Fluorescent magnetic PEI-PLGA nanoparticles loaded with paclitaxel for concurrent cell imaging, enhanced apoptosis and autophagy in human brain cancer

Accepted Manuscript Title: Fluorescent magnetic PEI-PLGA nanoparticles loaded with paclitaxel for concurrent cell imaging, enhanced apoptosis and autophagy in human brain cancer Authors: Xueqin Wang, Liping Yang, Huiru Zhang, Baoming Tian, Ruifang Li, Xuandi Hou, Fang Wei PII: DOI: Reference:

S0927-7765(18)30644-1 https://doi.org/10.1016/j.colsurfb.2018.09.033 COLSUB 9635

To appear in:

Colloids and Surfaces B: Biointerfaces

Received date: Revised date: Accepted date:

8-5-2018 9-8-2018 13-9-2018

Please cite this article as: Wang X, Yang L, Zhang H, Tian B, Li R, Hou X, Wei F, Fluorescent magnetic PEI-PLGA nanoparticles loaded with paclitaxel for concurrent cell imaging, enhanced apoptosis and autophagy in human brain cancer, Colloids and Surfaces B: Biointerfaces (2018), https://doi.org/10.1016/j.colsurfb.2018.09.033 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Fluorescent magnetic PEI-PLGA nanoparticles loaded with paclitaxel for concurrent cell imaging, enhanced apoptosis and autophagy

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in human brain cancer

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Xueqin Wang*a, Liping Yang a, Huiru Zhanga, Baoming Tianb, Ruifang Lia, Xuandi Houa, Fang Wei b*

College of Bioengineering, Henan University of Technology, Zhengzhou, Henan 450001, P.R .China

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School of Life Sciences, Zhengzhou University, Zhengzhou, Henan 450001,P.R.China

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* Corresponding authors: Xueqin Wang, E-mail address: [email protected], College of Bioengineering,

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Henan University of Technology, Zhengzhou, Henan 450001, P.R .China. Tel.: + 86 371 67756928; fax: + 86 371 67756928. Fang Wei, E-mail address: [email protected], School of Life Sciences, Zhengzhou University,

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Zhengzhou, Henan 450001,P.R.China. Tel.: + 86 371 67739513; fax: + 86 371 67739513.

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Graphical Abstract

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Highlights about the present work

A magnetic nanoplatform is fabricated for simultaneous cell imaging and drug

The fabricated nanoplatform could effectively inhibit cell growth and migration.

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delivery.

The fabricated nanoplatform could effectively induce cell apoptosis and autophagy.

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The fabricated nanoplatform could be a promising nanovehicle for brain tumor

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therapy.

Abstract 2

Magnetic nanoparticles are regarded as a promising drug delivery vehicle with the improved efficacy and lowered side effects for antitumor therapy. Herein, the poly lactic-coglycolic acid (PLGA) modified magnetic nanoplatform was synthesized using

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superparamagnetic γ-Fe2O3 nanoparticles (MNPs) as a core, and then labelled with polyethylenimine (PEI)-conjugated fluorescein isothiocyanate (FITC), and simultaneously

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loaded with antitumor drug paclitaxel (PTX) for theranostic analysis of antitumor effects

investigated in human brain glioblastoma U251 cells. As a result, the prepared PEI-PLGAMNPs showed a relatively round sphere with an average size of 80 nm approximately, and the

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FITC-labeling PEI-PLGA-MNPs were efficiently endocytosed by the U251 cells for cellular

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imaging. Moreover, the fabricated PEI-PLGA-PTX-MNPs also demonstrated an inhibition of

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the targeted cell proliferation and migration, and a programmed cell death, via both apoptosis modulating by a burst of reactive oxygen species (ROS) and autophagy with accumulation of

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autophagosomes and LC3-II signals detected in the treated glioblastoma U251 cells after

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uptaking. Therefore, the constructed nanoplatform could be effectively applied for simultaneous cellular imaging and drug delivery in human brain glioblastoma treatment in

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future.

Key words: Magnetic nanoparticles; Poly (Lactic-co-Glycolic Acid) (PLGA); Paclitaxel

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(PTX); Apoptosis; Autophagy; Fluorescein-labeling; Brain glioblastoma

1. Introduction

Drug delivery systems including physical entrapment and chemical covalent conjugation of 3

drugs have significant potential in improving drug loading and targeting [1, 2], reducing drug efflux from a surface amendable to chemical conjugation for targeting purposes [3]. The superparamagnetic iron oxide nanoparticles, particularly magnetite (Fe3O4) and maghemite

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(γ-Fe2O3), have proven as an efficient drug delivery carrier for both hydrophilic and hydrophobic drugs because of their nanosized diameter and outstanding superparamagnetic

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properties [4, 5], efficient drug loading and ease of biophysico-chemical modification [6, 7]. The most effective approach of drug delivery is the coating of the vesicle surface with biocompatible materials [8, 9]. Poly (Lactic-co-Glycolic Acid) (PLGA) is a preferential

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candidate as drug-carrier coating matrices material approved by the US Food and Drug

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Administration in biomedical applications due to its high stability, attractive biodegradability

applications and clinical translation.

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and biocompatibility [10,11], which ensures prolonged circulation time and safety for in vivo

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Paclitaxel (PTX) is a natural broad-spectrum anticancer drug, which has been extensively

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used in clinical treatments, and showed antitumor activity against various solid tumors such as breast, lung, gastric, ovarian, prostate cancers and glioma [12-15]. PTX induces tumor cells

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apoptosis by stabilizing microtubule assembly through non-covalent interactions with the cytoskeleton, thereby blocking cell division [16]. However, PTX has limited clinical

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application due to side effects, such as hydrophobicity, low therapeutic index, chemoresistance, highly invasive, and cellular resistance [17]. In order to overcome the side effects, the encapsulation of PTX into PLGA nanoparticles not only improves delivery of this relatively hydrophobic drug, but also strongly enhances its cytotoxic effect as compared to Taxol in vitro [18-20]. 4

Moreover, one critical challenge in early cancer diagnosis and treatment using nanotechnology is the development of multifunctional nanoparticles that simultaneously serve as sensitive and localized tumor treatments. The development of multifunctional nanocarriers

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has attracted increasing attention because of their advantageous properties [21, 22]. Fluorescent polymer/magnetic multifunctional nanocarriers that are a combination of

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fluorescent dye and polymer/magnetic nanoparticles have attracted great attention [23-25], and these nanoparticles possess three attractive features, namely, fluorescence,

biocompatibility and superparamagnetism, which allow their intracellular movements to be

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controlled by magnetic force and monitored by a fluorescence microscopic system

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simultaneously. These features lead to effective ways to probe specific functions of bioactive

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molecules in localized domains of living cells without disturbing other parts of the cell. Herein, we attempted to develop a multi-functional nanoplatform as drug delivery system

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using superparamagnetic γ-Fe2O3 nanoparticles (MNPs) as a core, which was further surface-

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modified with PLGA, and labeling with fluorescein isothiocyanate (FITC) through polyethylenimine (PEI) bridging, and simultaneously loaded with anti-tumor drug PTX to

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formulate PTX-loaded fluorescent MNPs through emulsion/solvent evaporation method [2629]. The prepared PEI-PLGA-PTX-MNPs were subjected to physico-chemical

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characterizations, and their antitumor effects and underlying mechanisms were then investigated in detail after delivered into glioblastoma U251 cells.

2. Materials and methods

2.1. Materials and reagents 5

Human glioblastoma cell line U251 was obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cell culture medium and fetal bovine serum (FBS) were purchased from Gibco Invitrogen Corporation (CA, USA).

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Polyethylenimine (PEI), polyvinylalcohol (PVA, MW = 30–70 kDa) , poly (lacticco-glycolic acid) (PLGA, lactide/glycolide molar ratio of 50: 50 MW = 7000–17000), paclitaxel (PTX),

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3-(4,5-dimethylthiazol-2-diphenyl-tetrazolium) bromide (MTT), potassium ferrocyanide (Perls reagent), fluorescein diacetate (FDA), propidium iodide (PI), RNase, dimethyl sulfoxide (DMSO), Triton X-100 solution, and paraformaldehyde were purchased from

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Sigma-Aldrich (St. Louis, MO, USA). Fluorescent dye FITC and 4,6-diamidino-2-

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phenylindole (DAPI) were purchased from Molecular Probes, Inc. (Eugene, OR, USA).

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Rhodamine phalloidin was obtained from Cytoskeleton, Inc. (Denver, CO, USA). Other reagents and chemicals were purchased from local commercial suppliers and were of

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analytical reagent grade, unless otherwise stated. De-ionized water (Milli-Q, Millipore,

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Bedford, MA) was used to prepare aqueous solutions.

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2.2. Principle of Fluorescein-labeling PEI-PLGA-PTX-MNPs for biological assays in tumor U251 cells

Generally, as illustrated in Scheme 1, supermagnetic γ-Fe2O3 nanoparticles (MNPs) were

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decorated with PLGA, and then fluorescently labelled with FITC through amino group reaction with PEI, which were simultaneously coupled with PTX through solvent evaporation method. The formulated fluorescein-labeling PEI-PLGA-PTX-MNPs were finally delivered into human brain glioblastoma U251 cells, and the anti-tumor effects and underlying 6

mechanism of these nanoparticles were investigated in detail. 2.3. Synthesis of PEI-PLGA-MNPs and PTX loading In the present study, magnetic γ-Fe2O3 nanoparticles i.e. MNPs were synthesized as

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magnetic nanocarrier cores through the chemical coprecipitation method [30, 31], and more information about synthesis of MNPs are provided in the Supplementary information (SI).

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The PLGA-PTX-MNPs were prepared using the solvent evaporation method as previously described [32, 33]. Briefly, 50 mg PLGA and/or 1 mg PTX were dissolved in 1mL

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dichloromethane, and added with 15 mg MNPs to obtain an organic dispersion, which was

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subsequently poured into 10 mL 3% polyvinylalcohol solutionto form a stable emulsion by a

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constant sonication. The formed PLGA-PTX-MNPs were firstly washed three times under

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magnetic field using DI water, and then centrifugated and lyophilized. Subsequently, 5 mg

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PLGA-PTX-MNPs were dissolved in 1 mL RPMI-1640 medium, and then added with 50 µL of 1 mg/mL PEI and slowly shaken for 2 h at 4 ℃. The prepared PEI-PLGA-PTX-MNPs

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were washed three times under magnetic field using RPMI-1640 medium and finally

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suspended in RPMI-1640 medium and stored at 4 ℃ until use. The drug loading capacity and encapsulation efficiency of PTX in PEI-PLGA-PTX-MNPs

were determined via a high performance liquid chromatography (HPLC) (Agilent 1100 series,

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Agilent Technologies, Diegem, BE) [34, 35]. The releasing of PTX from the PEI-PLGAMNPs was investigated in a PBS (pH 7.4) and acetate buffer (pH 4.5). In brief, 20 mg PEIPLGA -PTX-MNPs were placed in a dialysis bag (MWCO: 20 kDa) kept in 30 mL of the PBS (pH 7.4) and acetate buffer (pH 4.5) at 37 ℃ at 280 r/min for 96 h, respectively. At 7

predetermined time points, an equal amount of dialysates was taken out and replaced with same volume of fresh liquid. The obtained dialysates were extracted with dichloromethane, and the residue was dissolved in acetonitrile. The amount of extracted PTX was measured

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with the HPLC method. 2.5. Assay of anti-proliferation capacity

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The MTT assay was used to evaluate anti-proliferation capacity of the PTX-loaded PEIPLGA-MNPs in the treated glioblastoma U251 cells. Briefly, the nanoparticles with the a

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series of concentrations (0-400 µg/mL) were added to the microwell plates, in which the

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U251 cells were seeded a density of 1×104 cells/well and then incubated for 24 h.

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Subsequently, 200 µL of 0.5 mg/mL MTT solution was added into each well and incubated

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for another 4 h, and finally 100 µL of DMSO was added and incubated for another 15 min.

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The absorbance of the solutions was measured at 570 nm on a microplate spctrophotometer (Bio Tek Instrument Inc., USA).

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2.6. FITC labeling of PEI-PLGA-PTX-MNPs for cell imaging

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To visualize dynamic change of the PTX loaded PEI-PLGA-MNPs in glioblastoma U251

cells, the polymer magnetic nanoparticles were fluorescently labelled with FITC according to

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the protocol as previously reported [36]. The cellular imaging of FITC-PEI-PLGA-PTXMNPs was assayed in the treated glioblastoma U251 cells after endocytosis. Briefly, the glioblastoma U251 cells were firstly seeded at a density of 5 × 104 cells/well, and precultured for 48 h. Subsequently, the fabricated FITC-PEI-PLGA- PTX-MNPs with different concentrations were then incubated with the glioblastoma U251 cells for 4 h, and then the 8

residual nanoparticles were removed and rinsed three times using PBS (pH 7.4) to collect the treated cells, which were finally counterstained with DAPI dye (100 nM; Sigma-Aldrich) to reveal nuclei before observed under an inverted fluorescence microscope.

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2.7. Apoptosis assay Apoptosis of the glioblastoma U251 cells was analyzed after treatments with the fabricated

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nanopaticles for 12 h, followed by a fixation with 4% paraformaldehyde for 15 min and washing three times with PBS (pH 7.4). The prepared cells were stained with 2 µg/mL

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Hoechst H33258 solution for 10 min, and observed using an inverted fluorescence

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microscope equipped with a high-resolution CCD camera. Meanwhile, the treated cells were

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also analyzed with the TUNEL detection kit (Keygen Biotech, Nanjing, China) according to

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the manufacturer’s protocol. In addition, to detect apoptosis of treated U251cells at a

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molecular level, the apoptotic DNA ladder detection kit (Keygen Biotech, Nanjing, China) was used to detect DNA fragmentation for the treated glioblastoma U251 cells following the

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manufacturer’s instructions.

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2.8. Autophagy assay

The accumulating volume of acidic vesicular organelles (AVOs) as a marker of autophagy,

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could be detected with the lysosomotropic agent AO. The treated cells were stained with AO (5 μg/mL) at 37 °C for 1 min, and the cytoplasm and nucleus of the stained cells produced a bright green fluorescence, whereas the AVOs produced a bright red fluorescence when observed using an inverted fluorescence microscope. Meanwhile, another marker of autophagy LC3 was also detected with immunocytochemistry staining. The pretreated cells 9

were firstly incubated with anti-LC3A/B antibody (mouse monoclonal IgG1, 1:100) overnight at 4 °C following with washing three times with PBS (pH 7.4), and the treated cells were then incubated with PBS-diluted fluorescence-labeling secondary Alexa Fluor 488 goat anti-mouse

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IgG (1:20) at 37 °C for 1h. The cell nuclei were counterstained with DAPI (100 nM; SigmaAldrich) to reveal the nuclei.

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2.9. Measurement of cellular reactive oxygen species (ROS)

The intracellular ROS level of treated U251 cells was measured using ROS assay kit

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following the manufacturer’s instruction. The precultured U251 cells were firstly treated with

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different concentrations of nanoparticles for 12h, and then incubated with DCFH-DA (10

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μM/mL, Keygen Biotech, Nanjing, China) for 20 min at 37°C in dark. The membrane of

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treated cells was permeated with DCFH-DA probe, which was cleaved by esterase to yield

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DCFH. The produced DCFH was then oxidized in the presence of ROS to generate the highly fluorescent 2, 7-dichlorofluorescein (DCF). The fluorescence intensity was measured by

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flow cytometry (BD Biosciences, San Jose, CA).

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2.10. Immunofluorescent staining for actin cytoskeleton The morphological alternation in F-actin cytoskeletal structure of the treated U251 cells

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were investigated with rhodamine phalloidin. The precultured U251 cells were treated with different concentrations of nanoparticles for 12 h, and washed three times with PBS (pH 7.4), followed by fixation with 2.5% glutaraldehyde for 10 min at room temperature. The fixed cells were permeabilized for 5 min, and stained with 200 µL of 100 nM rhodamine phalloidin in dark for 30 min at room temperature. The prepared cells were finally counterstained with 10

DAPI dye (100 nM; Sigma-Aldrich) to reveal nuclei, before observed under an inverted fluorescence microscope equipped with a high-resolution CCD camera. 2.11. Cell cycle analysis

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The precultured U251 cells were firstly treated with different concentrations of nanoparticles for 12 h, and harvested and dissociated into a single-cell suspension. The treated

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cells were then fixed with 70% ice-cold ethanol at 4℃ overnight, and centrifuged to discard

the fixative, and resuspended in 2 mL of PBS (pH 7.4) solution. The collected cell suspension

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was stained with 50 µg/mL PI solution containing 20 µg/mL RNase in dark at 4℃ for 1 h,

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and finally analyzed with a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA).

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2.12. Western blot analysis

The treated glioblastoma U251 cells were lysed in lysis buffer (50 mM Tris-HCl, pH 7.4,

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150 mM NaCl, 1% NP-40, 0.5% sodium dexoycholate, 0.1% SDS, 2 mM PMSF) and then

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boiled in metal bath at 100 °C for 5 min. The total protein extracts (10 μg) were separated by 12% SDS-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride

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(PVDF) membrane (Millipore, USA), which was blocked with 8% (w/v) non-fat milk in PBSTween 20 (PBST; 0.05%) buffer for 1 h. The transferred membrane then incubated with

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primary antibodies (1:5000 or 1:10,000 in PBST) at 4 °C overnight, and incubated with the appropriate HRP-conjugated secondary antibodies (1: 10,000 or 1:20,000) for 1 h at room temperature after washed with PBST buffer. The immunoreactive bands were developed with the Pierce ECL (Thermo Fisher Scientific, Waltham, MA, USA) western blotting system. The relative quantity of the proteins was normalized to β-actin levels. 11

2.13. Image acquisition and analysis Fluorescence images were photographed using an inverted fluorescence microscope (Eclipse TE 2000-U, Nikon, Kyoto, Japan) equipped with a high-resolution CCD camera

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(CV-S3200, JAI Co., Japan). Software Image-Pro Plus® 6.0 (Media Cyternetics) and SPSS 12.0 (SPSS Inc.) were used to perform image analysis and statistical data analysis,

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respectively. Quantitative data are presented as mean ± standard deviation for each

experiment. All experiments were repeated at least three times, and the results presented were

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from representative experiments.

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3. Results and discussion

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3.1. Synthesis and characterizations of PEI-PLGA-MNPs and PTX loading

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Magnetic nanoparticles i.e. PEI-PLGA-MNPs were firstly formulated using a solvent evaporation method. TEM results showed the PEI-PLGA-MNPs were spherical with a

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relatively uniform size of 80 nm approximately (Fig. 1 B). The XRD pattern showed all the

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tested nanoparticles including MNPs, PLGA-MNPs and PEI-PLGA-MNPs had six characteristic peaks that distinctly match the standard γ-Fe2O3 reflections (Supplementary Fig. S1A-C), which indicated that crystallization of γ-Fe2O3 was not affected.

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The PTX loading onto PEI-PLGA-MNPs was accomplished using a solvent evaporation

method, and their composition was then analyzed by FT-IR. The FT-IR spectra (Fig. 1C) showed that the C–N stretching vibration of amide groups was located at 1380 cm−1, the C–O bond vibration of ether groups was located at 1096 cm−1, and the characteristic band (Fe–O) 12

of MNPs was located at 469cm−1. The peaks at 2943 cm−1 were attributed to the stretching vibrations of methylene. The characteristic band of –COOH was also observed at 3407 cm−1. Overall, these data indicated that the PEI-PLGA-MNPs were successfully loaded with the

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anti-tumor drug PTX. Fig.1 (D) illustrates the magnetization curves of the prepared MNPs and PEI-PLGA-MNPs

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were represented as a symmetrical hysteresis loop (Fig.1D), which indicates that the PEI-

PLGA-MNPs are readily magnetized in the presence of a magnetic field. The removal of the

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magnetic field results in minimal residual magnetization within the particles in different

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media, including DI water, PBS (pH 7.4), and RPMI-1640 culture medium (Fig. 1E).

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The dispersion stability of the fabricated PEI-PLGA-MNPs was evaluated with

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transmittance in RPMI-1640 medium, PBS solution (pH 7.4), and DI water at different time

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points. As presented in Fig. 1(F), the data indicated that the PEI-PLGA-MNPs could be uniformly dispersed and remained relatively steady in RPMI-1640 medium and DI water.

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Besides, the zeta potential of the prepared nanoparticles was examined and the data showed

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the PEI-PLGA-MNPs were positively charged at around +21.7 mV, but the PLGA-MNPs were negatively charged at around -16.9 mV (Supplementary Fig. S2). The results indicated

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that the fabricated PEI-PLGA-MNPs were probably ideal vehicle for drug delivery. 3.2. Drug loading, encapsulation efficiency and release

The ideal nanoparticles for drug delivery should have a high capacity for drug loading and encapsulation efficiency. The PTX loading onto MNPs was calculated about (1.93±0.2)%, and encapsulation efficiency was about (96.74±0.7)%, which indicated that the fabricated PEI13

PLGA-MNPs could be a moderate nanovehicle for PTX delivery. In addition, a controlled release of PTX from PEI-PLGA-MNPs was evaluated in the solution with representative PH value including acetate (pH 4.5) and PBS (pH 7.4)

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(Supplementary Fig. S3), and the results showed that about 33.4% of PTX were instantly released from nanoparticles during the first 12 h. The releasing of PTX was slowed down, and

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most PTX molecules was run over after 48 h. The sustained release of PTX from PEI-PLGAMNPs could be beneficial for enhancing long-term antitumor efficiency and improving drug

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accumulation at the targeted site.

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3.3. Proliferation, migration and invasion of the treated U251 cells

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The proliferative capacity of glioblastoma U251 cells was determined after treatment with

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different concentrations of nanoparticles for 24 h. As shown in Fig.2A, the results indicated

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that compared with the drug-free PEI-PLGA-MNPs, the applied PEI-PLGA-PTX-MNPs could effectively inhibit the growth of glioblastoma U251 cells in a dose-dependent manner.

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Meanwhile, the wound healing assay was used to evaluate cell migration by monitoring

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scratch closure at a different time (see Supplementary Information), and the results showed that the scratch closure was obviously disrupted (Fig.2B & Fig.S4), which indicated that the PTX-loaded nanoparticles could inhibit migration ability of the treated glioblastoma U251

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cells. Besides, the transwell invasion assays demonstrated that the number of the treated U251 cells that could migrate across 8 µm pore-sized membrane was dramatically decreased after treatment (Supplementary Fig. S5), which indicated that PTX-loaded nanoparticles could effectively inhibit glioblastoma U251 cell invasion. 14

3.4. Cell imaging of FITC-labeling PEI-PLGA-PTX-MNPs Cellular imaging was conducted by incubating the FITC-labeling PEI-PLGA-PTX-MNPs with glioblastoma U251 cells for 12 h. As shown in Fig. 2C, green fluorescence was visibly

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distributed in the cytoplasm and closely around the nuclei of treated U251 cells, indicating that the fluorescent nanoparticles were successfully endocytosed, and penetrated the nuclear

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membrane, and approached the nuclei of the glioblastoma U251 cells. Remarkably, much

stronger green fluorescence was observed around the nuclei, which indicated the PEI-PLGA-

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PTX-MNPs could be mostly enriched around the nuclei to guarantee a release of PTX around

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the nuclei of glioblastoma U251 cells.

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3.5. Apoptosis of the treated U251 cells

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Cell apoptosis is a typical programmed cell death, commonly considered as a positive sign

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of the improved treatment for targeted tumor cells [37,38], which is generally characterized by distinct morphological changes, including blebbing, cell shrinkage, nuclear

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fragmentation, chromatin condensation, and chromosomal DNA fragmentation [39]. To

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visualize nuclear fragmentation of apoptotic cells, the glioblastoma U251 cells were stained with Hoechst H33258, a fluorescent dye that binds to the AT-rich regions of DNA and allows

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detection and relative quantification of DNA of apoptotic cells [40]. The glioblastoma U251 cells were exposed to the fabricated nanoparticles with different concentrations for 12 h, and the results showed that the typical changes including chromatin condensation, nuclear peripheral aggregation and nuclear fragmentation were observed in the treated glioblastoma U251 cells in comparison with the controls (Fig. 3A & B). 15

TUNEL assay was also performed to assess apoptosis of the treated U251 cells, and the results showed that the number of apoptotic cells (TUNEL-positive) increased with higher concentrations of PEI-PLGA-PTX-MNPs applied (Supplementary Fig. S6 A&B). Therefore,

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these results indicated that PEI-PLGA-PTX-MNPs could efficiently induce apoptosis in brain glioblastoma U251 cells.

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In addition, at the molecular level one of important features for cell apoptosis is the

fragmentation of genomic DNA into integer multiples of 180-200 bp, which usually results in

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a characteristic ladder upon agarose gel electrophoresis [41]. As illustrated in Fig.4A, the

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results showed that the treated U251 cells presented a typical ladder-like DNA band after 12 h

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treatment with PEI-PLGA-PTX-MNPs, which indicated that PEI-PLGA-PTX-MNPs could

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induced apoptosis in glioblastoma U251 cells.

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Simultaneously, western blotting was used to determine apoptosis-related protein markers (Caspase 3, Bcl-2 and Bax) in the treated U251 cells, and the results showed that expression

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of Caspase 3 and Bax increased in a dose-dependent manner in the treated U251 cells,

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whereas the anti-apoptotic protein Bcl-2 was decreased (Fig. 4 B & C). Therefore, these results indicated that the fabricated PEI-PLGA-PTX-MNPs could induce apoptosis probably through fragmenting nuclear DNA and positively regulating the apoptotic proteins in treated

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glioblastoma U251 cells. 3.6. Autophagy of the treated U251 cells Autophagy is another typical programmed cell death initiated by formation of autophagosomes [42, 43], in which acidic vesicular organelles (AVOs) accumulated as a 16

characteristic of autophagy [44]. In addition, the microtubule-associated protein light chain 3I (LC3-I) was conjugated with phosphatidylamine to form LC3-phosphatidylamine (LC3-II), and the increasing ratio of LC3-II to LC3-I levels reflects the activation of autophagy [45].

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The cytoplasmic protein LC3 was thus detected with its anti-LC3A/B antibody. The results showed that a stronger AO staining signal was observed in the treated cells (Fig.5A&B), and

occurred in the treated cells in comparison with the controls.

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accumulating anti-LC3 puncta were also detected (Fig.5C&D), which indicated autophagy

Besides, the western blotting showed a conversion of LC3-I into LC3-II in the treated U251

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cells because the expression of LC3-II and Beclin 1 was positively up-regulated in a dose-

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dependent manner (Fig. 5 E &F). In addition, P62 involved in formation of autophagosome

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was constitutively degraded through specific binding to LC3, and thus a decrease in expression level of P62 could reflect an autophagy flux [46, 47]. The data showed that the

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expression level of P62 was decreased with increasing concentrations of PEI-PLGA-PTX-

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MNPs applied in glioblastoma U251 cells (Fig. 5E &F). Therefore, these results suggested that PEI-PLGA-PTX-MNPs could efficiently induce autophagy by initiating autophagosome

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formation in the treated U251 cells. 3.7. Overproduction of ROS in the treated U251 cells

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Excess reactive oxygen species (ROS) have been proved to damage tumor cells efficiently.

ROS overproduction is thus greatly desirable for antitumor therapy. The intracellular ROS was measured by monitoring fluorescent 2, 7-dichlorofluorescein (DCF) produced in the treated U251 cells, and the results showed the fluorescence intensity dramatically enhanced in 17

the treated cells with increasing dosages of PEI-PLGA-PTX-MNPs applied (Fig.6A&B), which indicated that PEI-PLGA-PTX- MNPs could accelerate the production of intracellular ROS in a dose-dependent manner.

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3.8. Actin cytoskeleton of the treated U251 cells Cytoskeleton microfilaments are commonly involved in normal cell attachment and

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morphology, and the altered appearance of F-actin in the treated cells could be considered as

highly cytotoxic. Rhodamine phalloidin could specifically bind to the polymerized form of F-

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actin cytoskeleton with high affinity in the treated glioblastoma U251 [36]. The

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immunofluorescent staining showed that F-actin cytoskeleton was obviously polymerized in

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the treated U251 cells compared with the controls, and the volume and size of the treated cells

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tubulin polymerization in cells [48].

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were also comparatively reduced (Fig. 6C), as the PTX could efficiently promote and stabilize

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3.9. Cell cycle distribution of the treated U251 cells Cell cycle distribution was examined in U251 cells after treatments with different

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concentrations of PEI-PLGA-PTX-MNPs. As shown in Fig. 7, the results showed that the number of U251 cells arrested at the G2/M phase increased after treatment with nanoparticles

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in a dose-dependent manner, which indicated that the PEI-PLGA-PTX-MNPs could target tubulin, block cell cycle progression at mitosis and induce apoptosis.

4. Conclusions

In this present study, we have successfully developed a fluorescence-labeling magnetic 18

PEI-PLGA nanoplatform loaded with anti-tumor drug PTX for paralleling cell imaging and antitumor therapy in human brain tumor cells. The results showed the fabricated FITClabeling PEI-PLGA-PTX-MNPs could be effectively endocytosed by the targeted U251 cells

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for cellular imaging, inhibition of cell growth and migration, and inducing cell apoptosis and autophagy. Therefore, the fabricated fluorescence-labeling PEI-PLGA-PTX-MNPs should be

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of great significance for cellular imaging and delivery of antitumor drugs in the personalized

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brain tumor treatments in future.

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Acknowledgments

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This work was supported by the National Natural Science Foundation of China (No. 314

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008 55), the Technology Research and Development Support Funds Project of Zhengzhou

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(No. 2014 YWQ Q15).

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City (No. 153 PXX CY 184), the Basal Research Fund of Henan University of Technology

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Figure captions

Scheme 1 Schematic representation of fabrication of PLX-loaded multifunctional magnetic

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nanoparticles and delivery into glioblastoma U251 cells for analysis of antitumor effects. Fig. 1. (A) Schematic representation of the preparation of PEI-PLGA-MNPs. (B) TEM image

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of PEI-PLGA-MNPs. (C) The magnetic hysteresis loop of MNPs, and PEI-PLGA-MNPs at 300 K. (D) FT-IR spectra of the PTX-loaded PEI-PLGA-MNPs. (E) The response to an

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external magnetic field by adding and removing an external magnetic field in different

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medium. (F) Stability assay in different medium including RPMI-1640 culture medium, PBS

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(pH 7.4) and DI water.

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Fig. 2. The growth of U251 cells after treated with differerent concentrations of nanoparticles

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(20 µg/mL to 400 µg/mL) for 24 h (A). PEI-PTX-PLGA-MNPs inhibit glioblastoma U251 cell migration, U251 cells wound closure after treatment 3h, 6h, and 12h(B, a: Control, b:

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PLGA-MNPs, c: PTX-PLGA-MNPs, d: PEI-PTX-PLGA-MNPs). Cell imaging of the

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formulated FITC-PEI-PLGA-PTX-MNPs within glioblastoma U251 cells after 12 hours’ incubation (Concentration: 100 µg/mL). Nuclei were located by counterstaining with DAPI

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(blue) (C, a: Control, b: FITC-PLGA-MNPs, c: FITC-PLGA-PTX-MNPs, d: FITC-PEIPLGA-MNPs, e:FITC-PEI-PLGA-PTX-MNPs). Scale bar =100 µm. Fig. 3. Hoechst H33258 staining of the treated U251 cells. (A) The observed nuclei of the treated glioblastoma U251 cells with different concentrations of nanoparticles, and (B) the statistic analysis of apoptotic U251 cells. Scale bar = 50 μm. 27

Fig.4. Molecular assay for apoptosis in the treated U251 cells with PEI-PLGA-PTX-MNPs, (A) The apoptotic DNA fragments were visualized for the treated cells with different concentrations of nanoparticles appled (400, 200, 100, 40, 20, 0 μg/mL, as numbered a, b, c,

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d, e, f); (B)Western blot analysis of apoptosis-related proteins in treated U251 cells and the relative expression of theses apoptosis-related proteins (C).

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Fig.5 Autophagy assay of the treated U251 cells. (A&B) AO staining in the treated brain glioblastoma U251 cells. In contrast with the untreated glioblastoma U251 cells, AO

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staining was clearly detected in the drug loaded PLGA-PTX-MNPs and PEI-PLGA-PTX-

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MNPs treated glioblastoma U251 cells for 12 h. (C&D)Accumulated cytoplasmic LC3 signal

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as a hallmark of autophagy in treated brain glioblastoma U251 cells. In contrast with cells

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treated with PEI-PLGA-MNPs, the LC3 signal was obviously detected in PLGA-PTX-MNPs

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and PEI-PLGA-PTX-MNPs treated cells. Scale bar =100 μm. (E)Western blot analysis of autophagy-associated proteins in the U251 cells treated with different concentration of PEI-

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PLGA-PTX-MNPs (20–400 μg/mL) for 12h, and statistical analysis of relative level of

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proteins in the treated cells (F). Fig. 6. (A)Analyses of ROS production in glioblastoma U251 cells treated with nanoparticles for 12 h, and statistical analysis of fluorescence intensity in the treated cells (B).

(C)

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Immunostaining of actin cytoskeleton of the treated U251 cells using the rhodamine phalloidin, and the nuclei were located by counterstaining with DAPI (blue) (red). The treated U251 cells were cultured with different nanoparticles for 12 h. Scale bar = 50 μm.

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Fig. 7 (A) Cell cycle distribution of the treated U251 cells. The untreated U251 cells were used as controls (a), and the U251 cells were treated with different concentrations of PEIPLGA-PTX-MNPs (20–400 μg/mL) for 12 h (b-f). The cells in each phase during cell cycle

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