2, *
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1
School of Life Sciences, Tsinghua University, Beijing 100084, China;
2
Division of Life and Health Sciences, Graduate School at Shenzhen, Tsinghua
University, Shenzhen 518055, P.R. China 3
Joint Department of Biomedical Engineering, University of North Carolina at
Chapel Hill and North Carolina State University, Raleigh, NC 27695, USA 4
Department of Pharmacological and Physiological Science and Center for
Neuroscience, Saint Louis University School of Medicine, St. Louis, Missouri, USA
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a
These authors contributed equally to this work.
*
Corresponding author.
Tel/Fax: +86 75526036736, E-mail:
[email protected] 23 24 25 26 1
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Molecular Pharmaceutics
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ABSTRACT
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Magnetite (iron oxide, Fe3O4) nanoparticles have been widely used for drug
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delivery and magnetic resonance imaging (MRI). Previous studies have shown that
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many metal-based nanoparticles including Fe3O4 nanoparticles can induce
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autophagosome accumulation in treated cells. However, the underlying mechanism is
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still not clear. To investigate the bio-safety of Fe3O4 and PLGA-coated Fe3O4
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nanoparticles, some experiments related to the mechanism of autophagy induction by
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these nanoparticles have been investigated. In this study, the results showed that
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Fe3O4, PLGA-coated Fe3O4 and PLGA nanoparticles could be taken up by the cells
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through cellular endocytosis. Fe3O4 nanoparticles extensively impair lysosomes and
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lead to the accumulation of LC3-positive autophagosomes, while PLGA-coated Fe3O4
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nanoparticles reduce this destructive effect on lysosomes. Moreover, Fe3O4
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nanoparticles could also cause mitochondrial damage, ER and Golgi body stresses,
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which induce autophagy, while PLGA-coated Fe3O4 nanoparticles reduce the
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destructive effect on these organelles. Thus, the Fe3O4-nanoparticle induced
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autophagosome accumulation may be caused by multiple mechanisms. The
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autophagosome accumulation induced by Fe3O4 was also investigated. The Fe3O4,
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PLGA-coated Fe3O4 and PLGA nanoparticle treated mice were sacrificed to evaluate
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the toxicity of these nanoparticles on the mice. The data showed that Fe3O4
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nanoparticle treated mice
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autophagosomes in the kidney and spleen in comparison to the PLGA-coated Fe3O4
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and PLGA nanoparticles. Our data clarifies the mechanism by which Fe3O4 induces
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autophagosome accumulation and the mechanism of its toxicity on cell organelles and
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mice organs. These findings may have an important impact on the clinical application
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of Fe3O4 based nanoparticles.
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Key Words: Fe3O4; PLGA; Autophagy; Lysosome; Nanomedicine
would lead to the extensive accumulation of
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INTRODUCTION
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Magnetite (Fe3O4) is a miraculous half-metallic material because it is magnetic.
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The external magnetic field allows Fe3O4 particles localize to a specific area, which
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makes Fe3O4 a promising material for medicine [1]. Moreover, nanosized Fe3O4
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changes some of these magnetic properties, such as making the nanoparticles own the
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superparamagnetic phenomena [1, 2]. Superparamagnetic nanoparticles have many
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promising applications in nanomedicine such as cell targeting, drug delivery,
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magnetic resonance imaging (MRI), hyperthermia, magnetofection and tissue repair
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[1]. Before the application to medicine, the particles have to be modified with some
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molecular layers to generate biocompatibility [3]. Inorganic materials such as gold,
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silica, and alumina have been extensively used to modify the Fe3O4 nanoparticles [1].
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Some organic polymeric materials based on poly(ethylene-co-vinyl acetate),
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poly(vinylpyrrolidone)
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poly(ethyleneglycol) (PEG), and poly(vinyl alcohol) (PVA) are also used to modify
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the Fe3O4 nanoparticles [1]. Linking ligands such as antibodies, proteins, peptides and
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small molecules to the polymer surfaces will make the particles target specific tissues
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and organs for drug delivery and diagnosis [4].
(PVP),
poly(lactic-co-glycolic
acid)
(PLGA),
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Autophagy involves the degradation of deleterious cellular components including
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aggregation-prone proteins and damaged organelles or invaded pathogens [5, 6]. An
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isolated double membrane (also termed phagophore), sequesters the cytoplasm
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components, including several organelles and cytosol, to form a vesicle (also referred
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as autophagosome) [6]. The autophagosome is fused with a lysosome and forms an
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auto-lysosome which degrades the “cargo” within it. Many previous publications have
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reported that autophagosomes accumulate within the cells after treating the cells with
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metal-based NPs such as gold NPs [7, 8], ferric oxide (Fe2O3 and Fe3O4) [9],
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manganese oxide (MnO), copper oxide (CuO) [10], neodymium oxide (Nd2O3) [11],
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titanium oxide (TiO2) [12] and silica oxide (SiO2) [10, 12]. Many cellular stressed
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activities could induce the initiation of autophagy in the cells. These stressful events
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include ER stress, Golgi body stress and mitochondrial damage [13]. Most of these 4
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Molecular Pharmaceutics
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NPs are found to be internalized by cells through endocytosis [4]. Internalization
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allows the NPs reach the subcellular locations and interact with diverse organelles
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such as mitochondria, Endoplasmic Reticulum (ER), lysosome, Golgi body, nuclear
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membrane, even chromosomes [4]. Thus, many cellular reactions including autophagy
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may be induced by interactions between the NPs and organelles. Thus, the presence of
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autophagosomes with the cell-induced metal-based nanoparticles may be caused by
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multiple mechanisms.
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However, Autophagy induction cannot only determined by the presence of
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autophagosome within the cells. Autophagy is a complex cellular process, the
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autophagosome is matured from the isolated membrane, and it finally fuse with
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lysosome,
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autophagosomes can result from true induction of autophagy or blockade of the
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turnover of autophagosome by lysosomes [14]. Previous research shows that
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autophagosomes are induced by treating the cells with the gold NPs [7]. However, a
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recent report shows that gold NPs could impair the function of the lysosomes, and
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lead to the accumulation of autophagosomes [15]. In other words, the gold NPs may
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not induce autophagy, instead they may impair the autophagic flux [15]. Autophagic
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substrates such as p62/SQSTM1 are required for establishing induction of autophagy
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[5]. When the protein levels are decreased, it indicates that autophagy may be induced.
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Conversely, when the protein level of p62 is increased, the autophagic flux may be
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blocked and the function of the lysosome may be impaired. Our work investigated the
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detailed relationship and mechanism between Fe3O4 nanoparticles and autophagy,
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which may have important implications for the development of Fe3O4 nanoparticles
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for biomedical applications.
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EXPERIMENTAL SECTION
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Materials
which
called
“autophagic
flux’’ [14].
Thus,
accumulation
of
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Coumarin-6, oleic acid, PLGA (acid terminated, lactide:glycolide 50:50,
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Mw24,000-38,000), oleylamine and Fe(acac)3 were purchased from Sigma-Aldrich
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(St. Louis, MO, USA). All other chemicals of highest quality were commercially
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available and used as received. Antibodies against EEA1, LC3, p62, mTOR, p-mTOR, 5
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ULK1 and p-ULK1 were from Cell Signaling Technology (Beverly, MA, USA).
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Antibodies against Tom20 were from Santa Cruz Biotechnologies. Antibody against
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actin was obtained from Abmart, Inc. (Shanghai, China). Golgi’s Complex Tracker
4
Red, ER’s Tracker Red and Lysosome Tracker Red probes were obtained from
5
Beyotimes (Jiangsu, China). JC-1 and ROS probes were also obtained from
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Beyotimes. The EGFP-LC3 and DsRed-LC3 plasmids were from our lab.
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DsRed-Rab5 and DsRed-Rab7 were from Addgene (Cambridge, MA, USA). All
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plasmids were confirmed by automated DNA sequencing. Cells were transiently
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transfected with the plasmids using lipofectamine 2000 (Invitrogen) according to the
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manufacturer’s instructions.
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Methods
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Synthesis of Fe3O4
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The iron oxide (Fe3O4) nanoparticles were prepared by solvent-free thermal
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decomposition method of Fe(acac)3 as done earlier. 2 mM of Fe(acac)3 was solved in
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a mixture of 10 ml oleic acid (90%, sigma-aldrich) and 10 ml oleylamine (70%,
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sigma-aldrich). Then the solution is magnetically stirred under a flow of argon gas. It
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was heated at 120 °C for 1 hour for dehydration, then heated to 300 °C quickly and
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kept it for 1 hour. The solution was cooled in room temperature for hours until it is
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down to room temperature. Then 20 ml of ethanol was added and IOs were collected
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by centrifugation at 10,000 rpm. After that, it is washed by ethanol three times.
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Finally the Fe3O4 particles were dispersed in THF.
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Formulation of Fe3O4-encapsulated and coumarin-6-loaded PLGA nanoparticles
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The IOs (Fe3O4)-encapsulated PLGA capsules were prepared using the
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membrane dialysis method [29]. Briefly, 100 mg of copolymer PLGA and 10 mg of
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IOs were dissolved in 10 mL of THF. With this mixture, 15 mL of deionized water
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was added dropwise under stirring. The resulting solution was transferred to dialysis
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bag (MWCO: 3500 Da, Spectra/Por® 6, Spectrum Laboratories, CA, USA) and
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dialyzed against deionized water for 24 h. Finally, these capsules were freeze-dried
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for two days. In addition, the coumarin-6-loaded PLGA capsules were prepared in the
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same way except the Fe3O4 was replaced by coumarin-6. 6
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Molecular Pharmaceutics
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Characterization of Fe3O4, PLGA-coated Fe3O4 and PLGA NPs
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The size and zeta potential of the nanoparticles were measured by Malvern
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Mastersizer 2000 (Zetasizer Nano ZS90, Malvern Instruments Ltd., UK). The
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morphology of the nanoparticles was observed by transmission electron microscopy
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(TEM, Tecnai G2 20, FEI Company, Hillsboro, Oregon, USA). Before observation,
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the sample was deposited onto a copper grid coated with carbon and dried at room
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temperature.
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Cell culture
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MCF-7 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)
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supplemented with 10% Fetal Bovine Serum (FBS).
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Cellular uptake of coumarin-6-loaded PLGA-coated Fe3O4 and PLGA NPs.
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Coumarin-6 was used as a model fluorescent molecule, which was formulated in
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PLGA-coated Fe3O4 and PLGA NPs. Cellular uptake of the coumarin-6 loaded
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PLGA-coated Fe3O4 NPs and PLGA NPs by MCF-7 cells were assessed. The
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non-transfected or DsRed-Rab5 and DsRed-Rab7 cells were incubated with 100
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µg/mL coumarin-6-loaded PLGA-coated Fe3O4 and PLGA NPs at 37 ℃ for 2 h. For
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lysosome detection, the cells were incubated with Lyso-Tracker Red for 1 h. Then the
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cells were washed with PBS for three times, and then fixed by 4% paraformaldehyde
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for 20 min. Cells were stained with DAPI for 15 min and washed three times with
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PBS. Confocal microscopy was performed on a FLUO-VIEW laser scanning confocal
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microscope (Olympus, FV1000, Olympus Optical, and Japan) in sequential scanning
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mode using a 60~100×objective.
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Autophagy assays
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The cells were transfected with EGFP-LC3 under indicated conditions and then
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fixed in 4% paraformaldehyde. The percentage of cells with fluorescent dots
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representing EGFP-LC3 translocations were counted by confocal microscopy as
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described previously [31].
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LC3II protein level was detected using the anti-LC3 antibody. Immunoblotting Immunoblotting analysis was performed as previously described [31]. For 7
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abbreviation, cell lysates were resolved on 12% SDS-PAGE and analyzed by
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immunoblotting using LC3, p62, mTOR, p-mTOR, ULK1 and p-ULK1 antibody,
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followed by enhanced chemiluminescence (ECL) detection (Thermo Scientific).
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Mitochondrial transmembrane potential (∆Ψm) detection
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JC-1 mitochondrial membrane potential assay kit was used to detect the
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mitochondrial (∆Ψm) according to the manufacturer’s protocol. Briefly, the MCF-7
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cells were seeded in 12-well plates, and treated with 100 µg/mL Fe3O4 NPs,
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PLGA-coated Fe3O4 (PLGA-Fe3O4) NPs and PLGA NPs for 24 h. Then, 100 µl JC-1
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staining solution was add to each well and incubated for another 15~30 min. The cells
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were washed with PBS for three times, and then fixed by 4% paraformaldehyde for 20
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min. After that, cells were stained with DAPI for 15 min and washed three times with
12
PBS. Confocal microscopy was performed on a FLUO-VIEW laser scanning confocal
13
microscope (Olympus, FV1000, Olympus Optical, and Japan) in sequential scanning
14
mode using a 60~100×objective.
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Reactive oxygen species (ROS) detection
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MCF-7 cells were seeded in 6-well plates. The cells were treated with 100 µg/mL
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Fe3O4 NPs, PLGA-coated Fe3O4 (PLGA-Fe3O4) NPs and PLGA NPs for 24 h, Then
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cells were incubated with DCFH-DA (Beyotime Biotechnology, China) according to
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the instruction. For quantitative analysis, the ROS levels were examined using a
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Partec PAS III flow cytometer (Partec, Munster, Germany).
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In vivo autophagy assay with NIH mice
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Animal experiments were approved by the Administrative Committee on Animal
23
Research in the Tsinghua University Shenzhen Graduate School. Female severe
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combined NIH mice were purchased from Guangdong Medical Laboratory Animal
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Center. Mice were detected by organ-size and weight. IOs (at a single dose of 10 mg
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IOs /kg), PLGA-coated Fe3O4 NPs (at a single dose of 10 mg IOs /kg) and PLGA NPs
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were injected via peritoneal cavity in PBS on days 0, 2, 4, 8, 10 and 12. Mice were
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sacrificed by cervical decapitation 20 days after treatment. The terminal organ
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damage was measured and utilized to evaluate the autophagy. The organ samples were
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used to make LC3. 8
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Molecular Pharmaceutics
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Immunofluorescence assay
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Immunohistochemical analysis was performed as previously described [20]. For
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abbreviation, the cells or tumor sections were incubated with primary antibodies. The
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TRITC and FITC labeled secondary antibody was used to detect the primary antibody.
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Statistical methodology
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All results are reported as the mean ± SD of three independent experiments.
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Comparisons were performed using a two-tailed paired Student’s t test (* P
