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Biomaterials 33 (2012) 1808e1820

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Biomaterials

journal homepage: www.elsevier .com/locate/biomateria ls

Mitochondrial targeting topotecan-loaded liposomes for treating drug-resistantbreast cancer and inhibiting invasive metastases of melanoma

Yang Yu, Zhao-Hui Wang, Liang Zhang, Hong-Juan Yao, Yan Zhang, Ruo-Jing Li, Rui-Jun Ju,Xiao-Xing Wang, Jia Zhou, Nan Li, Wan-Liang Lu*

State Key Laboratory of Natural and Biomimetic Drugs, and School of Pharmaceutical Sciences, Peking University, Beijing 100191, China

a r t i c l e i n f o

Article history:Received 13 September 2011Accepted 10 November 2011Available online 1 December 2011

Keywords:Mitochondrial targeting topotecan-loadedliposomesMultidrug resistanceMetastasesBreast cancerMelanoma

* Corresponding author. School of PharmaceuticalXueyuan Road 38, Beijing 100191, China. Tel./fax: þ86

E-mail address: luwl@bjmu.edu.cn (W.-L. Lu).

0142-9612/$ e see front matter � 2011 Elsevier Ltd.doi:10.1016/j.biomaterials.2011.10.085

a b s t r a c t

Multidrug resistance and cancer metastases are two obstacles to a successful chemotherapy andmetastases are closely associated with drug resistance. Mitochondrial targeting topotecan-loaded lipo-somes have been developed to overcome this resistance and resistance-related metastases. Investiga-tions were performed on breast cancer MCF-7 and resistant MCF-7/adr cells, MCF-7 and resistant MCF-7/adr tumor spheroids, resistant MCF-7/adr cell xenografts in nude mice, and a naturally resistant B16melanoma metastatic model in nude mice. The mitochondrial targeting topotecan-loaded liposomeswere approximately 64 nm in size, and exhibited the strongest inhibitory effects on MCF-7 cells andresistant MCF-7/adr cells. Mitochondrial targeting effects were demonstrated by co-localization inmitochondria, enhanced drug content in mitochondria, dissipated mitochondrial membrane potential,opening of mitochondrial permeability transition pores, release of cytochrome C, and activation of cas-pase 9 and 3. The targeting liposomes had a stronger inhibitory effect on the resistant tumor spheroidsin vitro, enhanced accumulation in resistant MCF-7/adr cell xenografts in mice, as well as being veryeffective on resistant MCF-7/adr cell xenografts in mice, and having a marked anti-metastastic effect onthe naturally resistant B16 melanoma metastatic model in mice. In conclusion, mitochondrial targetingtopotecan-loaded liposomes could be a promising strategy for treating resistant cancers and resistance-related metastases.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Multidrug resistance and cancer metastases are two obstacles tosuccessful chemotherapy [1,2]. Multidrug resistance involvesacquired and intrinsic resistance while metastases after chemo-therapy are closely related to drug resistance [3,4].

There are two basic approaches to destroying cancer cells bychemotherapy. One is to kill the cancer cells by directly exposingthem to drugs and the other is to induce the suicide of cancer cells,namely, apoptosis [5]. The acquired resistance to cancer may comefrom a drug stimulus, leading to the overexpression of ATP-bindingcassette (ABC) transporters, and subsequent efflux of anticancerdrugs from within cancer cells [3,6]. Intrinsic resistance is mainlyassociated with mitochondria, which are considered as the majorpowerhouse of the cells and play a central role in energy metabo-lism and in apoptosis [4].

Sciences, Peking University,10 8280 2683.

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There are two kinds of regulatory genes located on themembrane of mitochondria: pro-apoptotic and anti-apoptoticgenes [7]. Apoptotic resistance of cancer cells is associated withthe upregulation of anti-apoptotic proteins and/or the down-regulation of pro-apoptotic proteins [8]. The induction of apoptosisof cancer cells involves several factors, including the activation ofpro-apoptotic gene expressed proteins and inhibition of anti-apoptotic gene expressed proteins which initiate the apoptosis ofcancer cells, the opening of mitochondrial permeability transitionpores, which result in the release of cytochrome C from mito-chondria to the cytoplasm, and the activation of the apoptoticprotein enzyme, caspase 9, which leads to activation of down-stream caspase 3 [9,10].

In the present study, we hypothesized that mitochondrial tar-geting topotecan-loaded liposomes could circumvent the acquireddrug resistance due to the drug-induced overexpression of ABCtransporters on the cancer cell membrane, and overcome theintrinsic drug resistance due to the apoptotic resistance derivedfrom the mitochondria of cancer cells, thereby preventing potentialmetastases after chemotherapy. In actual fact, this strategy is aimed

Y. Yu et al. / Biomaterials 33 (2012) 1808e1820 1809

at overcoming the multidrug resistance and preventing the resul-tant cancer metastases after chemotherapy at two levels: inhibitingthe acquired resistance derived from membranes of cancer cells,and circumventing the intrinsic resistance derived from mito-chondria of cancer cells.

Dequalinium (DQA) is a delocalized lipophilic cationiccompound that can be selectively accumulated into the mito-chondria of cancer cells in response to the transmembrane electricpotential [11e13]. Therefore, DQA has been used for the selectivedelivery of drugs into mitochondria [14]. D-a-tocopheryl poly-ethylene glycol 1000 succinate (TPGS1000), known as vitaminE polyethylene glycol succinate, has been used to increasechemotherapeutic efficacy by inhibiting the drug efflux of ABCtransporters [15e17]. It is an interesting compound for constructingnanoparticle drug delivery systems, showing a high encapsulationefficiency and an enhanced cellular uptake of drugs by cancer cells[18]. In the present study, DQA and TPGS1000 were incorporatedinto the membrane of liposomes, and used as a mitochondrialtargeting molecule and a functional agent for inhibiting drug efflux,respectively.

Topotecan hydrochloride is a water-soluble derivative ofcamptothecin and a topoisomerase I inhibitor [19]. It has been usedin the treatment of ovarian cancer, lung cancer, and many othertypes of cancer including breast cancer [20,21]. However, topotecanis a substrate of ABC transporters like ABCB1 protein (P-gp), andABCG2 protein (breast cancer resistance protein, BCRP) and, thus,produces severe resistance in tumor cells [22e24]. In this study,topotecan was used as a model anticancer drug for constructingmitochondrial targeting liposomes.

The objectives of the present study were to develop mitochon-drial targeting topotecan-loaded liposomes, to define the targetingeffect, and to demonstrate the efficacy for treating resistant cancerand inhibiting cancer metastases in animals.

2. Materials and methods

2.1. Preparation of mitochondrial targeting topotecan-loaded liposomes

Mitochondrial targeting topotecan-loaded liposomes were prepared usinga film-dispersion method followed by an active loading method. Briefly, eggphosphatidylcholine (EPC), cholesterol (Chol), polyethylene glycol-distearoylphosphosphatidylethanolamine (PEG2000-DSPE, NOF Corporation, Japan),TPGS1000 (SigmaeAldrich, USA) and dequalinium (Hangzhou Sanhe Chemicals, Co.,Ltd, Hangzhou, China) (60:29:2:2:7, mmol ratio) were used as liposome-formingmaterials. The materials were dissolved in chloroform and methanol (3:1, v/v) ina pear-shaped flask. The chloroform and methanol were then removed by evapo-ration in a rotary vacuum evaporator, and the lipid film was hydrated with 250 mMammonium sulfate by sonication for 5 min in a water bath, followed by sonicationfor 9 min using a probe-type sonicator, to produce blank liposomes which were thenextruded twice through polycarbonate membranes (Millipore, Bedford, MA) witha pore size of 400 nm and 200 nm, respectively. After dialysis (MWCO 14 kDa) threetimes in a solution of Hepes buffered saline (HBS, 25 nM Hepes/150 nM NaCl), theblank liposomes were mixed with an appropriate volume of topotecan hydrochlo-ride (Chengdu Furunde Enterprise, Co., Ltd, Sichuan, China) in HBS buffer (liposomematerials: topotecan ¼ 15: 1, w/w), incubated at 60 �C in a water bath and thenshaken intermittently for 20 min, to produce mitochondrial targeting topotecan-loaded liposomes.

Mitochondrial targeting 6-coumarin-loaded liposomes, mitochondrial targetingrhodamine-123-loaded liposomes, and mitochondrial targeting cyanine 7 (Cy7)loaded liposomes were prepared using the same liposome-forming materials. Toprepare mitochondrial targeting 6-coumarin-loaded liposomes, 6-coumarin wasadded with the liposome-forming materials, dissolved, dried for forming the lipidfilm, and hydrated with phosphate buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl,8 mM Na2HPO4, 2 mM KH2PO4, pH 7.4). To prepare mitochondrial targetingrhodamine-123-loaded liposomes or mitochondrial targeting Cy7-loaded lipo-somes, rhodamine-123 or Cy7 was dissolved and added with the PBS (pH7.4). Theseliposomes were used as fluorescently labeled probes.

In addition, topotecan-loaded liposomes were prepared (EPC: Chol: PEG2000-DSPE ¼ 60: 29: 2, mmol ratio) using the same protocol of mitochondrial targetingtopotecan-loaded liposomes except that TPGS1000 and dequaliniumwere not used inthe procedure, and used as a control.

Furthermore, 6-coumarin-loaded liposomes, rhodamine-123-loaded liposomesand Cy7-loaded liposomeswere prepared using the same protocols of mitochondrialtargeting 6-coumarin-loaded liposomes, mitochondrial targeting rhodamine-123-loaded liposomes, and mitochondrial targeting Cy7-loaded liposomes, respec-tively. However, TPGS1000 and dequalinium were not used in the procedure, andused as controls.

2.2. Particle size, encapsulation efficiency and drug release

The particle sizes and zeta potentials of the topotecan-loaded liposomes andmitochondrial targeting topotecan-loaded liposomes were measured using a NanoSeries Zen 4003 Zeta Sizer (Malvern Instruments, Ltd, UK).

The encapsulation efficiency of topotecan was estimated from the formula:EE ¼ (Wdialysis/Wtotal) � 100%, where EE is the encapsulation efficiency of topotecan,Wdialysis is the measured amount of topotecan in the liposome suspensions afterdialysis, and Wtotal is the measured amount of topotecan in an equal volume ofliposome suspension before dialysis. The drug-loaded liposomes were dialyzed 3times (each for a period of 12 h) against a 10-fold volume of HBS to remove theunloaded topotecan. Tomeasure the content of topotecan, the liposome suspensionswere dissolved by adding methanol. The topotecan in various liposomal formula-tions was measured using fluorospectrophotometry as reported previously [25,26].The excitation and emission wavelengths were set at 381 and 531 nm, respectively.

Mitochondrial targeting topotecan-loaded liposomes were passed througha Sephadex G-50 column (SigmaeAldrich Corporation, Beijing local agent, China) toremove the unmodified dequalinium. Dequalinium was determined using an ODScolumn (Dikma, 5 mm, 250 � 4.6 mm) by high performance liquid chromatography(HPLC) system with UV detection (Agilent Technologies Inc., Cotati, CA, USA).The mobile phase consisted of acetonitrile, 0.2 M NaH2PO4, methanol and triethyl-amine (34: 66: 2.4: 0.3, v/v). The flow rate was set at 1.0 ml/min and the detectionwavelengthwas 237 nm. Themodifying rate of dequaliniumwas calculated from theformula: MR ¼ (Wmodifying/Wtotal) � 100%, where MR is the modifying rate ofdequalinium, Wmodifying is the measured amount of dequalinium in the liposomesuspension after passing through the column, and Wtotal is the measured amount ofdequalinium in an equal volume of liposome suspension before passing through thecolumn. The modifying efficiency of dequalinium was calculated from the formula:ME ¼ nDQA/nlipid, where ME is the modifying efficiency of dequalinium, nDQA is theamount of dequalinium (mmol), and nlipid is the amount of membrane material(mmol).

In vitro drug release of topotecan-loaded liposomes was performed by dialysis(MWCO 14 kDa) against a release medium of phosphate buffered saline (PBS,137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 2 mM KH2PO4, pH 7.4) containing 10%fetal bovine serum. A volume of 4 ml of liposome suspension in dialysis tubing wasimmersed in 20.0 ml of the release medium, and agitated in a shaker at a rate of 100oscillations per minute at 37 �C. Then, 0.5 ml samples of release mediumwere takenat 0, 0.5, 1, 2, 4, 8, 12, 24, and 48 h and immediately replacedwith a similar volume offresh release medium. The topotecan content of the release medium was measuredby fluorospectrophotometry. The release rate was calculated from the formula:RR ¼ (Wi/Wtotal) � 100%, where RR is the drug release rate (%), Wi is the measuredamount of topotecan at the ith h in the release medium, and Wtotal is the totalamount of topotecan in an equal volume of liposome suspension prior to dialysis.

2.3. Cell culture

Human breast cancer MCF-7 cells were obtained from the Institute of MateriaMedica (Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing, China), doxorubicin-resistant breast cancer MCF-7/adr cells were obtainedfrom the Institute of Hematology and Blood Diseases Hospital (Chinese Academy ofMedical Sciences and Peking Union Medical College, Tianjin, China), and the murineB16 melanoma cell line was obtained from the Cell Bank of Type Culture Collectionof the Chinese Academy of Sciences (Shanghai, China). The culture medium wasprepared with RPMI 1640 (Macgene Biotech Ltd, Beijing, China) supplemented with10% heat-inactivated fetal bovine serum (FBS, Macgene Biotech), 100 U/ml penicillin(Macgene Biotech), and 100 mg/ml streptomycin (Macgene Biotech). To maintain thedrug-resistant phenotype of MCF-7/adr cells, 2 mM doxorubicin was added to themedium and doxorubicin-free mediumwas used for 1 week before the experimentswere started. Cell culture was performed in an incubator maintained at 37 �C ina humidified containing 5% CO2.

2.4. Antiproliferative activity in vitro

MCF-7 or MCF-7/adr cells were seeded in a 96-well plate at a density of 5000cells per well. After cells were cultured in the incubator for 24 h, fresh mediumcontaining serial concentrations of different drug formulations was added to theplate wells, including blank culture medium (blank control), free topotecan(0e10 mM), topotecan-loaded liposomes (0e10 mM) and mitochondrial targetingtopotecan-loaded liposomes (0e10 mM). After further incubation for 48 h, theantiproliferative activity was measured by sulforhodamine B (SRB) staining assay byreading the absorbance on a microplate reader (Bio-Rad model 680, Bio-Rad Labo-ratories, Shanghai, China) at a wavelength of 540 nm, as reported previously [27].

Y. Yu et al. / Biomaterials 33 (2012) 1808e18201810

The survival rate was calculated from the following formula: survival (%) ¼ (A540 nm

for the treated cells/A540 nm for the control cells) � 100%, where A540 nm is theabsorbance value and the dose-effect curves were plotted. All the experiments wereperformed in triplicate.

2.5. Mitochondrial targeting effects in vitro

2.5.1. Co-localization in mitochondriaA confocal laser scanning fluorescent microscope with Leica confocal software

(Leica, Heidelberg, Germany) was used to observe the co-localization of mitochon-drial targeting liposomes. Briefly, MCF-7 or MCF-7/adr cells were seeded in cham-bered coverslips at a density of 2.5�104 cells per well. After incubation for 24 h, thecells were treated with free 6-coumarin, 6-coumarin-loaded liposomes or mito-chondrial targeting 6-coumarin-loaded liposomes in an atmosphere of 5% CO2 at37 �C. The final concentration of 6-coumarin was 10 mM. Following further incu-bation for 4 h, the cells were washed with PBS (pH 7.4) and then stained withmitotracker red FM (Molecular Probe, Eugene, OR) for 30min in an atmosphere of 5%CO2 at 37 �C. The concentrations of mitotracker were set at 0.5 mM for MCF-7 cellsand 2 mM for MCF-7/adr cells. Then, the cells were washed twice with PBS andobserved with a laser scanning confocal microscope.

2.5.2. Drug content in the mitochondrial fractionMCF-7 or MCF-7/adr cells were harvested after applying blank culture medium,

free 6-coumarin, 6-coumarin-loaded liposomes or mitochondrial targeting 6-coumarin-loaded liposomes for 6 h at a final 6-coumarin concentration of 10 mM.The isolation of the mitochondria was performed according to the guide for the cellmitochondria isolation kit (Beyotime Institute of Biotechnology, Haimen, China).Briefly, cells were reacted withmitochondria extraction reagent (provided in the kit)and stirred in a homogenizer. The suspensions were centrifuged at 600 g for 10 min.The supernatants were collected and further centrifuged at 3500 g for 10 min. Themitochondria were collected from the precipitates, and a FACScan flow cytometer(Becton Dickinson FACS Calibur, Mountain View, CA, USA) was used to quantify thedrug content in the mitochondrial fraction with the events collected 1 � 104.

2.5.3. Mitochondrial membrane potential (Djm)MCF-7 or MCF-7/adr cells were seeded in six-well plates at 5�105 cells per well.

After incubation for 24 h, the cells were exposed to different formulations for 6 h,including free topotecan (10 mM), topotecan-loaded liposomes (10 mM), mitochon-drial targeting topotecan-loaded liposomes (10 mM) and fresh medium as a control.Cells were harvested, washed twice with PBS (pH 7.4), and stained with rhodamine-123 at 37 �C for 30 min. Rhodamine-123 is a fluorescent dye which can be incor-porated into mitochondria and retained proportionally by the transmembranepotential, thus being used to measure the Djm [28]. Considering that rhodamine-123 is a substrate of ABC proteins, the staining of MCF-7/adr was performed inthe presence of the MDR inhibitor verapamil hydrochloride (100 mM). The fluores-cence intensity of rhodamine-123 accumulated in mitochondria was observed usinga FACScan flow cytometer (Becton Dickinson FACS Calibur, Mountain View, CA, USA)with the events collected 1 � 104.

2.5.4. Opening of mitochondrial permeability transition poresThe opening of the mitochondrial permeability transition pores was directly

assessed by co-loading calcein acetoxymethyl ester (calcein AM) with CoCl2, as re-ported previously [29]. The principle is based on the fact that calcein AM ispermeable to intact membranes but not to intact mitochondrial membranes, andthat the opening of mitochondrial permeability transition pores allows calcein toenter into mitochondria, thereby allowing monitoring of the calcein fluorescence inmitochondria. A confocal laser scanning fluorescent microscope was used todetermine the opening of the mitochondrial permeability transition pores afterapplying mitochondrial targeting topotecan-loaded liposomes. Briefly, MCF-7 orMCF-7/adr cells were seeded in chambered coverslips at a density of 2.5 � 104 cellsper well. After incubation for 24 h, the cells were treated with free topotecan,topotecan-loaded liposomes and mitochondrial targeting topotecan-loaded lipo-somes for 12 h in an atmosphere of 5% CO2 at 37 �C. The final concentration oftopotecan was 10 mM. Following further incubation for 12 h, the cells were washedwith PBS (pH 7.4) and then co-loaded with 1 mM calcein AM and 1 mM CoCl2 inmodified Hanks’ balanced solution supplemented with 10 mM Na-HEPES (pH 7.3)without phenol red and NaHCO3 (Sigma). The cells were then maintained in thissolution at room temperature for 30 min and washed with PBS (pH 7.4) to removethe outside calcein and CoCl2 of the cells. Calcein fluorescence was monitored usinga laser scanning confocal microscope (Leica, Heidelberg, Germany) [30].

2.5.5. Release of cytochrome CThe translocation of cytochrome C from mitochondria to the cytosol was

examined by immunohistochemical staining using a streptavidin-peroxidaseimmunohistochemical kit (Zhongshan Goldenbridge Biotechnology, Co. Ltd, Bei-jing, China). Briefly, after incubation for 24 h, MCF-7/adr cells were exposed to freetopotecan (10 mM), topotecan-loaded liposomes (10 mM), mitochondrial targetingtopotecan-loaded liposomes (10 mM) and fresh medium as a control. After incuba-tion for another 12 h, the cells were treated with 3% H2O2, blocking buffer (provided

in the kit), primary antibody anti-cytochrome C (Nanjing KeyGen Biotechnology, Co.,Ltd, Nanjing, China), enhanced secondary antibody (provided in the kit) and thenenhanced streptavidin HRP conjugate (provided in the kit). After color development,the release of cytochrome C was observed under a light microscope.

2.5.6. Caspase activationCaspase 9 and caspase 3 activities in MCF-7/adr cells were determined using

peptide substrates that emit fluorescence once cleaved by the specific protease [31].Briefly, MCF-7/adr cells were cultured for 24 h, and then treated with free topotecan(10 mM), topotecan-loaded liposomes (10 mM) and mitochondrial targetingtopotecan-loaded liposomes (10 mM). Controls were performed by adding blankmedium. After a 12 h incubation, the cells were harvested and lysed. The cell lysateswere centrifuged at 10,000 rpm at 4 �C for 1 min and the supernatants wereremoved and treated with caspase 9 and caspase 3 substrates (Nanjing KeyGenBiotechnology), respectively. Caspase 9 and caspase 3 activities were measured at405 nm on amicroplate reader, and the activity ratiowas calculated according to thekit instructions. Each assay was carried out in triplicate.

2.6. Apoptosis-inducing effect in vitro

Apoptotic cell death was determined by staining using an Annexin V-FITCapoptosis detection kit (Nanjing KeyGen Biotechnology) according to the manu-facturer’s protocol using a flow cytometer. Briefly, MCF-7 cells or MCF-7/adr cellswere seeded in a 6-well flat-bottomed tissue culture plate at a density of 5 � 105

cells/well in 2 ml growth medium. After 24 h, the cells were treated with freshmedium (as a blank control), free topotecan, topotecan-loaded liposomes andmitochondrial targeting topotecan-loaded liposomes for 12 h in an atmosphere of5% CO2 at 37 �C. The concentration of topotecan was 20 mM. Then the cells wereharvested and suspended in the provided binding buffer, and 5 ml Annexin V-FITCwas added to the cell suspensions. The mixture was incubated at room temperaturein the dark for 15 min, and then 5 ml PI (propidium iodide, provided in the kit) wasadded. The cells were immediately analyzed using a FACScan flow cytometer withthe events collected 1 � 104. Each assay was carried out in triplicate.

2.7. Inhibitory effect on resistant tumor spheroids

2.7.1. Inhibitory effect on the volumeMulticellular tumor spheroids were formed with MCF-7 or MCF-7/adr cells

using the hanging drop method as described previously [32]. Agarose solution(2%, w/v) was prepared in serum-free RPMI 1640 by heating it at 80 �C for 30 minand then sterilizing in an autoclave. Each well of the 24-well culture plates wascoatedwith a thin layer (0.2 ml) of this sterilized solution followed by the addition of900 ml culture medium. Then, 20 ml aliquots of the cell suspensions containing 500cells were suspended on the lid of the 24-well culture plate. After an intervalrequired for cell aggregation (at 72 h), the spheroids were transferred to the bottomof the well containing 900 ml culture medium. Multicellular tumor spheroids werethen allowed to grow for 2 days, and the spheroids with a diameter of 200 mmwereused in the experiments.

To evaluate the inhibitory effect, MCF-7 or MCF-7/adr tumor spheroids wereincubated with serum-free RPMI 1640 (as a blank control), free topotecan (20 mM),topotecan-loaded liposomes (20 mM) and mitochondrial targeting topotecan-loadedliposomes (20 mM). Growth inhibition was monitored by measuring the size of thetumor spheroids using an inverted phase microscope (Chongqing Optical & Elec-trical Instrument, Co., Ltd., Chongqing, China). The major (dmax) and minor (dmin)diameters of each spheroid were determined, and the spheroid volume was calcu-lated using the following formula: V ¼ (p� dmax � dmin)/6. The MCF-7 or MCF-7/adrtumor spheroid volume change ratio was calculated from the formula: R ¼ (Vday i/Vday 0)� 100%, where the Vday i is the tumor spheroid volume at the ith day (day 1, 2,3, 4, 5) after applying drug, and Vday 0 is the tumor spheroid volume prior totreatment.

To observe the spheroid morphology MCF-7/adr tumor spheroids at day 0 weresimilarly treated with serum-free RPMI 1640 (as a blank control), free topotecan(20 mM), topotecan-loaded liposomes (20 mM) and mitochondrial targetingtopotecan-loaded liposomes (20 mM). At day 3, the spheroids were fixed in 2.5%glutaraldehyde for 60 min, rinsed three times in 0.1M PBS, then dehydrated andcoated with gold. These tumor spheroid specimens were viewed under a scanningelectronmicroscope (SEM, JSM-5600 LV, JEOL, Japan) at a magnification of 200� and1000�.

2.7.2. Penetration assayTo evaluate the penetration ability, MCF-7/adr tumor spheroids were incubated

with free rhodamine-123, rhodamine-123-loaded liposomes, and mitochondrialtargeting rhodamine-123-loaded liposomes at a final concentration of 10.0 mMrhodamine-123 for 12 h. Before transfer to a chambered coverslip, the spheroidswere rinsed with PBS and examined using a laser scanning confocal microscope(Leica SP2, Heidelberg, Germany). Z-stack images were obtained by scanning thetumor spheroid step by step. The scanning began from the top of a spheroid to theequatorial plane. Each scanning layer was 10 mm in thickness and the total scan was80 mm in depth in a spheroid.

Table 1Characterization of the liposomes.

Measurements Topotecan-loadedliposomes

Mitochondrial targetingtopotecan-loadedliposomes

Particle size (nm) 88.47 � 1.30 64.84 � 0.56Zeta potential (mV) �0.72 � 0.07 �0.52 � 0.08PDI 0.206 � 0.012 0.194 � 0.003Encapsulation efficiency (%) 96.95 � 0.75 96.38 � 0.45Modifying rate of DQA (%) N/A 91.20 � 0.51Modifying efficiency of DQA

(mmol DQA/mmol lipids)N/A 0.0717 � 0.0004

Data are presented as mean � standard deviation (n ¼ 3). N/A, not applicable.

Y. Yu et al. / Biomaterials 33 (2012) 1808e1820 1811

2.8. In vivo imaging in the resistant breast cancer xenografts in mice

To observe the real-time distribution and tumor accumulation ability of fluo-rescently labeled mitochondrial targeting Cy7-loaded liposomes in MCF-7/adr cellxenografts in mice, non-invasive optical imaging systems were used. All animalexperiments were performed in accordance with the principles for the care and useof laboratory animals, and were approved by the Institutional Animal Care and UseCommittee of Peking University. Female BALB/c nude mice (17e19 g, obtained fromPeking University Health Science Center) were used for the in vivo imaging exper-iments. Subcutaneous tumormodels were established by inoculating 8.0� 106MCF-7/adr cells into the right flanks of the nude mice. When tumors reached approxi-mately 500 mm3 in volume, the mice were randomly divided into three treatmentgroups (5 animals each). The mice were then given an intravenous injection via thetail vein of physiological saline, free Cy7 (2 mg/kg, diluted in PBS) or mitochondrialtargeting Cy7-loaded liposomes (2 mg/kg). Dose of liposomal formulation wasequivalent to native Cy7. Themicewere anaesthetized by intraperitoneal injection ofpentobarbital (60 mg/kg), and scanned at 0.5 h, 1 h, 3 h, 7 h, 12 h and 24 h usinga Kodak multimodel imaging system (Carestream Health, Inc., USA) with an exci-tation band pass filter at 730 nm and an emission at 790 nm. The exposure time was30 s per image. After in vivo imaging, the mice were sacrificed at 24 h, and hearts,livers, spleens, lungs, kidneys and tumors were excised. The near-infrared fluores-cence signal intensities in different tissues were measured.

2.9. Efficacy in treating resistant breast cancer xenografts in mice

Female BALB/c nude mice (initially weighing 17e19 g) were used to investigatethe antitumor efficacy in vivo. Briefly, approximately 8 � 106 MCF-7/adr cells werere-suspended in 200 ml serum-free RPMI 1640 culture medium, and injectedsubcutaneously into the right flanks of the nude mice. When tumors reached150e180 mm3 in volume, the mice were randomly divided into four treatmentgroups (5 animals each). At day 11, 13, 16 and 19 post-inoculation, physiologicalsaline, free topotecan (5 mg/kg), topotecan-loaded liposomes (5 mg/kg) and mito-chondrial targeting topotecan-loaded liposomes (5 mg/kg) were injected into themice via the tail vein. Dose of liposomal formulation was equivalent to native top-otecan. The mice were then monitored every day using calipers to measure thetumor progression. Tumor volumes were calculated as length � width2/2 (mm3).The tumor volume inhibitory rate at day 25 was calculated from the formula:Rv ¼ 100%-(Vdrug/Vsaline) � 100%, where Vdrug is the tumor volume after drugtreatment, and Vsaline is the tumor volume after treatment with physiological saline.

2.10. Efficacy in treating melanoma metastasis

Female BALB/c nude mice (initially weighing 17e19 g) were used to investigatethe efficacy in treating melanoma metastasis in vivo. Briefly, 8 � 106 B16 melanomacells in a volume of 200 ml mediumwere injected into a mouse via the tail vein. Micewere randomly divided into four groups (3 animals each). At day 11, 15 or 19 post-inoculation, physiological saline, free topotecan (5 mg/kg), topotecan-loaded lipo-somes (5 mg/kg) and mitochondrial targeting topotecan-loaded liposomes (5 mg/kg) were administered once daily via the tail vein. Dose of liposomal formulationwas equivalent to native topotecan. The mice were sacrificed at day 27 and the lungswere removed and photographed. The tumor colonies on the same surface of the leftlung were counted, and the percentage of metastasized melanoma colonies wascalculated from the following formula: metastasized melanoma colonies(%) ¼ (tumor colony numbers after treatment)/(tumor colony numbers of blankcontrol) � 100%.

2.11. Statistics

Data are presented as the mean � standard deviation. One-way analysis ofvariance (ANOVA) was used to determine significance among groups, after whichpost hoc tests with the Bonferroni correction were used for multiple comparisonsbetween individual groups. A value of P < 0.05 was considered to be statisticallysignificant.

3. Results

3.1. Characterization of the liposomes

Table 1 shows the average particle sizes, the potential values andencapsulation efficiencies of topotecan-loaded liposomes andmitochondrial targeting topotecan-loaded liposomes. The meanparticle sizes of topotecan-loaded liposomes and mitochondrialtargeting topotecan-loaded liposomes were 88.47 � 1.30, and64.84 � 0.56 nm, respectively. Accordingly, the potential valueswere �0.72 � 0.07 and �0.52 � 0.08 mV. For all liposomesprepared, the encapsulation efficiency of topotecan was �95%. For

mitochondrial targeting topotecan-loaded liposomes, the modi-fying rate of dequalinium was �90% and the modifying efficiencywas 0.072 mmol dequalinium/mmol lipid material. The PDI valuesfor all liposomes were approximately 0.2.

Fig. 1 shows the in vitro topotecan release rates from the drug-loaded liposomes in PBS containing 10% fetal bovine serum. Theresults showed that the topotecan release rates of all liposomeswere less than 7% at 2 h and approximately 10% at 24 h. The releaserates of topotecan-loaded liposomes and mitochondrial targetingtopotecan-loaded liposomes were all less than 15% at 48 h.

3.2. Inhibitory effect on resistant breast cancer cells in vitro

Fig. 2 shows the inhibitory effect of different topotecan formu-lations on MCF-7 (A) and MCF-7/adr cells (B). In inhibiting MCF-7cells, the IC50 values of free topotecan, topotecan-loaded lipo-somes, and mitochondrial targeted topotecan-loaded liposomeswere 4.52 mM, 4.53 mM and 2.04 mM, respectively. In inhibitingMCF-7/adr cells, the IC50 values of free topotecan, topotecan-loaded liposomes, and mitochondrial targeted topotecan-loadedliposomes were 5.39 mM, 1.54 mM, and 1.13 mM, respectively. Afterapplying fresh medium, free topotecan, topotecan-loaded lipo-somes or mitochondrial targeting topotecan-loaded liposomes, themitochondrial targeting topotecan-loaded liposomes exhibited thestrongest inhibitory effect on the proliferation of MCF-7 cells orMCF-7/adr cells. In addition, free topotecan exhibited some drugresistance in MCF-7/adr cells compared with topotecan-loadedliposomes.

3.3. Mitochondrial targeting effects in vitro

3.3.1. Co-localization in mitochondriaFig. 3 shows the confocal laser scanning microscopic images of

the subcellular localization of drugs in MCF-7 cells (A) and in MCF-7/adr cells (B). Mitochondria had a red fluorescence when stainedwith mitotracker red, and 6-coumarin was used as a green fluo-rescent probe to indicate the subcellular localization of drugs. Thebright yellow fluorescence, consisting of red and green fluores-cence, was used to indicate the co-localization of 6-coumarin andmitotracker. According to the images, mitochondrial targeting 6-coumarin-loaded liposomes showed a punctuate distribution, andwere selectively accumulated in the mitochondria with clear brightyellow fluorescence. However, after applying free 6-coumarin or 6-coumarin-loaded liposomes, no bright yellow fluorescence wasobserved in the cells.

3.3.2. Drug content in the mitochondrial fractionFig. 4 shows the 6-coumarin content in the isolated mitochon-

dria after applying blank culture medium, free 6-coumarin,6-coumarin-loaded liposomes or mitochondrial targeting6-coumarin-loaded liposomes to MCF-7 (A) andMCF-7/adr cells (B)

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Y. Yu et al. / Biomaterials 33 (2012) 1808e18201812

for 6 h. The fluorescence intensity of the mitochondria afterapplying mitochondrial targeting 6-coumarin-loaded liposomeswas clearly higher than that after applying free 6-coumarin or6-coumarin-loaded liposomes. In the mitochondria of MCF-7 cells,the fluorescence intensity of mitochondrial targeting 6-coumarin-loaded liposomes was 21.2-fold higher than that of free6-coumarin, and 12.9-fold higher than that of 6-coumarin-loadedliposomes. In the mitochondria of MCF-7/adr cells, the fluorescenceintensity of mitochondrial targeting 6-coumarin-loaded liposomeswas about 7.0-fold higher than that of free 6-coumarin, and 8.9-foldhigher than that of 6-coumarin-loaded liposomes.

3.3.3. Mitochondrial membrane potential (Djm)Fig. 5 shows the dissipation of mitochondrial membrane

potential (Djm) indicated by the fluorescent peak of rhodamine-123 accumulated in the mitochondria after applying differenttopotecan formulations. Data from flow cytometry showed that theexposure of mitochondrial targeting topotecan-loaded liposomesresulted in a clear loss of the mitochondrial membrane potential ofMCF-7 (A) or MCF-7/adr cells (B) compared with topotecan-loadedliposomes, free topotecan or blank control.

3.3.4. Opening of mitochondrial permeability transition poresFig. 6 shows the opening of the mitochondrial permeability

transition pores in MCF-7 (A) or MCF-7/adr cells (B) indicated bythe confocal laser scanning microscopic images after applyingblank culture medium (A1, B1), free topotecan (A2, B2), topotecan-loaded liposomes (A3, B3), and mitochondrial targeting topotecan-loaded liposomes (A4, B4) for 12 h. The fluorescence intensity ofcalcein in the mitochondria after applying mitochondrial targetingtopotecan-loaded liposomes was clearly higher than that afterapplying free topotecan or topotecan-loaded liposomes. Theimages showed a direct evidence of the opening of mitochondrialpermeability transition pores through observation of the fluores-cence of calcein in mitochondria. The fluorescence of calcein incytosol was quenched by adding Co2þ.

3.3.5. Release of cytochrome CFig. 7 shows the immunohistochemical staining results of cyto-

chrome C translocated from mitochondria to cytosol in the MCF-7/adr cells after applying blank culture medium (A), free topotecan(B), topotecan-loaded liposomes (C), and mitochondrial targetingtopotecan-loaded liposomes (D). The results showed that the mito-chondrial targeting topotecan-loaded liposomes induced a largeamount of cytochrome C release frommitochondria (D) as shown bythe brown staining. In contrast, the images in control groups

exhibited fewer brown regions in the cytosol when cells wereexposed to free topotecan (B) or topotecan-loaded liposomes (C).

3.3.6. Caspase activationFig. 8 shows the activation of caspase 9 (A) and caspase 3 (B) in

MCF-7/adr cells. After applying free topotecan, topotecan-loadedliposomes and mitochondrial targeting topotecan-loaded lipo-somes, the caspase 9 activity ratios in MCF-7/adr cells were 1.3-,1.4- and 3.7-fold higher than that after applying blank control.Accordingly, the caspase 3 activity ratio in MCF-7/adr cells wasabout 1.9-, 2.3- and 3.5-fold higher than that after applying blankcontrol. The results showed that the activities of caspase 9 andcaspase 3 most significantly increased after applying mitochondrialtargeting topotecan-loaded liposomes.

3.4. Apoptosis-inducing effect in vitro

Fig. 9 shows the in vitro apoptosis-inducing effects in MCF-7 (A)and MCF-7/adr cells (B) after applying blank culture medium(A1,B1), free topotecan (A2, B2), topotecan-loaded liposomes (A3, B3),and mitochondrial targeting topotecan-loaded liposomes (A4, B4).The apoptosis-inducing effect was evaluated by counting theapoptotic percentage during the early period plus the apoptoticpercentage during the late period. After applying free topotecan,topotecan-loaded liposomes, and mitochondrial targetingtopotecan-loaded liposomes, the induced apoptotic percentages inMCF-7 cells were 19.7%, 15.8%, and 33.5%; and those in MCF-7/adrcells were 10.4%, 13.6%, and 24.0%, respectively.

Fig. 3. Co-localization of mitochondrial targeting topotecan-loaded liposomes into mitochondria of MCF-7 (A) or MCF-7/adr breast cancer cells (B) was observed by laser scanningmicroscopy. Live MCF-7 or MCF-7/adr cells were stained with mitotracker red, and visualized at 4 h after applying free 6-coumarin(a, d), 6-coumarin-loaded liposomes (b, e), andmitochondrial targeting 6-coumarin-loaded liposomes (c, f), respectively. Notes: 1, red channel: mitotracker red stained mitochondria; 2, green channel: 6-coumarin (a fluorescentprobe); 3, composite images of 1 and 2. The bright yellow fluorescence in image of c3 or f3 indicates that the mitochondrial targeting 6-coumarin-loaded liposomes are co-localizedinto the mitochondria of the breast cancer cells. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Y. Yu et al. / Biomaterials 33 (2012) 1808e1820 1813

Fig. 4. Drug content of the mitochondrial fraction in MCF-7 cells (A) and in MCF-7/adrcells (B) after applying different formulations were observed by flow cytometry. Theordinate represents the cell counts and the abscissa indicates the fluorescent intensityof a 6-coumarin formulation internalized by the cancer cells. Notes: A1 and B1, blankcontrol; A2 and B2, free 6-coumarin; A3 and B3, 6-coumarin-loaded liposomes; A4 andB4, mitochondrial targeting 6-coumarin-loaded liposomes.

Fig. 5. Dissipation of mitochondrial membrane potential (Djm) measured by flowcytometry. Live MCF-7 (A) and MCF-7/adr cells (B) were stained with rhodamine-123after applying different formulations for 6 h, including blank control (A1, B1), freetopotecan (A2, B2), topotecan-loaded liposomes (A3, B3) and mitochondrial targetingtopotecan-loaded liposomes (A4, B4). Compared with other controls, mitochondrialtargeting topotecan-loaded liposomes lead to a clear reduction in the Djm value.

Y. Yu et al. / Biomaterials 33 (2012) 1808e18201814

3.5. Inhibitory effect on resistant tumor spheroids

3.5.1. Inhibitory effect on spheroid volumeFig. 10A and B show the inhibitory effects on the volume of MCF-

7 and MCF-7/adr tumor spheroids. After applying free topotecan,topotecan-loaded liposomes and mitochondrial targetingtopotecan-loaded liposomes, the spheroid volume change ratios ofMCF-7 tumor spheroids at day 5 were 86.7 � 3.1%, 81.9 � 4.7%, and48.3� 4.0%; and those of MCF-7/adr tumor spheroids at day 5 were136.6 � 25.7%, 91.5 � 11.4% and 54.1 � 9.7%, respectively. Theseresults showed that the mitochondrial targeting topotecan-loadedliposomes exhibited the strongest inhibitory effect on MCF-7 orMCF-7/adr tumor spheroids.

Fig. 10C shows the SEM observations on the MCF-7/adr tumorspheroids at day 3. After applying free topotecan, the surface of theMCF-7/adr spheroids changed slightly (b1, b2). When applyingtopotecan-loaded liposomes, the spheroids were markedly alteredby destroying the cells surrounding the spheroids (c1, c2). Afterapplying mitochondrial targeting topotecan-loaded liposomes, thespheroids collapsed, lost their three-dimensional structure anddisintegrated (d1, d2).

3.5.2. Penetration abilityIn Fig. 10D, the confocal images show the penetration of drug

into MCF-7/adr tumor spheroids. For MCF-7/adr tumor spheroids

treated with free rhodamine-123, fluorescence could be observedonly primarily on the spherical periphery. After applyingrhodamine-123-loaded liposomes and mitochondrial targetingrhodamine-123-loaded liposomes, the fluorescence in thespheroids could be observed at a depth of 50 mm and 80 mm,respectively.

3.6. In vivo imaging in resistant breast cancer xenografts in mice

Fig. 11 shows the real-time distribution and tumor accumulationability of mitochondrial targeting Cy7-loaded liposomes in theMCF-7/adr xenografts in BALB/c nude mice.

After applying mitochondrial targeting Cy7-loaded liposomes(Fig. 11A), a strong Cy7 fluorescence signal was observed in theblood circulation during the initial 12 h, and the fluorescence signalwas maintained up to 24 h in the tumor tissue. In contrast, aftergiving free Cy7, the fluorescence signal was only observed in theblood circulation and the tumor tissue during the initial 3 h.

Ex vivo images were collected immediately after whole-bodyoptical imaging at 24 h, when the strongest signal was found inthe tumor of the mitochondrial targeting Cy7-loaded liposomesgroup, while no clear fluorescence was detected in the tumors ofmice treated with free Cy7. In addition, fluorescence signals in themajor organs, including heart, liver, spleen, lung, and kidney of themice, were all undetectable at 24 h.

Fig. 6. The opening of the mitochondrial permeability transition pores in MCF-7 (A) or MCF-7/adr breast cancer cells (B) after applying different formulations. Live MCF-7 and MCF-7/adr cells were exposed to RPMI 1640 culture medium (A1,B1), free topotecan (A2, B2), topotecan-loaded liposomes (A3, B3), and mitochondrial targeting topotecan-loadedliposomes (A4, B4) for 12 h, and observed under laser scanning microscopy by monitoring the fluorescence of mitochondrial entrapped calcein. The fluorescence in the cytosolwas quenched by adding Co2þ.

Y. Yu et al. / Biomaterials 33 (2012) 1808e1820 1815

3.7. Efficacy in treating resistant breast cancer xenografts

Fig. 12 shows the therapeutic effects on the MCF-7/adr cellxenografts in nude mice. The tumor volume inhibitory rates at day25 were 40.86 � 7.81% for free topotecan, 75.25 � 6.51% fortopotecan-loaded liposomes and 93.45 � 1.00% for mitochondrialtargeting topotecan-loaded liposomes. These results showed thatthe mitochondrial targeting topotecan-loaded liposomes achievedsuperior antitumor effects compared with free topotecan ortopotecan-loaded liposomes.

3.8. Efficacy in treating melanoma metastases

Fig. 13A shows the photographs of the metastasized B16 mela-noma on the lung surface of the BALB/c nude mice after treatingwith physiological saline, free topotecan, topotecan-loadedliposomes and mitochondrial targeting topotecan-loaded lipo-somes. In view of the metastatic status, the rank order for anti-metastatic effect was as follows: mitochondrial targetingtopotecan-loaded liposomes > topotecan-loaded liposomes > freetopotecan > physiological saline (blank control).

Fig. 13B shows the quantitative results related to the efficacy intreating metastasis of B16 melanoma by counting the tumor colo-nies on the topical surface of a lung. The metastasized melanomacolonies (%) at day 27 after treating with different formulationswere 61.58� 4.60% for free topotecan, 51.22�10.18% for topotecan-loaded liposomes and 23.17 � 2.79% for mitochondrial targetingtopotecan-loaded liposomes compared with that after treating

Fig. 7. Immunohistochemical staining results of cytochrome C translocated from mitochondcontrol (A), free topotecan (B), topotecan-loaded liposomes (C), and mitochondrial targetina large release of cytochrome C.

with physiological saline (100%, as a blank control). In comparison,metastasizedmelanoma colonies in the lungs weremost effectivelyinhibited after treating with mitochondrial targeting topotecan-loaded liposomes.

4. Discussion

Multidrug resistance and invasive metastasis are two mainreasons for poor prognosis in chemotherapy.Multidrug resistance ofcancer may come from many factors including cancer cellmembrane-related drug resistance due to overexpression of ATP-binding cassette (ABC) transporters and the mitochondria-relateddrug resistance involved in the overall resistance to chemo-therapy. In addition, more and more evidence has demonstratedthat invasive metastases could be associated with multidrug resis-tance. In the present study, a form of novel mitochondrial targetingtopotecan-loaded liposomes was developed to overcome the drugresistance deriving from the cancer cell membrane and mitochon-dria, consequently inhibiting invasive metastases due to resistance.

The smaller particle size and higher encapsulation efficiency ofmitochondrial targeting topotecan-loaded liposomes enable top-otecan to be transported across the vasculature in tumor tissues,thus allowing concentration in the tumor by an enhanced perme-ability and retention (EPR) effect [33,34]. Delayed drug releaseduring the initial 12 h (approximately 10%) would be beneficial forpreventing rapid leakage during the delivery process. TPGS1000 hasbeen recognized as an inhibitor of ABC transporter-mediatedmultidrug resistance in tumor cells, and was used in this study to

ria to cytosol in MCF-7/adr cells after applying different formulations, including blankg topotecan-loaded liposomes (D). The broad brown regions in the image D indicate

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Y. Yu et al. / Biomaterials 33 (2012) 1808e18201816

block drug efflux to reduce the drug resistance [35,36]. DQA, asa mitochondria-tropic molecule with a delocalized charge center,has been used to determine the membrane potential of mito-chondria, and used to deliver DNA to mitochondria of living cells[14]. In the present study, DQA was used as a targeting moleculeand modified the surface of the liposomes to transfer the

Fig. 9. In vitro apoptosis-inducing effects on MCF-7 (A) and MCF-7/adr breast cancer celliposomes (A3, B3), and mitochondrial targeting topotecan-loaded liposomes (A4,B4). The cthen stained with annexin V-FITC/PI, and examined by flow cytometry.

mitochondrial targeting topotecan-loaded liposomes across themitochondrial membrane of cancer cells.

In the cytotoxicity assay, mitochondrial targeting topotecan-loaded liposomes exhibited the strongest inhibitory effect onboth MCF-7 cells (Fig. 2A) and MCF-7/adr cells (Fig. 2B).Themechanism for the enhanced inhibitory effect could involve thefollowing factors: punctuate mitochondria co-localization (Fig. 3Aand B) and higher mitochondrial uptake (Fig. 4A and B). In view ofthe cytotoxicity to both MCF-7 cells and MCF-7/adr cells, theinhibitory effects are consistent with the results of mitochondrialuptake, indicating that the mitochondrial targeting topotecan-loaded liposomes are able to increase the delivery of topotecan tothe mitochondria, thus increasing the toxicity and apoptosis-inducing effect of topotecan.

Laser confocal microscopy of live cells reveals the localization of6-coumarin-labeled mitochondrial targeting liposomes in mito-chondria (Fig. 3A and B), suggesting that the 6-coumarin-labeledmitochondrial targeting liposomes are punctuated and transportedinto mitochondria of cancer cells. In contrast, free 6-coumarin or 6-coumarin-loaded liposomes do not significantly accumulate in themitochondria. This is associated with the mitochondrial targetingability of DQA. The mitochondrial fraction uptake assay (Fig. 4A andB) further demonstrates the mitochondrial targeting effect of theliposomes. In view of the drug content in the mitochondrial frac-tion, the mitochondrial targeting drug-loaded liposomes internal-ized by the cancer cells are not simply attached to the surface of themitochondria but are further endocytosed by the mitochondria.The mechanism for the enhanced mitochondrial fraction uptake ofmitochondrial targeting liposomes may be related to the phago-cytosis of mitochondria or the opening of mitochondrial perme-ability transition pores. This deserves further investigation.

The mitochondrial targeting effect of mitochondrial targetingtopotecan-loaded liposomes is further confirmed by the reducedDjm (Fig. 5A and B) and the release of cytochrome C from mito-chondria (Fig. 7). It is known that the collapse of Djm is the result ofa mitochondrial leak [37] by opening the permeability transition

ls (B) after applying blank medium (A1,B1), free topotecan (A2,B2), topotecan-loadedoncentration of topotecan was 20 mM. Cells were incubated with the drugs for 12 h,

Fig. 10. Inhibitory effect on MCF-7 or MCF-7/adr tumor spheroids. A. Inhibitory effect on the growth of MCF-7 tumor spheroids after applying different formulations (n ¼ 3); B.Inhibitory effect on the growth of MCF-7/adr tumor spheroids after applying different formulations (n ¼ 3); C. Scanning electronic microscopic images of MCF-7/adr tumorspheroids after applying different formulations; D. Confocal images of MCF-7/adr tumor spheroids after applying different rhodamine-123 formulations for 12 h. Z-stack imageswere obtained from the top toward the tumor spheroid equatorial plane in 10 mm thickness. a, P < 0.05, versus blank; b, P < 0.05, versus free topotecan; c, P < 0.05, versustopotecan-loaded liposomes; a1 and a2 (enlarged image of a1), spheroids at day 3 after applying culture medium; b1 and b2 (enlarged image of b1), spheroids at day 3 afterapplying free topotecan; c1 and c2 (enlarged image of c1), spheroids at day 3 after applying topotecan-loaded liposomes; d1 and d2 (enlarged image of d1), spheroids at day 3 afterapplying mitochondrial targeting topotecan-loaded liposomes.

Y. Yu et al. / Biomaterials 33 (2012) 1808e1820 1817

pores (Fig. 6A and B) [38]. The mitochondrial targeting topotecan-loaded liposomes clearly cause the dissipation of Djm in bothMCF-7 and MCF-7/adr cells (Fig. 5A and B), consequently leading tocell death by initiating the apoptotic pathway.

Changes in mitochondrial membrane potential, release ofcytochrome C, and activation of caspase 9 followed by activation ofcaspase 3 are crucial steps in defining the role of mitochondriaduring apoptosis. The results from the immunohistochemicalstaining indicate that the mitochondrial targeting topotecan-loaded liposomes produce a significant release of cytochrome C(Fig. 7). In contrast, no clear or slight release of cytochrome C wasobserved in free topotecan or topotecan-loaded liposomes treated

cells, thereby confirming the targeting effect of mitochondrial tar-geting topotecan-loaded liposomes. The release of cytochrome C isdue to the opening of the mitochondrial permeability transitionpores by swelling of the mitochondrial matrix or rupture of theouter membrane.

To demonstrate the inhibitory effect and penetration ability ofmitochondrial targeting topotecan-loaded liposomes into solidtumor tissues, three-dimensional MCF-7 and MCF-7/adr tumorspheroids were constructed. The tumor spheroids are built fromaggregates of cells in close contact using the organized extracellularmatrix consisting of fibronectin, laminin, collagen and glycosami-noglycans (GAG), exhibiting the characteristics of solid tumor

Fig. 11. A. In vivo non-invasive near-infrared fluorescence (NIRF) images in the MCF-7/adr cell xenografts in BALB/c nude mice after intravenously administering physiological saline(blank control), free Cy7 and mitochondrial targeting Cy7-loaded liposomes via tail vein; B. The ex vivo NIRF images of tumors and organs after the above tumor-bearing mice weresacrificed at 24 h. Notes: Cy 7 represents cyanine 7, which is used as a fluorescent probe.

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Fig. 12. Antitumor efficacy of mitochondrial targeting topotecan-loaded liposomes in the MCF-7/adr xenografts in female nude mice. Approximately 8 � 106 MCF-7/adr breastcancer cells per animal were subcutaneously injected into the right flanks of the nude mice, and animals were treated at day 11, 13, 16 and 19 once daily with physiological saline,free topotecan (5 mg/kg), topotecan-loaded liposomes (5 mg/kg), and mitochondrial targeting topotecan-loaded liposomes (5 mg/kg), respectively. Data are presented as themean � standard deviation (n ¼ 5). a, P < 0.05, versus physiological saline; b, P < 0.05, versus free topotecan; c, P < 0.05, versus topotecan-loaded liposomes.

Y. Yu et al. / Biomaterials 33 (2012) 1808e18201818

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masses in vivo. The results obtained indicate that themitochondrialtargeting topotecan-loaded liposomes have the most significantinhibitory effect on the growth of both spheroids (Fig. 10A and B).After applying mitochondrial targeting topotecan-loaded lipo-somes, SEM observations show that the tumor spheroids lose theirthree-dimensional structure which results in partial collapse of thespheroids (Fig. 10C d1, d2).

In the spheroid penetration study, rhodamine-123-labeledmitochondrial targeting liposomes were able to reach the centralnecrotic region of the MCF-7/adr tumor spheroids after a 12 hincubation (Fig. 10D). In contrast, free rhodamine-123 or rhoda-mine-123-loaded liposomes demonstrate a weaker penetrationinto the spheroids. The permeation of mitochondrial targetingrhodamine-123-loaded liposomes could be related to two mecha-nisms: the action of TPGS1000 which is able to inhibit drug efflux,and the role of cationic dequalinium which enhances the electro-static interactions between mitochondrial targeting rhodamine-123-loaded liposomes and the negative functional groups in thetumor spheroids. In addition, the smaller size of mitochondrialtargeting rhodamine-123-loaded liposomes may lead to a deeperpenetration into tumor tissues.

The near-infrared fluorescent probe Cy7 is similarly encapsu-lated into the same size of mitochondrial targeting liposomesaimed at showing the distribution of the liposomes in the tumor-bearing mice with less background interference at the designatedwavelengths (excitation 730 nm/emission 790 nm), while

Fig. 13. Efficacy of mitochondrial targeting topotecan-loaded liposomes in treatingmetastasis of murine B16 melanoma cells to the lungs of BALB/c female nude mice.A. Images for metastasized melanoma colonies on the lungs; B. Metastasized mela-noma colonies (%) on the surface of the left lung. Approximately 8 � 106 tumor cellsper animal were injected via the tail vein, and animals were treated once daily at day11, 15, 19 with physiological saline (1), free topotecan (5 mg/kg) (2), topotecan-loadedliposomes (5 mg/kg) (3), and mitochondrial targeting topotecan-loaded liposomes(5 mg/kg) (4), respectively. The mice were sacrificed at day 27, and then immediatelyphotographed and the metastasized melanoma colonies were counted. a, P < 0.05,versus control; b, P < 0.05, versus free topotecan; c, P < 0.05, versus topotecan-loadedliposomes.

topotecan cannot be detected at these wavelengths. The in vivoimaging results demonstrate that the mitochondrial targetingCy7-loaded liposomes prolong the blood circulation, and areaccumulated to a higher degree in the tumors (Fig.11A). The ex vivofluorescent images of the excised organs further confirm the higheraccumulation of mitochondrial targeting Cy7-loaded liposomes inthe tumors (Fig. 11B). These mechanisms are explained by the factthat the PEG constituents of TPGS1000 and PEG-DSPE2000 anchoredto the surface of mitochondrial targeting liposomes are able toavoid rapid uptake by the reticuloendothelial system (RES), thusprolonging the circulation time and resulting in a higher accumu-lation of the liposomes in the tumor vasculature or mass. Further-more, the particle size of nanoliposomes plays an important role inthe increased accumulation by the EPR effect as discussed above.

The therapeutic efficacy on the resistant MCF-7/adr xenograftednude mice demonstrate that the mitochondrial targetingtopotecan-loaded liposomes exhibit the most significant antitumoractivity compared with the other formulations at comparable dosesof topotecan, and the tumor growth was markedly inhibited(Fig. 12). The enhanced efficacy of mitochondrial targetingtopotecan-loaded liposomes is associated with the followingaspects: firstly, the addition of dequalinium and TPGS1000 inmitochondrial targeting topotecan-loaded liposomes enhance thepermeability and uptake of the resistant tumor; secondly, mito-chondrial targeting topotecan-loaded liposomes improve thepharmacokinetic profile of topotecan due to the pegylated mate-rials [39,40] which ultimately results in a higher accumulation intumors, and the increased accumulation is confirmed by whole-body optical imaging (Fig. 11); thirdly, mitochondrial targetingtopotecan-loaded liposomes having a smaller size may result indeeper penetration into tumor tissues, as confirmed by confocalmicroscopic images (Fig. 10D); and, finally, topotecan in aqueoussolution is reversibly hydrolyzed in a pH-dependent reactioninvolving the active lactone ring to form an inactive carboxylate inalkaline medium and, accordingly, topotecan-loaded liposomes arebeneficial for the active lactone species by suppressing hydrolysisbecause of the weakly acidic medium inside the constructed lipo-somes, thereby possibly increasing the therapeutic efficacy [20].

To observe the efficacy in treatingmetastasis of breast cancer, wepreviously used MCF-7/adr expressed luciferase to establish a lungmetastatic model in nude mice by inoculation via the tail vein.However, metastatic colonies were not successfully attached to thelungs as shownby the in vivo bioluminescent imaging observations.Consequently, B16 melanoma cells were used to develop a lungmetastaticmodel. B16melanoma is a highlymetastatic cell linewithoverexpressing ABC transporters, and our previous study success-fully established metastasis of B16 melanoma on the lung of micefollowing intravenous administration [41]. The results of thepresentstudy demonstrate that the mitochondrial targeting topotecan-loaded liposomes exhibit the strongest anti-metastastic efficacy inthe experimental model of murine B16 melanoma cells comparedwith other controls (Fig. 13). The mechanisms are also associatedwith the same causes as those described above.

5. Conclusions

In the present study, new mitochondrial targeting topotecan-loaded liposomes have been successfully developed to overcomethe multidrug resistance of cancers and to prevent their possiblemetastases due to acquired and intrinsic drug resistance. Mito-chondrial targeting topotecan-loaded liposomes have a smallparticle size (64 nm), with a high encapsulation efficiency fortopotecan (�95%), and are stable in blood component-containingsystems with minimal leakage. The marked efficacy of mitochon-drial targeting topotecan-loaded liposomes has been demonstrated

Y. Yu et al. / Biomaterials 33 (2012) 1808e18201820

in treating breast cancer MCF-7 cells, treating resistant MCF-7/adrcells, treating resistant MCF-7/adr xenografts in nude mice, andinhibiting a naturally resistant melanoma in the lung metastaticmodel in nude mice. The mechanisms of action of mitochondrialtargeting topotecan-loaded liposomes involve the followingfactors: (1) the internal weak acidic environment in the liposomes,stable drug carriers, smaller particle size, and pegylated materialsbenefiting the pharmacokinetic properties, and the EPR effectincreasing the therapeutic effect in tumor masses; (2) TPGS1000 inmitochondrial targeting topotecan-loaded liposomes inhibiting thedrug efflux of ABC transporters; and (3) targeting apoptosis-inducing effect increasing the therapeutic effect via the mito-chondrial signaling pathway. Therefore, mitochondrial targetingtopotecan-loaded liposomes could offer a promising chemotherapystrategy for treating the acquired resistance-related to the drug-induced overexpression of ABC transporters on the membrane ofcancer cells and for treating the intrinsic resistance-related to themitochondria of cancer cells, consequently preventing possiblecancer metastases derived from the resistance.

Acknowledgments

This study was supported by the Key Grant of Beijing NaturalScience Foundation (No. 7091005), and by National Natural ScienceFoundation of China (No. 81172991).

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