Nanomedicine: Nanotechnology, Biology, and Medicine11 (2015) 947–958

Original Article

Rational design of cancer-targeted selenium nanoparticles to antagonizemultidrug resistance in cancer cells

Ting Liu, PhD, Lilan Zeng, MS, Wenting Jiang, MS, Yuanting Fu, MS,Wenjie Zheng, PhD, Tianfeng Chen, PhD⁎

Department of Chemistry, Jinan University, Guangzhou, China

Received 5 July 2014; accepted 22 January 2015

nanomedjournal.com

Abstract

Multidrug resistance is one of the greatest challenges in cancer therapy. Herein we described the synthesis of folate (FA)-conjugatedselenium nanoparticles (SeNPs) as cancer-targeted nano-drug delivery system for ruthenium polypyridyl (RuPOP) exhibits strongfluorescence, which allows the direct imaging of the cellular trafficking of the nanosystem. This nanosystem could effectively antagonizeagainst multidrug resistance in liver cancer. FA surface conjugation significantly enhanced the cellular uptake of SeNPs by FA receptor-mediated endocytosis through nystain-dependent lipid raft-mediated and clathrin-mediated pathways. The nanomaterials overcame themultidrug resistance in R-HepG2 cells through inhibition of ABC family proteins expression. Internalized nanoparticles triggered ROSoverproduction and induced apoptosis by activating p53 and MAPKs pathways. Moreover, FA-SeNPs exhibited low in vivo acute toxicity,which verified the safety and application potential of FA-SeNPs as nanodrugs. This study provides an effective strategy for the design ofcancer-targeted nanodrugs against multidrug resistant cancers.

From the Clinical Editor: In the combat against hepatocellular carcinoma, multidrug resistance remains one of the obstacles to be overcome.The authors designed and synthesized folate (FA)-conjugated selenium nanoparticles (SeNPs) with enhanced cancer-targeting capability.This system carried ruthenium polypyridyl (RuPOP), an efficient metal-based anti-cancer drug with strong fluorescence. It was shown thatthis combination was effective in antagonizing against multidrug resistance in vitro.© 2015 Elsevier Inc. All rights reserved.

Key words: Multidrug resistance; Nanodrug delivery; Cancer targeting; Selenium nanoparticles

Multidrug resistance is becoming one of the most importantobstacles for cancer therapy. P-glycoprotein (P-gp or ABCB1),adenosine triphosphate (ATP)-dependent active efflux pump, is oftenoverexpressed in the plasma membrane of most multidrug resistantcancer cells.1-3 Oneof themost commonmalignancies in theworld ishepatocellular carcinoma. Importantly, a serious obstacle for thesuccessful treatment of liver cancer is the development of drugresistance. Nowadays, doxorubicin (DOX)-based combinationchemotherapy is the main therapeutic strategy for hepatocellular

No conflict of interest was reported by the authors of this article.Research Support: This work was supported by National High Technology

Research and Development Program of China (SS2014AA020538), ScienceFoundation for DistinguishedYoung Scholars (S2013050014667) of GuangdongProvince, the Foundation for High-level Talents in Higher Education ofGuangdong, Natural Science Foundation of China and Research Fund for theDoctoral Program of Higher Education of China (20114401110004), ShenzhenBasic Research Grant (JCYJ20130401152508660) and Yang Fan Innovative &Entrepreneurial Research Team Project (201312H05).

⁎Corresponding author.E-mail address: tchentf@jnu.edu.cn (T. Chen).

http://dx.doi.org/10.1016/j.nano.2015.01.0091549-9634/© 2015 Elsevier Inc. All rights reserved.

Please cite this article as: Liu T, et al, Rational design of cancer-targeted seNanomedicine: NBM 2015;11:947-958, http://dx.doi.org/10.1016/j.nano.2015.0

carcinoma, but it failed to treat drug resistance cancers.4 What ismore, some drugs, such as cyclosporin-A (a calcium channelblocker)5 and verapamil (an immunosuppressive peptide)6 are thetwomost studied agents to reverse the drug resistance.However, theyare not effective and specific to P-gp overexpressing cancer cells.Until now, no effective treatment is available for end-stagehepatocellular carcinoma.7 Therefore, developing new therapeuticagents which can overcome drug resistance for hepatocellularcarcinoma cancer patient is urgently needed.

In order to overcome the multidrug resistant and reduce theside effect, targeted nanodrug delivery systems were widely usedby improving the stimuli-triggered drug release and cancer-targeted drug delivery to minimize the side effects.8,9 Significantly,the cancer targeting ligands could bind to their receptor on thecancer cell membrane, which could enhance the selectiveaccumulation and uptake of the nanoparticles in the tumor-bearing organs, and reduce the toxicity toward normal cells at thesame time.Nanotechnology is nowwidely used, as a drug carrier forcancer therapy.8,10 So far, many nanosystems with differentfunctions had been reported for cancer therapy, such as oxides,

lenium nanoparticles to antagonize multidrug resistance in cancer cells.1.009

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metals, polymers, mesoporous silica and semiconductors.10-13

Among them, selenium nanoparticles (SeNPs) receive more andmore attention as nanocarriers due to their biocompatibility,straightforward synthesis, low-toxicity, degradability in vivo,excellent antioxidant activity and chemopreventative effects, suchas SeNPs used as 5-fluorouracil (5-FU) and DOX carriers.12,14 Wehad also reported cancer-targeted nanoparticles enhanced anticancereffects.10,14 But there was no report about multidrug resistance inour previous work. The folate receptor (FAR) is frequentlyoverexpressed on cancers cells and has been used for targeteddelivery of FA inked liposomes to cancer cells in vitro.15 FARtransports the captured drugs into the cell by receptor-mediatedendocytosis and this has found use in cancer therapy by enhancingthe concentration of drugs in the cancer cells.16 Studies also foundthe FA-targeted drugs could target specific cancer cells andnon-targeted the normal cells.17 Therefore, FA could be linked toSeNPs to target FAR-overexpressing cancers.

The major limitation of cisplatin is the side effects in normaltissues, which include neurotoxicity, ototoxicity, nausea andvomiting, and especially nephrotoxicity.18 The serious limita-tions of cisplatin-based treatments have spurred scientists tosearch for alternative metal-based anticancer drugs.19,20 Spe-cially, ruthenium (Ru) displays several favorable propertiessuitable for drug design and medicinal applications. Studies haveshown that Ru complexes exhibited low cytotoxicity towardnormal cells and high activity against tumor metastasis.21-23 Tillnow, a number of Ru complexes have been synthesized andidentified as novel anticancer agents.24 Among them, KP109 andNAMI-A have already entered clinical trials.21,23,25,26 Previouslywe found the Ru complex RuPOP exhibited higher anticanceractivity and lower toxicity than cisplain.21 However, the use anddevelopment of the RuPOP complex were limited by its pooraqueous solubility. Therefore this study aimed to construct a drugdelivery system for hydrophobic Ru complexes conquering theirdrawbacks. Following our investigation, we found that pluronicF-127 was a good surface modification agent. The pluronic is agroup of block copolymers consisting of propylene oxide andethylene oxide with central hydrophobic poly (propylene oxide)flanked by two hydrophilic chains of poly (ethylene oxide).27,28

Pluronics could absorb hydrophobic drugs by intermolecularforces and thus increase the loading rate of RuPOP. Our resultsconfirmed that as-synthesized FA-SeNPs nanosystem could beused as a cancer-targeted carrier of RuPOP to enhance anticancerefficacy against multidrug resistant cancer cells. The underlyingmechanisms of FA-SeNPs were also elucidated. Taken together,this study may provide an effective strategy for the design anddevelopment of nanodrugs against multidrug resistant cancers.

Methods

Preparation of FA-SeNPs

The solution of 20 mM vitamin C, 5 mM of sodium selenite(Na2SeO3) solution and 0.8 mg/mL of chitosan (CS) solutionwas freshly prepared before the experiment. Pluronic F-127(2.5 g) was activated by 4-nitrophenyl chloroformate (4-NPC)(125 mg) in dichloromethane (10 ml) at room temperature for12 h.29 The activated pluronic F-127 solution was dialyzed

against distilled water (DW) for 24 h and then the solution wasfreeze-dried to powder for further use. Then added thioglycolicacid (150 μL) to the amine terminated pluronic in DW for 12 hwith stirring. The resultant solution was dialyzed against DW for24 h and freeze-dried. The preparation of RuPOP-loaded SeNPswas as described in our previous works.14 2.5 g thiolatedpluronic dissolved in 1 mL methanol and 500 μL 0.1 M CS-FAwere added in stirring for 12 h at room temperature then dialyzedagainst DW for 24 h.

Characterization of FA-SeNPs

The FA-SeNPs were characterized by different methodsincluding Zetasizer particle size, Fourier transform infraredspectroscopy (FT-IR), transmission electron microscope (TEM),UV-vis spectroscopy, and fluorescence spectroscopy analysis.The TEM images were obtained at an accelerating voltage at80 kV on Hitachi (H-7650). Zetasizer Nano ZS particle analyzer(Malvern Instruments Limited) was used to measure sizedistribution and zeta potential of the nanoparticles.

Determination of loading rate of RuPOP in FA-SeNPs

The concentration of Se and RuPOP was determined byICP-AES analysis.

Hemolysis activity examinations

The hemolysis properties of FA-SeNPs were examined byspectrophotometry as reported.30,31 For the studies of erythro-cyte agglutination, each sample was treated to a hemolysis assayfor 1 h (5 μM SeNPs, 5 μM RuPOP and 5 μM FA-SeNPs),placed on a glass slide, covered by a cover slip and analyzed by aphase contrast microscope (Life technologies, EVOS FL auto).

Cell lines and cell culture

HepG2 hepatocellular carcinoma cells, L02 human hepaticcells, and R-HepG2 drug-resistant hepatocellular carcinoma cellswere purchased from American Type Culture Collection (ATCC,Manassas, VA, USA). HepG2 and L02 were incubated in DMEM,but R-HepG2 were incubated in 1640 with 100 U/ml penicillin,50 U/ml streptomycin and 10% fetal bovine serum (FBS) in ahumidified incubator at 37 °C with 5% CO2 atmosphere.

MTT assay

The effects of FA-SeNPs on a series of cancer cells wereexamined by MTT assay.32

In vitro cellular uptake of FA-SeNPs

The in vitro cellular uptake of FA-SeNPs was quantitativelydetermined bymeasuring the fluorescence intensity ofRuPOP-loadednanoparticles inside the cells by using SpectraMaxM5Microplatereader (Bio-Tek).14

Folate competing assay

FA-SeNPs and excess amount of FA competed for bindingFARs on R-HepG2 cells. The uptake of FA-SeNPs was thenmeasured by using SpectraMaxM5Microplate reader (Bio-Tek).14

Figure 1. The characterization of FA-SeNPs and hemocompatibility of SeNPs, FA-SeNPs and RuPOP. (A) The diagrammatic figure of FA-SeNPs and thestructure of RuPOP. (B) The TEM image and (C) the zeta potential of SeNPs and FA-SeNPs. (D) The FT-IR spectra of FA-SeNPs, RuPOP, FA and CS-FA. (E)Hemolysis induced by SeNPs. (F, G)Hemolysis induced by FA-SeNPs and RuPOP after 10 and 60 min. Significant difference between RuPOP and FA-SeNPsunder the same concentration is indicated at P b 0.01 (**) level. (H) Agglutination of red blood cells observed by phase microscopy after 1 h of incubation withSeNPs 5 μM, RuPOP 5 μM and FA-SeNPs 5 μM. Each value represents means ± SD (n=3).

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Intracellular colocalization of FA-SeNPs

Based on the strong fluorescence of RuPOP, fluorescencemicroscopy was used to monitor the intracellular traffickingof the nanosystem. Briefly, 8 × 04 R-HepG2 cells/mL wereseeded in 2-cm dish and allowed to incubate for 24 h.Lyso-tracker DND-99 was added to each dish and incubatedfor 2 h. The cells were then incubated with 1 μg/mL of DAPIH33342 (Sigma-Aldrich) for 30 min. After rinsing by PBSfor 3 times, the cells were exposed to 2 μM RuPOP-loadedFA-SeNPs for different times and observed under fluores-cence microscopy.

Mechanisms of cellular uptake of FA-SeNPs

The mechanisms of cellular uptake of FA-SeNPs in R-HepG2cells were investigated by endocytosis inhibitors.14

In vitro drug release of FA-SeNPs

FA-SeNPs (10 mg) were suspended in 10 ml PBS with pHvalues at 5.3 and 7.4 respectively, with constantly shaking in darkat 37 °C. After different periods of time, 0.3 ml of solution wastaken out from the tubes and centrifuged. The RuPOP concentra-tion in the supernatant was detected by fluorescence microplate

Table 1Cytotoxic effects of FA-SeNPs and RuPOP.

Material IC50 (μM)

HepG2 R-HepG2 L02 RI

FA-SeNPs 0.33 ± 0.02 0.24 ± 0.02 1.40 ± 0.13 0.73RuPOP 0.71 ± 0.03 1.04 ± 0.12 0.90 ± 0.08 1.47DOX 0.28 ± 0.02 2.84 ± 0.29 0.36 ± 0.02 10.1

RI (resistance index): the ratio of IC50 (R-HepG2) against IC50 (HepG2).

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reader (Spectra Max M5, Bio-Tek) with excitation and emissionwavelength set as 450 nm and 602 nm respectively.

Flow cytometric analysis

The flow cytometry was used to analyze the cell cycledistribution.32 DNA histogram displays the proportion of cells inG0/G1, S, and G2/M phases, and the sub-G1 peak was quantifiedin cell cycle pattern to determine the apoptotic cells withhypodiploid DNA content.

Measurement of intracellular ROS generation

Intracellular ROS overproduction induced by FA-SeNPs inR-HepG2 cellswasmeasured by staining cells withDHE as a probewith excitation and emission wavelength set as 300 and 600 nm.14

Western blot analysis

Western blot analysis was used to analyze the effects ofFA-SeNPs on the expression levels of cellular proteins altered bytreatments of the nanoparticles.32

Acute toxicity experiments

ICR mice (body weight of 18-22 g) used in this study wereprovided by Shanghai Super-B&K Laboratory Animal Corp.Ltd. The mice were housed in cages in a room with controlledtemperature of 20-25 °C and humidity of 40%-70%. The micewere fed Se-deficient formula feed and allowed ad libitumaccess to diet and water. One hundred and fifty mice wererandomly divided into 15 groups with 10 mice per group.FA-SeNPs, SeMet or selenite was fed orally at the dosesindicated in Table 2 for 14 consecutive days. All mice groupswere sacrificed at 14 days. Then the livers were taken out forH&E staining. Cumulative mortality within 14 days after thetreatment was used for the calculation of LD50. Forty mice wererandomly divided into five groups with 8 mice per group. Theyreceived saline as control, FA-SeNPs, SeMet or selenite at thedose of 10 mg Se/kg orally respectively. All mice groups weresacrificed at 6, 12, 24, and 48 h. All animal experiments wereapproved by the Animal Experimentation Ethics Committee ofJinan University.

Statistical and synergistic analysis

All experiments were carried out at least in triplicate andresults were expressed as mean ± SD. Difference betweentwo groups was analyzed by two-tailed Student’s t test.Statistical analysis was performed using SPSS statisticalprogram. The difference between three or more groups was

analyzed by one-way ANOVA multiple comparisons. Diffe-rence with P b 0.05 (*) or P b 0.01 (**) was consideredstatistically significant.

Results

Preparation and characterization of FA-SeNPs

In the study, FA-SeNPs were successfully prepared asillustrated in Figure 1, A. As shown in Figure 1, B, theFA-SeNPs were highly uniform and monodisperse sphericalparticles with a 180 nm diameter. In contrast, SeNPs were easilyaggregated and precipitated in aqueous solutions in Figure 1, B.From the Figure 1, C, we know that the FA-SeNPs are morestable than SeNPs. We confirmed the S-Se bond and thesuccessful conjugation of FA-CS and RuPOP-pluronic F-127 toSeNPs by FTIR. As shown in Figure 1, D, the absence of an S-Hstretching mode in the IR spectrum of FA-SeNPs indicated theformation of the HS-Se bond between the ligands and the SeNPssurface; the peaks at 1490 and 679 cm−1 in FA-SeNPs wereassigned to the bending vibration of N-H and the symmetricalbending vibration of amino groups, respectively. The spectrumof FA-SeNPs retained the special peaks of SeNPs 528 cm−1. Thepeak AT 1213 cm−1 was assigned to stretching vibration of C-Nfrom RuPOP. The UV-Vis and fluorescence spectra ofFA-SeNPs and RuPOP further verified the successful loadingof RuPOP into the nanosystem (Figure S1).

Hemocompatibility of FA-SeNPs

Hemocompatibility can serve as a marker for membraneactivity and predict potential side effects of exogenous drugs andmaterials.33,34 The hemolysis of red blood cells induced bySeNPs, FA-SeNPs and RuPOP was shown in Figure 1, E, F andG. SeNPs alone induced slight hemolysis both after 10 min and2 h. In contrast RuPOP induced slight hemolysis after 10-minincubation, but it induced higher hemolysis after 2 h with highestconcentration. Compared with SeNPs and RuPOP, FA-SeNPsdidn’t induce hemolysis of erythrocyte. Moreover, SeNPs andRuPOP induced prominent agglutination of erythrocytes afterincubation for 1 h, while no agglutination was observed inerythrocytes exposed to FA-SeNPs (Figure 1, H).

Antagonizingmultidrug resistance in R-HepG2Cells byFA-SeNPs

MTT assay was used to study the effects of FA-SeNPs oncancer cells. From Table 1, we found that the FA-SeNPsdisplayed significant anticancer activities against R-HepG2 andHepG2 cancer cells and showed low toxicity toward L02 normalcells. Especially, the IC50 of R-HepG2 was 0.24 ± 0.02 μM,while that of HepG2 was 0.33 ± 0.02 μM, which suggests that theFA-SeNPs can antagonize multidrug resistant cancer. FA-SeNPsat the concentrations of 0.2-0.8 μM exhibited higher growthinhibition on R-HepG2 cells than HepG2 cells and L02 cells(Figure 2, A and Figure S2). Generally, the effects of FA-SeNPson cancer cells were much higher than those on normal cells(Figure 2, A). Therefore, the FA-SeNPs could well antagonizemultidrug resistant cancer cells (R-HepG2).

Figure 2. Cell growth inhibition and selective cellular uptake of FA-SeNPs. (A) The cell viability of R-HepG2, HepG2 and L02 exposed in differentconcentrations of FA-SeNPs and the schematic of selective celluar uptake. Significant difference between control and treatment groups is indicated at P b 0.05(*) or P b 0.01 (**) level. (B)Quantitative analysis of cellular uptake of FA-SeNPs in R-HepG2 and HepG2 cells with 10 μMFA-SeNPs for 0.5, 1, 2, 4 and 8 h.Significant difference between R-HepG2 and HepG2 groups at the same time point is indicated at P b 0.05 (*) or P b 0.01 (**) level. (C) Inversed fluorescentmicroscope images of R-HepG2 and HepG2 cells treated with 2 μMFA-SeNPs for 2, 4, 8, and 12 h. (D)Western blot analysis of FAR in R-HepG2, HepG2 andL02 cells. (E) The fluorescence intensity of R-HepG2 cells after treatment with different concentrations of FA for 2 h and incubation with 20 μMFA-SeNPs foranother 2 h. Each value represents means ± SD (n=3).

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Selective cellular uptake of FA-SeNPs

Cellular uptake was a main factor for the bio-activity ofnanoparticles. In Figure 2, B, the cellular uptake of R-HepG2cells was higher than HepG2 cells after incubation with 20 μMFA-SeNPs for 0.5 h, 1 h, 2 h, 4 h and 8 h. The cellular uptakewas quantified by measuring the fluorescence intensity ofRuPOP. The fluorescent intensity of RuPOP in R-HepG2 cellswas significantly stronger than that in HepG2 cells for the sametime point (Figure 2, C). It is possible that, the different cellularuptake of R-HepG2 cells and HepG2 cells is regulated by FAR.Therefore, we examined the expression level of FAR in differentcells. As shown in Figure 2, D, the expression levels of FAR in

the tested cell lines were in the order of R-HepG2 N HepG2 NL02. We also investigated the importance of FAR in cellularuptake of the nanosystem by FA competing assay. As shown inFigure 2, E, FA dramatically inhibited the cellular uptake ofFA-SeNPs in cancer cells in a dose-dependent way. These resultsdemonstrate the important role of FAR in the cellular uptake ofFA-SeNPs in cancer cells.

Intracellular translocation of FA-SeNPs and pH-mediateddrug release

The intracellular translocation of the nanoparticles wasmonitored by fluorescence microscopy. As shown in Figure 3,

Figure 3. Intracellular localization, endocytosis mechanism of FA-SeNPs, pH-mediated release of RuPOP and the western blot analysis of ABC family. (A)Colocalization of RuPOP (red fluorescence), Lyso-tracker (green fluorescence) and DAPI (blue fluorescence) in HepG2 cells. (B) Intracellular uptake ofFA-SeNPs in R-HepG2 cells with different endocytosis-inhibited conditions. Significant difference between treatment and control groups is indicated at P b 0.05(*) or P b 0.01 (**) level. (C) The percentage of released RuPOP at different times. (D) Western blot analysis of ABC family in R-HepG2, HepG2 and L02cells. (E) Western blot analysis of ABC family in R-HepG2 cells by a dose-.dependent manner. β-Actin was used as loading control. Each value representsmeans ± SD (n=3).

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A, FA-SeNPs entered the cytoplasm by endocytosis after 1-hincubation. Under the acid environments, RuPOP wasreleased from FA-SeNPs after 4 h and then entered cyto-plasm in a time-dependent manner. These results suggest thatendocytosis is the major uptake mechanism of FA-SeNPs incancer cells.

To further dissect the FA-SeNPs endocytosis mechanism, weused different endocytosis inhibitors to treat the cells before theaddition of FA-SeNPs. As shown in Figure 3, B, treatments ofNaN3 and DOG markedly inhibited the internalization ofFA-SeNPs to 66.4% ± 5.5% (Pb0.01) of control. The resultsshowed that sucrose strongly decreased the FA-SeNPs internali-zation to 59.7% ± 6.5% (Pb0.01) of the control. Moreover,nystatin and dynasore induced the uptake of FA-SeNPs to 63.9% ±1.7% (Pb0.01) and 70.8% ± 2.8% (Pb0.01) of the control,respectively. Dynamin, a GTP-binding protein, is essential forreceptor-mediated endocytosis.35,36

Drug release was studied to simulate the acidic environmentsof lysosomes and normal body blood environments. As shown inFigure 3, C, the released amount of RuPOP was 17.7% ± 1.6% atpH 5.3 and 8.6% ± 0.9% at pH 7.4 within 2 h, and the finalreleased amount of RuPOP was 72.5% ± 2.7% at pH 5.3 and16.4% ± 1.2% at pH 7.4 after 48 h treatment. Therefore, RuPOPwas released from FA-SeNPs in a pH-dependent manner. It ispossible that the SeNPs were expanded to snowflake particlesunder acidifying stimulus, which led to enhanced drug-releaseover prolonged periods.37 Moreover, in acidic solution, theproton competes with RuPOP to bind to oxygen atom of pluronicF-127, which would promote the drug release from SeNPs.

Inhibition of ABC family protein expression by FA-SeNPs

First, we analyzed the expression levels of ABC familyprotein in R-HepG2 cells, HepG2 cells and L02 cells without

Figure 4. Induction of cancer cell apoptosis by FA-SeNPs. Flowcytometric analysis of FA-SeNPs treated (A) R-HepG2 cells and (B) HepG2 cells for 72 h.(C) Western blot analysis for the expression of Caspase-9, Caspase-8, Caspase-3, and PARP. β-Actin was used as loading control.

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FA-SeNPs. As shown in Figure 3, D, the expression levels ofABCB1, ABCC1 and ABCG2 in R-HepG2 cells were higherthose in HepG2 cells and L02 cells. Then we further studied theexpression levels of ABC family protein in R-HepG2 cells in adose-dependent manner. As shown in Figure 3, E, expressionlevels of ABC family proteins in R-HepG2 cells were decreasedwhen the concentration of FA-SeNPs increased.

FA-SeNPs induces apoptotic cell death

The effects of FA-SeNPs on the cell cycle distribution ofcancer cells were examined by flow cytometric analysis. Asshown in Figure 4, A, the R-HepG2 cells exposed to FA-SeNPsfor 72 h demonstrated a significant dose-dependent increase in

sub-G1 cell population from 5.8% to 94.8%. In contrast, treatedHepG2 cells demonstrated lower increase in sub-G1 cellpopulation from 0.7% to 26% (Figure 4, B).

As shown in Figure 4, C, the expression levels of caspase-8(extrinsic death receptor-mediated pathways) and caspase-9(intrinsic mitochondria-mediated pathways), two initiator caspases,as well as caspase-3, an effector caspase, were decreased byFA-SeNPs dose-dependently in R-HepG2 cells. Moreover, thetruncation of caspases triggered the proteolytic cleavage of PARP.

Activation of ROS-mediated signaling pathways

As shown in Figure 5, A, FA-SeNPs trigged the production ofROS after 10 min and reached the maximum value; the amount

Figure 5. ROS overproduction activates intracellular apoptotic signaling pathways. (A) R-HepG2 cells were exposed to different concentrations of FA-SeNPsand RuPOP for 100 min and the levels of the intracellular ROS were analyzed by DHE fluorescence intensity. (B) R-HepG2 cells were incubated with 10 μMDHE for 30 min, then exposed to 0.96 μM FA-SeNPs and RuPOP for 100 min. Pictures were taken at 0, 5, 10, 20, 60, and 100 min. Western blot analysis forthe expression of (C) P53, p-P53, p-ATM, p-Histone, and (D) p-P38, P38, p-ERK, ERK, p-JNK, JNK, p-AKT, andAKT.β-Actin was used as loading control. (E) Themain signaling pathway of FA-SeNPs-induced apoptosis. Each value represents means ± SD (n=3).

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of ROS declined to 186% ± 1.62% with 0.96 μMFA-SeNPs after100 min. RuPOP induced a trace of the generation of ROS inwhole process. We could find that the fluorescent intensity ofDHE in R-HepG2 cells treated with FA-SeNPs was muchstronger than RuPOP in Figure 5, B.

Overproduction of ROS could trigger various downstreamsignaling to activation cells apoptosis (Figure 5, C-E). As shownin Figure 5, C, FA-SeNPs induced the elevation of total p53 andits phosphorylation at Ser 15 site. Moreover, p-ATM, p-BRCA1and phosphorylated histone H2A.X (an important biochemicalmarker of DNA damage) were also increased in cells exposed toFA-SeNPs. Taken together, these results indicate that ROS-mediated p53 phosphorylation plays an essential role inapoptosis induced by FA-SeNPs in cancer cells.

To examine the roles of MAPKs and AKT in FA-SeNPs-induced apoptosis, we examined the level of MAPKs and AKTin treated cells. As shown in Figure 5, D, FA-SeNPs exhibiteddifferential effects on the phosphorylation of p38, JNK, ERK, andAKT. The phosphorylation of pro-apoptotic kinases p38 and JNKdisplayed a trend of up-regulation in a dose-dependent manner. Incontrast, the phosphorylation of anti-apoptotic kinases ERK andAKT was effectively suppressed by FA-SeNPs.

Acute toxicity of FA-SeNPs

As shown in Table 2, selenomethionine (SeMet) and selenitecaused 100% mortality at a dose of 50 mg Se/kg. However,FA-SeNPs caused only 10%mortality at a dose of 373.3 mg Se/kg.

Table 2Acute lethal effect of FA-SeNPs, SeMet and selenite in mice.

FA-SeNPs SeMet Selenite

Se dose(mg Se/kg)

MouseMortality(%)

Se dose(mg Se/kg)

MouseMortality(%)

Se dose(mg Se/kg)

MouseMortality(%)

1800 100 50 100 50 1001440 90 40 80 40 801152 70 32 60 32 40921.6 40 25.6 20 25.6 30373.3 10 20.48 10 20.48 10

Table 3Half lethal dose of FA-SeNPs, SeMet and selenite in mice.

Sample LD50 (mg/kg) 95% Confidence limits

FA-SeNPs 1003.8 ± 52.0 943.7-1047.7SeMet 30.5 ± 3.0 27.5-33.5Selenite 31.1 ± 4.3 27.0-35.6

Each value represents means ± SD (n=3).

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As calculated by Bliss method using NDST software, the LD50

values of FA-SeNPs, SeMet and selenite were found to be 1000.3 ±52.0, 30.5 ± 3.0 and 31.1 ± 4.3 mg Se/kg (Table 3), respectively.Therefore, the acute toxicity of SeMet and selenite was aboutthirty-two fold larger than that of FA-SeNPs. As shown in Figure 6,A, B and C after 48 h treatment, FA-SeNPs induced lower ALT,AST and LDH enzymatic activities than those of SeMet andselenite, indicating the lower injury of the nanomaterials of themiceliver. Furthermore, H&E staining was also used to verify the toxiceffects of FA-SeNPs, SeMet and selenite on the liver. As shown inFigure 6, D, E, F and G compared with normal group, FA-SeNPscaused slight hydropic and fatty degeneration liver cells. In contrast,SeMet induced significant degeneration and apoptosis of liver cells,while selenite caused hypertrophy and fatty degeneration.

Discussion

Many kinds of nanomaterials have been synthesized andapplied in cancer therapy.12,13,29,38 Among of them, SeNPs havesignificant anticancer activity and low toxicity to normal cells thatattracted a great deal of attention as a novel anticancer agent anddrug carrier. However, the poor selectivity between differentcancer cells and normal cells and the low drug loading rate havelimited its future clinical application. This study described thesynthesis of FA-conjugated SeNPs as cancer-targeted drugdelivery systems for RuPOP to antagonize multidrug resistancein liver cancer. The introduction of pluronic F-127 greatlyincreased the loading rate of RuPOP into the nanomaterials andincreased the hydrophilic of drugs.

Although the combination of different chemotherapeuticagents is often employed to prevent drug resistance in cancerpatients, the ability of the cancer cells to adapt and developmultidrug resistance ultimately leads to the treatment failure inmost cancer patients.39,40 However, multidrug resistance is acomplicated process with the involvement of acquired andmultiple multidrug resistant mechanisms as a result of theexpression of drug efflux pumps, up-regulation of antiapoptoticproteins, and increase in regulators of drug metabolism.41 ABCfamily proteins are often overexpressed in the plasma membraneof most multidrug resistant cancer cells and decreased cellularuptake of drugs.42 Therefore, we designed a cancer-targeted drugdelivery system for RuPOP to antagonize multidrug resistance inR-HepG2 cells by enhancing cellular uptake of drugs. Firstly, thephysicochemical properties of the drug are very important. Its

high stability enabled the nanoparticles to enter the cells byendocytosis. Secondly, FA-SeNPs were able to target R-HepG2cells overexpressing FAR, then enter and accumulate inR-HepG2 cells through endocytosis after 1-h incubation.Under the acid environments, RuPOP was released fromFA-SeNPs after 4 h and then entered cytoplasm in a time-dependent manner. It is possible that the SeNPs were expandedto snowflake particles under acidifying stimulus, which led toenhanced drug-release over prolonged periods.37 Moreover, inacidic solution, the proton competes with RuPOP to bind tooxygen atom of pluronic F-127, which would promote the drugrelease from SeNPs. The high expression levels of FAR in cancercells effectively increased the cellular uptake of FA-SeNPs incancer cells, and improved its selectivity between cancer andnormal cells, thus finally enhancing the anticancer efficacy.

Further studies were carried out to elucidate the anticancermechanisms of FA-SeNPs. Many studies have shown thatcaspases, a family of cysteine proteases, play a vital role inapoptosis.43 Therefore, Western blot analysis was performed toanalyze the expression and roles of caspase-8, caspase-9 andcaspase-3 in FA-SeNPs-induced apoptosis. Moreover, activationof caspases triggered the proteolytic cleavage of PARP, animportant biochemical marker of apoptosis. These resultsindicated that FA-SeNPs induced liver cancer cell apoptosis bytriggering intrinsic and extrinsic apoptotic pathways. Consis-tently, our previous studies also showed that 5FU-SeNPs,12

Tf-SeNPs14 and SeNPs@ATP44 induced apoptosis also incancer cells through PARP cleavage and caspases activation.

ROS has been identified as an essential chemical signalingmolecule that could regulate the apoptotic signaling pathwaytriggered by various anticancer agents, including cisplatin and Rucomplexes.23,45 ROS overproduction can activate various kinasesincluding MAPK and AKT, consequently causing proteinmodification and DNA damage and inducing cells apoptosis.Our previous work also indicated that modified SeNPs couldinduce apoptosis by ROS-mediated MAPK and AKT pathways.46

And various multifunctional SeNPs also develop their anticancereffect by these pathways including 5FU-SeNPs12 and Tf-SeNPs.14

In this study, the combination of SeNPs and RuPOP significantlyincreased the intracellular ROS generation, which led to markedimprovement in anticancer efficacy. And the MAPKs and AKTpathways played an important role in apoptosis induced byFA-SeNPs with the generation of ROS.

Se is an essential trace element with many biologicalfunctions, but exhibits a narrow margin between beneficial andtoxic effects. Therefore, toxicity examination is always a crucialconcern for development of Se-based anticancer drugs. Manystudies have demonstrated that SeNPs have less toxicity, higherbioavailability and stronger biological activities than inorganic

Figure 6. Acute toxicity of FA-SeNPs. Induction of acute liver injury: Mice were orally fed FA-SeNPs, SeMet or selenite at a dose of 10 mg/kg. Time-relatedchanges of the hepatic metabolic enzymes for 48 h: (A) ALT activity, (B) AST activity and (C) LDH activity. Pathological changes induced by FA-SeNPs,SeMet and selenite:Micewere orally fed 1152 mg/kg FA-SeNPs, 32 mg/kg SeMet or 32 mg/kg selenite once a day for 14 consecutive days respectively. (D)Normalliver. (E) FA-SeNPs, (F) SeMet and (G) selenite-treated group. Each value represents means ± SD (n=3).

956 T. Liu et al / Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 947–958

or organic selenocompounds47,48 In this study, we havecompared the cytotoxicity of FA-SeNPs against several humancancer and normal cell lines, and our results demonstrate thatFA-SeNPs exhibited a broad spectrum of growth inhibitionagainst human cancer cells, but showed the lowest cytotoxicitytoward human normal cells. These results suggested thatFA-SeNPs possess great selectivity between cancer and normalcells and display potential application in treatment of humancancers. Moreover, the acute toxicity studies were carried out forsafety evaluation. The results showed that the acute toxicity ofSeMet and selenite was about 2-3 folds larger than that ofFA-SeNPs and the lower injury of the nanomaterials of the miceliver. Consistently, previous studies also showed that SeNPswere less toxic than SeMet and selenite.48,49 Therefore, theFA-SeNPs could reduce the acute toxicity of SeNPs. Takentogether, these results demonstrate that FA-SeNPs exhibit muchlower acute toxicity than the common Se positive controls SeMetand selenite, which indicate that FA-SeNPs is an efficient andlow toxicity drug delivery system for anticancer drugs.

In summary, herein we have demonstrated the design ofcancer-targeted nanoparticles loaded with RuPOP complex.

Importantly, the FA-SeNPs increase the sensibility to cancer cellsoverexpressing FARand against themultidrug resistant of R-HepG2cells by inhibition of ABC family proteins expression. InternalizedFA-SeNPs induce cells apoptosis through up-regulating the level ofROS in cells to trigger MAPKs and AKT pathways. Moreover,FA-SeNPs exhibit lower in vivo acute toxicity than selenomethio-nine and selenite, which verify the safety and application potential ofFA-SeNPs as nanodrug system. We would expect that this studymay provide an effective strategy for the design and development ofnanodrugs against multidrug resistant cancers.

Appendix A. Supplementary data

Supplementary data to this article can be found online athttp://dx.doi.org/10.1016/j.nano.2015.01.009.

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