TAT peptide-modified cisplatin-loaded iron oxide nanoparticles for reversing cisplatin-resistant nasopharyngeal carcinoma

Biochemical and Biophysical Research Communications 511 (2019) 597e603

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TAT peptide-modified cisplatin-loaded iron oxide nanoparticles for reversing cisplatin-resistant nasopharyngeal carcinoma Huanhuan Weng a, Naveen Kumar Bejjanki b, Juan Zhang a, Xiangwan Miao a, Ying Zhong a, Hailiang Li b, Huifen Xie c, Siqi Wang a, Quanming Li b, Minqiang Xie a, b, * a b c

Department of OtolaryngologyeHead and Neck Surgery, Zhujiang Hospital, Southern Medical University, Guangzhou, 510282, Guangdong, China Department of OtolaryngologyeHead and Neck Surgery, Zhuhai People's Hospital, Kangning Road, Zhuhai, 519000, Guangdong, China Department of OtolaryngologyeHead and Neck Surgery, Shenzhen University General Hospital, Shenzhen, Guangdong, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 18 February 2019 Accepted 21 February 2019 Available online 28 February 2019

As chemo-radiotherapy continues to increase the lifespan of patients with nasopharyngeal carcinoma (NPC), adverse reaction and drug resistance remain two major problems when using cisplatin (CDDP). In this study, we took the lead in designing a dual-mechanism anti-cancer system modified with cellpenetrating peptide on the surface of superparamagnetic iron oxide nanoparticles (SPION) to enhance CDDP delivery efficacy to NPC cells, especially CDDP resistant NPC cells. The combinatorial delivery of CDDP and iron oxide nanoparticles showed an unexpected effect on reversal of CDDP resistance due to the Fenton reaction with an average decrease in the half maximal inhibitory concentration (IC 50) of 85% and 94% in HNE-1/DDP and CNE-2/DDP resistant cells respectively compared to CDDP alone. On this basis, modification with TAT peptide (YGRKKRRQRRR) significantly improved tumor intracellular uptake, devoting to better curative effects and minimized side effects by reducing CDDP therapeutic doses. Furthermore, we specifically labelled CDDP with fluorescence for detection of intracellular nanoparticles uptake and mechanism research through drug tracing. This novel compound provides a promising therapy for reducing chemotherapy side effects and reversing CDDP-resistant nasopharyngeal carcinoma. © 2019 Elsevier Inc. All rights reserved.

Keywords: TAT peptide Iron oxide nanoparticles Nasopharyngeal carcinoma Cisplatin-resistance Fenton effect

1. Introduction Nasopharyngeal carcinoma (NPC) is a regional malignant tumor that commonly occurred in south China [1]. Concurrent/adjuvant platinum-based chemo-radiotherapy has been regarded as the gold standard in locoregionally advanced NPC therapy according to the latest NCCN guidelines [2]. However, toxicity and resistance have become two major problems when using CDDP [3]. Approximately 30% patients suffered relapses are less sensitive to platinum while the recurrent rate of NPC is around 15% [4,5]. The imbalance between DNA damage and DNA repair, the failure intracellular aggregation as well as the disconnection between CDDP and cancer cells are three significant mechanisms responsible for CDDP resistance [3]. To overcome such problems, many researchers dedicated to studying the dysregulation of genetic or protein expression

* Corresponding author. Department of OtolaryngologyeHead and Neck Surgery, Zhujiang Hospital, Southern Medical University, No. 253 Industrial Road, Guangzhou, 510282, Guangdong, China. E-mail address: [email protected] (M. Xie). https://doi.org/10.1016/j.bbrc.2019.02.117 0006-291X/© 2019 Elsevier Inc. All rights reserved.

related to drug resistance such as BST2 [6], cyclooxygenase-2 (COX2) [7], STAT3 [8] and so on. These diversified results suggest that the resistant mechanism may be possibly influenced by polygene regulations, which are difficult to be altered by making changes in a single gene. Intensity-modulated radiotherapy (IMRT) is considered as an improved reirradiation method to prolong distant survival rate of locally recurrent NPC patients but it notably results in 20%e30% mortality [9,10]. Therefore, there is an evident need to move towards novel therapeutic regimen to decrease side effects and combat platinum-resistance. In recent years, nanoparticles (NPs) composed of silver, gold or superparamagnetic materials have attached great attentions in the field of anti-cancer therapy with the advantages of facile preparation, hypotoxicity, biosecurity and easy modification [11,12]. These NPs are internalized into cytoplasm or nucleus through endocytosis, leading to cell apoptosis or programmed cell death with the mechanism of reactive oxygen species (ROS), macrophages activation, angiogenesis inhibition or magnetic mediated hyperthermia [13]. Compared with gold and silver, superparamagnetic NPs such as iron oxide are considerably economical and easy to synthesize,

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some of which have been approved by US Food and Drug Administration (FDA) as imaging contrast agent and medicine against hypoferric anemia [14,15]. However, the therapeutic use of iron oxide is often restricted by its colloidal instability. With regard to this limitation, a variety of biocompatible materials are applied to modification, including polyethylene glycol (PEG), dextran, poly (ethylenimine) (PEI) and so on [16,17]. And PEG has been proved far superior biosecurity, which has been repeatedly used in our research [18,19]. In our previous work, sodium alginate (SA), as a natural polysaccharide organism, was supposed to be the effective surface modifier because of its high stability and easy modification [20]. On account of containing plentiful carboxyl, SA is inclined to combine with cationic materials, contributing to subsequent synthesis of different compounds. Thus we had successfully synthesized the activated sodium alginate modified iron oxide nanoparticles before. Cell-penetrating peptides (CPPs), known as short peptides, have shown great capabilities in transporting various substances including some small molecules, proteins, genes and nanoparticles into cell nucleus and cytoplasm. The underlying mechanism could be grouped into three types: 1) the CPPs is targeted to cancer cells through electrostatic effect or the hydrophobic combination; 2) the CPPs enter cells via endocytosis; 3) they cross the membrane by forming a new kind of membrane structure [21]. Human immunodeficiency virus-1 transcription activator (HIV-1 TAT) characterized with nontoxicity and high transmembrane efficiency, is one of the most ancient CPPs initially found in 1988 [22]. Recently, significant attention has been afforded into the field of CPP-related drug delivery and tumor therapy [23,24]. However, most of the researchers focused on the combinatorial delivery of CPPs with only genes or nanoparticles with or without chemotherapeutic drug like docetaxel (DTX) or doxorubicin (Dox). Seldom combination between CPPs and CDDP for cancer therapy could be found even though CDDP is the initial therapy for a large quantity of cancers, possibly reflecting the difficulties in combination of CDDP with other nanoparticles. Fortunately, our team have successfully synthesized CDDP-loaded iron oxide nanoparticles and it could be modified with CPPs for resistant NPC treatment [20]. The aim of this study is to synthesize and characterize TATmodified CDDP-loaded SPION for the treatment of NPC especially with CDDP resistance. What's more, such a novel compound can improve the survival rate and simultaneously decrease the longterm side effects with the reduced dosage of CDDP. 2. Methods 2.1. Nanoparticle synthesis 2.1.1. Synthesis of CDDP-loaded aldehyde-SA modified magnetic nanoparticles (ASA-MNP-CDDP) The detailed method was reported by our team before [25]. 2.1.2. Synthesis of FITC labelled CDDP The method was modified and improved based on R. J. Heetebrij's work [26]. CDDP was activated with 0.95 equivalents of AgNO3 in DMF. The system was stirred for 16 h at room temperature avoiding light and then filtered to remove AgCl. This activated complex was subsequently incubated with 0.8 equivalents of NH2(CH2)4NHtBoc and after work-up to obtain white powder, which was subsequently deprotected by treating in 200 mM HCl at 50
C for 16 h. The intermediate was then afforded after pH adjustment to neutral, filtration and drying and finally conjugated with equivalent of FITC to generate the fluorescent CDDP analogues FITC-CDDP.

2.1.3. Synthesis of TAT-PEG-NH2 1) 7.164 g Na2HPO4$12H2O (0.2 M) and 3.121 g NaH2PO4$2H2O (0.2 M) were separately dissolved in 100 ml deionized water and the two solution were mixed together at a volume ratio of 81:19 to get the phosphate buffer at a concentration of 0.2 M (pH ¼ 7.4). 2) 1 g TAT-SH and 1.28 g mal-PEG2000-NH2 (molar ratio ¼ 1:1) were dissolved in 10 ml pre-made phosphate buffer under magnetic stirring for 24 h. The products were dialyzed for 1 d (Mw: 3000 Da) to remove the phosphate and unreacted materials. Finally, we got the dried TAT-PEG2000-NH2 by effectively using the vacuum pump system. The products were keep in 20
C refrigerator. 2.1.4. Synthesis of the final product TAT-PEG-ASA-MNP-CDDP 1) first, sodium borate buffer (0.1 M) was prepared by adding 1.91 g Na2B4O7$10H2O in 50 ml deionized water under magnetic stirring for half an hour. 2) Based on Schiff base reaction, the final products were synthesized by dissolving 0.1 g ASA-MNP-CDDP and 0.1 g TAT-PEG2000-NH2 in 4 ml sodium borate buffer and magnetically stirred for 0.5 h (1000 rpm). To confirm the products, 0.1 g sodium borohydride was introduced to the medium to react overnight for reduction of double bond. After that, the system was dialyzed for 3 d (Mw: 14000 Da) to remove the free unreacted TATPEG2000-NH2 and the product was keep in room temperature for reservation. 2.1.5. Synthesis of FITC modified nanoparticles The whole process was the same as the methods above except that the CDDP was replaced by CDDP-FITC. 2.2. Characterization of nanoparticles Hydrogen nuclear magnetic spectra (1H NMR) of TAT-PEG-NH2 and intermediates of CDDP-FITC were performed with Superconducting Fourier Transform Nuclear Magnetic Resonance Spectrometry (Bruker, Germany), using deuteroxide as a solvent, contributing to demonstration of TAT-PEG-NH2 synthesis. Fourier transform infrared spectrometer (FT-IR) (Bruker, Germany) was used to evaluate the chemical bone breaking and formation among reactions using a VERTEX 70 FTIR spectrometer in the range of 4000e400 cm1. After chemosynthesis, dynamic light scattering (DLS) was detected by Laser Scattering Particle Size Distribution Analyzer (HORIBA, Japan) to determine the hydrodynamic diameter and z-potential of the particles at the concentration of 0.1 mg/ ml at RT. The shape and size of the nanoparticles were observed through transmission electron microscopy (TEM) (JEM-2100F, Japan). The Fe/Pt concentration of nanoparticles was measured and calculated through Inductively coupled plasma emission spectrometer (ICP-OES) (PerkinElmer, American) after adding 5% aqua regia to products and diluting with water to 25 ml, heating to 200
until the metal compound changed to ionic formula. 2.3. Nanoparticle effect on NPC cells with or without resistance 2.3.1. Cell culture Human nasopharyngeal carcinoma cell lines HNE-1 and CNE-2 and human renal epithelial cell line were preserved by our laboratory. CDDP-resistant HNE-1 cells (HNE-1/DDP) and CDDPresistant CNE-2 cells (CNE-2/DDP) were developed by gradually increasing doses of CDDP (up to 1 mg/ml). All cells were cultured at 37
C with 5% CO2 in a constant temperature incubator using RPMI1640 medium (containing 10% fetal bovine serum and 1% penicillin/ streptomycin). 2.3.2. Cell-counting kit 8 (CCK-8) assay To compare the cytotoxicity of CDDP, ASA-MNP-CDDP, TAT-PEG-

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ASA-MNP-CDDP and TAT-PEG-ASA-MNP, CCK-8 assay was performed in different tumor cells. HNE-1 and CNE-2 cells, HNE-1/DDP and CNE-2/DDP resistant cells were seeded on 96-well plates at 3  103 per well incubated with 100 ml normal culture media for 24 h. they were then treated with CDDP, ASA-MNP-CDDP, TAT-PEGASA-MNP-CDDP based on the gradient concentrations of CDDP (0, 0.1, 1, 2, 4, 8, 16 mg/ml) and TAT-PEG-ASA-MNP on the gradient concentrations of Fe (0, 0.1, 1, 10, 50, 100 mg/ml) for another 48 h and 72 h. The absorbance values and inhibition rate were observed and recorded through Universal Microplate Spectrophotometer and cell viability was calculated. 2.3.3. Cell apoptosis assay To determine cell apoptosis involved in the cell uptake of different nanoparticles, the cell apoptosis assay was conducted through Flow Cytometry (FCM). 5  105 HNE-1 and CNE-2 cells, HNE-1/DDP and CNE-2/DDP resistant cells were introduced to sixwell plates followed by the addition of CDDP, ASA-MNP-CDDP and TAT-PEG-ASA-MNP-CDDP at the CDDP concentration of 5 mg/ml for 24 h and 48 h. Annexin V-FITC/PI Apoptosis Detection Kit was employed to test the cell apoptosis on a FACS Calibur Flow Cytometer (Becton Dickinson, New Jersey, USA). 2.3.4. Prussian blue iron stain assay HNE-1, HNE-1/DDP, CNE-2, CNE-2/DDP cells were cultured in 24-well plates (1  104 cells/well) overnight. The complete medium was replaced by serum-free medium for starvation culture for 4 h, followed by treatment of ASA-MNP-CDDP and TAT-PEG-ASA-MNPCDDP at the Fe concentration of 5 mg/ml for 6 h. The following staining procedures were operated according to the instruction. 2.3.5. Confocal laser scanning microscopy (CLSM) HNE-1/DDP and CNE-2/DDP cells were cultured in multi confocal dish (5  105 cells/well) and incubated overnight. After totally adhesion, the cells were cultured with CDDP-FITC, ASAMNP-CDDP-FITC, TAT-PEG-ASA-MNP-CDDP-FITC for 2 h and 6 h, followed by addition of Hoechst 33342 for nuclear staining for 20 min. The intracellular uptake of nanoparticles was determined by Inverted laser scanning confocal microscope (LSM 880 with Airyscan, Carl Zeiss, Germany). 2.3.6. Fluorescence uptake assay Intracellular uptake of CDDP, ASA-MNP-CDDP, TAT-PEG-ASAMNP-CDDP was performed by fluorescence uptake experiment. 4  105 HNE-1 and CNE-2 cells, HNE-1/DDP and CNE-2/DDP resistant cells were introduced to six-well plates followed by the addition of CDDP-FITC, ASA-MNP-CDDP-FITC and TAT-PEG-ASAMNP-CDDP-FITC at CDDP concentration of 5 mg/ml for 6 h. The intensity of FITC was detected by FCM. 2.4. Statistical analysis All data were presented as mean ± standard deviation. The means between two groups were compared by independent sample T-test and difference among multiple groups were analyzed through one-way analysis of variance. The Statistical Package for the Social Sciences (SPSS version 20.0; IBM Corporation, Armonk, NY, USA) was used to perform all statistical analyses. Statistical significance was defined as P < 0.05. 3. Results and discussion 3.1. Synthesis and characterization of nanoparticles The detailed reaction equations for the preparation of TAT-PEG-

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ASA-MNP-CDDP were shown in Fig. S1. The TAT peptide “YGRKKRRQRRRC” was tethered to the maleimide end group of PEG to obtain the TAT peptide-functionalized polymer TAT-NH2. 1H NMR analysis was used to confirm the synthesis of TAT-PEG-NH2: (the spectrum and relevant chemical structural formula were shown in Fig. S3C). The characteristic peaks at 3.66 ppm (a) were referred to eCH2CH2O group of PEG, and after conjugation, the double bond of the maleimide (6.7 ppm, peak b) diminished in the 1 H NMR spectrum. The conjugation efficiency of TAT-SH to the maleimide group was measured by integrate the peak intensity before and after the reaction (90%). The responsible functional groups of various nanoparticle were further confirmed by FT-IR spectroscopy. The terminal NH2 group of TAT-PEG-NH2 was reacted with the aldehyde groups of ASA-MNP via imino reduction. As shown in Fig. S4C, TAT-PEG groups displayed a broad peak at 3350e3250 cm1, which refers to eNH2 and eNHe groups. The peaks at 1606-1460 cm1 correspond to the benzene ring of TAT. The FT-IR spectra of aldehyde groups (eCHO) were characterized at 1629 cm1 in ASA-MNP and ASA-MNP-CDDP. After reduction, the complete disappear of CHO groups were observed at 1629 cm1 and it can demonstrate the imino reduction and which declares the conjugation of TAT-PEG. Additionally, we took the lead in synthesizing CDDP-FITC (Fig. S2), which are specifically adapted to act as analytical probes to quantize cell uptake ratio, target drug location and better dissect the mechanisms of cytotoxicity. In the first step, the chloride ligand of CDDP was displaced by a DMF ligand to afford compound 2. This activated complex is subsequently incubated with 0.8 equivalents of NH2(CH2)4NHtBoc to get [Pt(NH3)2(NH2(CH2)6NH-tBoc)Cl](NO3) (195 Pt NMR: 2632 ppm) in an average yield of 74% and purity 90% (based on 1H and 195Pt NMR analysis). (1H NMR (D2O): d ¼ 3.11 (2H), 2.71 (4H), 2.63 (2H), 1.69 (2H), 1.52 (2H), 1.46 (9H), 1.39 (4H) ppm) (Fig. S3A). Next, compound 3 was subsequently deprotected by treatment HCl and neutralized by NaOH to form [Pt(NH3)2(NH2(CH2)4 NH3)Cl]Cl2 (1H NMR (D2O): d ¼ 3.11 (2H), 2.71 (4H), 2.63 (2H), 1.69 (2H), 1.52 (2H), 1.46 (9H), 1.39 (4H) ppm) (Fig. S3B). Finally, the FITC was connected with compound 5 to afford CDDP-FITC, which was proved successfully synthesized in vitro.s. As can be noticed from the TEM image, TAT-PEG-ASA-MNPCDDP iron nanoparticles were successfully coated with organic materials and well-separated with ellipsoidal shapes. It was characterized with a size of 49.42 ± 9.5 nm in TEM (Fig. S4A) and an average hydrodynamic diameter of 96.2 nm in DLS (Fig. S4E). which means that it could largely avoid to be trapped by macrophage phagocytosis in the liver and spleen due to the average particle size was less than 100 nm and could escape from renal clearance because it was larger than 10 nm [27]. The effective coating of TATPEG-NH2 labelled SPIONs was also confirmed by the decrease in their zeta potential value from 75.4 mV in ASA-MNP to approximately 33.6 Mv in TAT-PEG-ASA-MNP-CDDP in aqueous suspensions at pH 7.4 (Fig. S4D). This decrease is concomitant with the positively charged amine groups. A zeta potential around 30 mV (positive or negative) is considered to be sufficient for electrostatic stabilization of NPs. and the neutral PEG shell protects ASA-MNP from aggregation in normal cells. The stability of ASA-MNP-CDDP, TAT-PEG-ASA-MNP-CDDP nanomaterials was evaluated by continuous monitoring their suspension in neutral buffer medium for half a year. No sediment in the latter dispersion and only a few precipitate in the previous one were seen during observation period. The stability of TAT-PEG-ASAMNP-CDDP might be due to the simultaneous addition of nonionic water-soluble ASA and PEG to the nanostructure and the electrostatic interactions between the negative charged particles. This result indicates that TAT-PEG-ASA-MNP-CDDP nanoparticles show

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satisfactory stability and long-time blood compatibility for delivering CDDP to the tumor site.

addition of TAT, which could be identified through the following experiments. 3.3. In vitro apoptosis assessments

3.2. In vitro cytotoxicity assessments To investigate the toxicity effects of TAT-PEG-ASA-MNP to the viability of NPC cells and to explore the pharmaceutical effects of CDDP, ASA-MNP-CDDP and TAT-PEG-ASA-MNP-CDDP chemotherapeutic products at various CDDP concentrations on the treatment of NPC, four kinds of tumor cells were analyzed using CCK-8 assay and the half maximal inhibitory concentrations (IC 50) were calculated using SPSS software. The results are depicted in Fig. S5 and Table 1. Obviously, the general tendency of cytotoxic effects of CDDP, ASA-MNP-CDDP, TAT-PEG-ASA-MNP-CDDP were more obvious with increased CDDP concentrations no matter on HNE-1/ CNE-2 parent cells or HNE-1/DDP and CNE-2/DDP resistant cells. The IC 50 value of HNE-1/DDP resistant cells with CDDP treatment for 48 h is 7.8 times higher than that in HNE-1 sensitive cells, which is 3.2 times higher when compared CNE-2/DDP resistant cells with CNE-2 sensitive cells. Correspondingly, the fold changes of IC 50 were 8.2 and 18.3 respectively when treated with CDDP for 72 h. The results confirm the development of CDDP resistance in HNE-1 and CNE-2 cell lines. Furthermore, it is noteworthy that ASA-MNPCDDP induced higher toxicity than single CDDP intervention especially in two CDDP resistant cells. The IC 50 of HNE-1 cells and CNE-2 cells treated with ASA-MNP-CDDP for 48 h were decrease by 35.7% and 60.4% in average compared to those with CDDP, and it was reduced by 87.9% and 94.5% in HNE-1/DDP and CNE-2/DDP resistant cells for 48 h. The average decline were 39.3%, 38.8%, 84.1% and 94.1% in 4 cell lines mentioned above respectively when treated for 72 h. The result demonstrates that ASA-MNP-CDDP is much more effective in reducing CDDP resistance in resistant NPC cells. However, the higher concentrations of the ASA-MNP did not induce any toxicity up to 400 mg/ml based on iron (Fe) concentration according to our previous research [25], suggesting that the huge improvement in curative effect might be due to the Fenton Reaction, a unique mechanism of iron oxide greatly contributing to anti-cancer therapy. In cancer cells, CDDP tends to generate H2O2, which could be catalyzed by Fe2þ/Fe3þ to produce ROS such as hydroxyl radical (OH) or superoxide radical (O2-) [28]. The combinatorial delivery of toxic components provide a fast approach to cancer cell death while the redox equilibrium keeps normal cells from being damaged, which has been proved in localized brain cancer [29]. However, the specific mechanism for larger toxicity effect on resistant cells compared to sensitive cells needs further study. Compared to ASA-MNP-CDDP, TAT-PEG-ASA-MNP-CDDP nanoparticles develop an evident advantage on tumor killing effects with an average IC 50 value decrease by 80.3% and 93.8% for 48 h and 72 h in 4 cell lines. However, TAT-PEG-ASA-MNP rarely show toxicity up to the Fe concentration of 100 mg/ml with cell viability of larger than 90% and 75% respectively (Fig. S5B). The antitumor activity of TAT-PEG-ASA-MNP-CDDP nanoparticles appears to be largely owning to the internalized toxic ASA-MNP-CDDP by

The effectiveness of TAT-PEG-ASA-MNP-CDDP on NPC cells was further investigated by cell apoptosis assay (Fig. 1). The 4 types of cells after treatment with PBS, CDDP, ASA-MNP-CDDP and TATPEG-ASA-MNP-CDDP for 24 h or 48 h were stained with a combination of Annexin V-FITC and propidium iodide (PI) to quantify the apoptotic or necrotic cell population by FCM. The necrosis and apoptosis population after drug treatment was increased overtime, particularly when treated with TAT-PEG-ASA-MNP-CDDP, which was 2e4 fold than that of PBS and 1e3 fold than that of CDDP. The necrosis and apoptosis rates in HNE-1 and CNE-2 sensitive cells when treated with CDDP for 48 h were almost twice than that in HNE-1 and CNE-2 CDDP resistant cells, the gap has significantly closed when treated with TAT-PEG-ASA-MNP-CDDP especially in HNE-1/DDP resistant cell lines. The results further confirmed the toxicity of TAT modified CDDP loaded iron nanoparticles. 3.4. Nanoparticles intracellular distribution assay For a more intuitive observation of the distribution of nanoparticles on tumor cells, iron staining assay and confocal laser scanning were considered as effective methods for nanoparticles qualitative and localization detection in cells. Prussian blue iron stain assay revealed that SPIONs were more efficiently taken up by tumor cells when treated with TAT-PEG-ASA-MNP-CDDP compared to ASA-MNP-CDDP, especially in HNE-1 cells and HNE-1/DDP resistant cells (Fig. S6). On the other hand, the successful synthesis of CDDP-FITC is convenient for us to further target drug location and make a comparison of cellular uptake among CDDP, ASA-MNPCDDP and TAT-PEG-ASA-MNP-CDDP. The result was shown in Fig. 2A. It was obvious that the fluorescence intensity of TAT-PEGASA-MNP-CDDP was stronger than that of CDDP and ASA-MNPCDDP with increasing treatment time. However, the difference between CDDP and ASA-MNP-CDDP was not clear. The result further demonstrated that the toxicity caused by TAT-PEG-ASAMNP-CDDP was related to the increased intracellular uptake of CDDP, while the toxicity of ASA-MNP-CDDP is irrelevant. 3.5. Cellular uptake of different nanoparticles Further quantitative detection on cellular uptake of CDDP, ASAMNP-CDDP and TAT-PEG-ASA-MNP-CDDP was evaluated by FACS (Fig. 2B). As expected, TAT-PEG-ASA-MNP-CDDP in all 4 kinds of cells showed the highest fluorescence intensity with a significant average increased cellular uptake of 74% compared to CDDP alone. The fluorescence intensity of ASA-MNP-CDDP seems higher than that of CDDP, however, no significant differences were found. The result well-illustrates that the higher anti-cancer ability of TATPEG-ASA-MNP-CDDP was due to the increased intracellular

Table 1 IC 50 of different nanoparticles in 4 types of NPC cells. 48 h (mg/ml)

HNE-1 cells CNE-2 cells HNE-1/DDP cells CNE-2/DDP cells

72 h (mg/ml)

CDDP

AMC

TAT-AMC

CDDP

AMC

TAT-AMC

5.04 ± 0.37 14.04 ± 2.81 39.42 ± 7.04 44.50 ± 10.84

3.24 ± 0.75 5.56 ± 0.97 4.77 ± 0.24 2.46 ± 0.40

0.377 ± 0.07 0.86 ± 0.26 0.98 ± 0.11 0.93 ± 0.07

1.45 ± 0.20 1.78 ± 0.48 11.85 ± 3.93 32.56 ± 11.63

0.88 ± 0.09 1.09 ± 0.04 1.88 ± 0.12 1.93 ± 0.16

0.03 ± 0.01 0.09 ± 0.03 0.08 ± 0.01 0.16 ± 0.01

AMC: ASA-MNP-CDDP; TAT-AMC: TAT-ASA-MNP-CDDP.

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Fig. 1. Cellular apoptosis of different nanoparticles. Apoptosis induced by PBS (I), CDDP (II), ASA-MNP-CDDP (III) and TAT-PEG-ASA-MNP-CDDP (IV) in 4 types of cells for 24 h and 48 h (Left) and the corresponding percentages of viable, apoptotic, and necrotic cell assessed by Annexin V/FITC-PI staining (Right).

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Fig. 2. Intracellular uptake of different nanoparticles. (A) Confocal laser micrographs of HNE-1/DDP and CNE-2/DDP resistant cells incubated with CDDP-FITC or ASA-MNP-CDDPFITC or TAT-PEG-ASA-MNP-CDDP-FITC for 2 h and 6 h. Scale bars 20 nm. (B) FACS analysis of intracellular uptake of different nanoparticles by HNE-1 cells, CNE-2 cells, HNE-1/DDP and CNE-2/DDP resistant cells.

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uptake of CDDP. While on the other side, no difference between CDDP and ASA-MNP-CDDP on intracellular amounts of CDDP further confirm the Fenton effect theory. In current study, we prepared and characterized novel TAT modified CDDP loaded Fe3O4 nanoparticles, providing promising therapy for improving therapeutic outcomes, reducing chemotherapy side effects and reversing CDDP-resistant nasopharyngeal carcinoma. Conflicts of interest None. Acknowledgments This study was supported by funding from the National Natural Science Foundation of China (No. 81673013 & NO.81372477), and partially supported by Science and Technology Planning Project of Guangdong Province of China (No. 2017B080701038). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.02.117. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.02.117. References [1] J. Ferlay, I. Soerjomataram, R. Dikshit, et al., Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012, Int. J. Canc. 136 (2015) E359eE386. [2] A.D. Colevas, S.S. Yom, D.G. Pfister, et al., NCCN guidelines insights: head and neck cancers, version 1.2018, J. Natl. Compr. Canc. Netw. 16 (2018) 479e490. [3] L. Amable, Cisplatin resistance and opportunities for precision medicine, Pharmacol. Res. 106 (2016) 27e36. [4] A.W. Lee, B.B. Ma, W.T. Ng, et al., Management of nasopharyngeal carcinoma: current practice and future perspective, J. Clin. Oncol. 33 (2015) 3356e3364. [5] M. Markman, R. Rothman, T. Hakes, et al., Second-line platinum therapy in patients with ovarian cancer previously treated with cisplatin, J. Clin. Oncol. 9 (1991) 389e393. [6] C.M. Kuang, X. Fu, Y.J. Hua, et al., BST2 confers cisplatin resistance via NFkappaB signaling in nasopharyngeal cancer, Cell Death Dis. 8 (2017) e2874. [7] C. Shi, Y. Guan, L. Zeng, et al., High COX-2 expression contributes to a poor prognosis through the inhibition of chemotherapy-induced senescence in nasopharyngeal carcinoma, Int. J. Oncol. 53 (2018) 1138e1148. [8] J. Gao, Z. Shao, M. Yan, et al., Targeted regulation of STAT3 by miR-29a in mediating Taxol resistance of nasopharyngeal carcinoma cell line CNE-1, Cancer Biomark. 22 (2018) 641e648.

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[9] A.W. Lee, S.Y. Tung, R.K. Ngan, et al., Factors contributing to the efficacy of concurrent-adjuvant chemotherapy for locoregionally advanced nasopharyngeal carcinoma: combined analyses of NPC-9901 and NPC-9902 Trials, Eur. J. Canc. 47 (2011) 656e666. [10] Y.H. Leong, Y.Y. Soon, K.M. Lee, et al., Long-term outcomes after reirradiation in nasopharyngeal carcinoma with intensity-modulated radiotherapy: a meta-analysis, Head Neck 40 (2018) 622e631. [11] E. Ahmadian, S.M. Dizaj, E. Rahimpour, et al., Effect of silver nanoparticles in the induction of apoptosis on human hepatocellular carcinoma (HepG2) cell line, Mater. Sci. Eng. C Mater. Biol. Appl. 93 (2018) 465e471. [12] D.S. Salem, M.A. Sliem, M. El-Sesy, et al., Improved chemo-photothermal therapy of hepatocellular carcinoma using chitosan-coated gold nanoparticles, J. Photochem. Photobiol., B 182 (2018) 92e99. [13] H. Chugh, D. Sood, I. Chandra, et al., Role of gold and silver nanoparticles in cancer nano-medicine, Artif. Cells Nanomed. Biotechnol. (2018) 1e11. [14] Y.X. Wang, Current status of superparamagnetic iron oxide contrast agents for liver magnetic resonance imaging, World J. Gastroenterol. 21 (2015) 13400e13402. [15] J.W. Bulte, In vivo MRI cell tracking: clinical studies, AJR Am. J. Roentgenol. 193 (2009) 314e325. [16] A. Lechanteur, T. Furst, B. Evrard, et al., PEGylation of lipoplexes: the right balance between cytotoxicity and siRNA effectiveness, Eur. J. Pharm. Sci. 93 (2016) 493e503. [17] K. Nam, S. Jung, J.P. Nam, et al., Poly(ethylenimine) conjugated bioreducible dendrimer for efficient gene delivery, J. Contr. Release 220 (2015) 447e455. [18] H. Li, C. Fu, X. Miao, et al., Multifunctional magnetic co-delivery system coated with polymer mPEG-PLL-FA for nasopharyngeal cancer targeted therapy and MR imaging, J. Biomater. Appl. 31 (2017) 1169e1181. [19] S.J. Chen, H.Z. Zhang, L.C. Wan, et al., Preparation and performance of a pHsensitive cisplatin-loaded magnetic nanomedicine that targets tumor cells via folate receptor mediation, Mol. Med. Rep. 13 (2016) 5059e5067. [20] M. Xie, S. Chen, X. Xu, et al., Preparation of two kinds of superparamagnetic carriers-supported cis-platinum complexes and the comparison of their characteristics, Chin. Sci. Bull. 51 (2006) 151e157. [21] Z. Guo, H. Peng, J. Kang, et al., Cell-penetrating peptides: possible transduction mechanisms and therapeutic applications, Biomed. Rep. 4 (2016) 528e534. [22] A.D. Frankel, C.O. Pabo, Cellular uptake of the tat protein from human immunodeficiency virus, Cell 55 (1988) 1189e1193. [23] L. Li, L. Yang, M. Li, et al., A cell-penetrating peptide mediated chitosan nanocarriers for improving intestinal insulin delivery, Carbohydr. Polym. 174 (2017) 182e189. [24] H. Zhao, M. Wu, L. Zhu, et al., Cell-penetrating peptide-modified targeted drug-loaded phase-transformation lipid nanoparticles combined with lowintensity focused ultrasound for precision theranostics against hepatocellular carcinoma, Theranostics 8 (2018) 1892e1910. [25] P. Huang, L.L. Yang, C.Y. Wang, et al., Preparation and characterization of folate targeting magnetic nanomedicine loaded with gambogenic acid, J. Nanosci. Nanotechnol. 15 (2015) 4774e4783. [26] R.J. Heetebrij, E.G. Talman, M.A. v Velzen, et al., Platinum(II)-based coordination compounds as nucleic acid labeling reagents: synthesis, reactivity, and applications in hybridization assays, Chembiochem 4 (2003) 573e583. [27] Q. Feng, Y. Liu, J. Huang, et al., Uptake, distribution, clearance, and toxicity of iron oxide nanoparticles with different sizes and coatings, Sci. Rep. 8 (2018) 2082. [28] P. Ma, H. Xiao, C. Yu, et al., Enhanced cisplatin chemotherapy by iron oxide nanocarrier-mediated generation of highly toxic reactive oxygen species, Nano Lett. 17 (2017) 928e937. [29] Z. Shen, T. Liu, Y. Li, et al., Fenton-reaction-acceleratable magnetic nanoparticles for ferroptosis therapy of orthotopic brain tumors, ACS Nano 12 (2018) 11355e11365.