feng li lab of pharmaceutics and engineering - doxorubicin and … · 2018. 8. 28. · doxorubicin...

Doxorubicin and Lapatinib Combination Nanomedicine for TreatingResistant Breast CancerHuiyuan Wang,† Feng Li,*,‡ Chengan Du,‡ Huixin Wang,† Ram I. Mahato,§ and Yongzhuo Huang*,†

†Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Hai-ke Rd, Shanghai 201203, China‡Department of Pharmaceutical Sciences, School of Pharmacy, Hampton University, Hampton, Virginia 23668, United States§Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, Nebraska 68198, United States

ABSTRACT: Our objective was to design a polymeric micelle-baseddoxorubicin and lapatinib combination therapy for treating multidrugresistant (MDR) breast cancers. Poly(ethylene glycol)-block-poly(2-methyl-2-benzoxycarbonylpropylene carbonate) (PEG-PBC) polymers weresynthesized for preparing doxorubicin and lapatinib loaded micelles usinga film dispersion method. Micelles were characterized by determiningcritical micelle concentration (CMC), particle size distribution, and drugloading. The anticancer effects were determined in vitro with MTT assays aswell as with lactate dehydrogenase (LDH) release studies. In addition, thecellular uptake of drug-loaded micelles was determined with fluorescencemicroscopy and flow cytometry. Finally, in vivo anticancer activity andtolerance of developed formulations were evaluated in resistant breast tumorbearing mice. PEG5K-PBC7K polymer synthesized in this study had a lowCMC value (1.5 mg/L) indicating an excellent dynamic stability. PEG-PBC micelles could efficiently load both doxorubicin andlapatinib drugs with a loading density of 21% and 8.4%, respectively. The mean particle size of these micelles was 100 nm andwas not affected by drug loading. The use of lapatinib as an adjuvant sensitized drug resistant MCF-7/ADR cells to doxorubicintreatment. Cellular uptake studies showed enhanced doxorubicin accumulation in MCF-7/ADR cells in the presence of lapatinib.The doxorubicin and lapatinib combination therapy showed a significant decrease in tumor growth compared to doxorubicinmonotherapy. In conclusion, we have developed PEG-PBC micelle formulations for the delivery of doxorubicin and lapatinib.The combination therapy of doxorubicin plus lapatinib has a great potential for treating MDR breast cancer.

KEYWORDS: polymeric micelles, breast cancer, multidrug resistance, combination therapy, lapatinib, doxorubicin, nanomedicine

1. INTRODUCTION

Breast cancer is the most common cancer and the secondleading cause of cancer related death in women in the UnitedStates, with an estimated 232 340 new cases of invasive breastcancer and 39 620 deaths in 2013.1 Chemotherapy is animportant treatment method among currently availabletherapeutic approaches. Anthracyclines and taxanes are thefirst-line chemotherapeutic agents for advanced metastaticbreast cancer.2,3 However, the use of these agents inevitablyinduces drug resistance and results in the relapse of breastcancer. Breast cancer develops chemo-resistance via multiplemechanisms including overexpression of MDR transporters,defective cell apoptosis, and the presence of cancer stem cells(CSCs).4,5 MDR transporter overexpression is primarilyresponsible for drug resistance and eventually leads to thefailure of breast cancer therapy. P-glycoprotein (Pgp), breastcancer resistance protein (BCRP), and multiple drug resistanceprotein (MRPs) are major MDR transporters which increasedrug efflux and reduce drug accumulation in tumor cells.5 Anumber of clinical studies have demonstrated that chemo-therapy could induce overexpression of MDR transporters inbreast cancers and MDR transporter overexpression correlatedwith a worse response to the treatment.6−8 Due to the lack of

effective clinical interventions, patients with resistant breastcancers have a poor prognosis. Thus, there is an urgent need foreffective therapies to overcome MDR and treat resistant breastcancer.Inhibitors of drug efflux pump have been investigated in

clinical trials for MDR reversal and considered as a promisingsolution to overcome MDR in cancers.9,10 However, aformidable obstacle to clinical success is that the dose ofinhibitors for reversing MDR usually causes unacceptable sidetoxicity, because high concentrations are needed to effectivelyinhibit MDR transporters. Lapatinib (LAPA) is a dual kinaseinhibitor against epidermal growth factor receptor (EGFR) andhuman epidermal receptor two (HER-2). It has been approvedby the FDA since 2007 for use in combination withcapecitabine to treat HER-2 positive refractory breast cancerswhich have progressed following previous chemotherapy with

Special Issue: Drug Delivery and Reversal of MDR

Received: November 14, 2013Revised: January 7, 2014Accepted: January 9, 2014Published: January 9, 2014

Article

pubs.acs.org/molecularpharmaceutics

© 2014 American Chemical Society 2600 dx.doi.org/10.1021/mp400687w | Mol. Pharmaceutics 2014, 11, 2600−2611

pubs.acs.org/molecularpharmaceutics

anthracyclines, taxanes, and trastuzumab (Herceptin).11,12 Inaddition to the activities as an EGFR and HER-2 inhibitor,lapatinib has also shown inhibitory activities on MDRtransporters.13−15 Lapatinib inhibits MDR transporters througha direct interaction with the substrate-binding site and has beenreported for its effectiveness to overcome MDR.16−18 Mostimportantly, the safety of lapatinib has already been well-documented. Its recommended daily dose is 1500 mg formetastatic breast cancer. Therefore, lapatinib may be an idealdrug efflux inhibitor not only for its good safety at a high dosebut also for its intrinsic therapeutic effect targeting multipleanticancer mechanisms. In our previous studies, we have usedlapatinib and paclitaxel combination therapy to effectively treatresistant prostate cancers.18 Being encouraged by theseprevious studies, we have extended our research and developeda combination therapy using doxorubicin (DOX) and lapatinibto treat MDR breast cancers.In the current study, we developed a polymeric micelle for

improved delivery of the poorly water-soluble doxorubicin andlapatinib. A poly(ethylene glycol)-block-poly(2-methyl-2-ben-zoxycarbonylpropylene carbonate) (PEG-PBC) copolymer wassynthesized for preparing polymeric micelle formulations.Polymeric micelle is a promising drug delivery carrier that issuitable for the delivery of hydrophobic anticancer drugs, asdemonstrated in our or other previous studies.19−26 Poly-(ethylene glycol) (PEG)-based amphiphilic block copolymersself-assemble in aqueous solution to form core−shell structuredpolymeric micelles. The hydrophilic PEG block forms ahydrophilic shell surrounding the micelle. It provides aneffective steric stabilization and prevents the uptake of micellesby reticuloendothelial system (RES).27 The hydrophobic blockforms a hydrophobic inner core which serves as a drug loadingreservoir. Amphiphilic polymers assemble into micelles andload hydrophobic drugs through hydrophobic interaction,hydrogen binding, and electrostatic interaction.28−31 Polymericmicelles can efficiently encapsulate hydrophobic drugs andsignificantly improve the drug solubility. The poor solubility of

anticancer drugs is a big challenge for their delivery andformulation. Polymeric micelles can effectively address thisissue. Because of the small particle size and long circulation inblood, polymeric micelles can specifically deliver drugs intosolid tumors through the enhanced permeability and retention(EPR) effects (passive tumor targeting).32−34 Therefore, weused polymeric micelles for enhanced delivery of hydrophobiclapatinib and doxorubicin for treating MDR breast cancer(Scheme 1). Formulations were optimized and characterized.Then, both in vitro assays and in vivo studies were performed toevaluate anticancer activities of developed formulations onMDR breast cancer cells.

2. MATERIALS AND METHODS

2.1. Materials. Doxorubicin and lapatinib were purchasedfrom Melone Pharmaceutical Co., Ltd., China. 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU), methoxypoly(ethyleneglycol) (mPEG, Mn = 5000, PDI = 1.03), and other reagentswere obtained from Sigma Aldrich and used without furtherpurification. 2-Methyl-2-benzyloxycarbonyl propylene carbo-nate (MBC) was synthesized as described.35

2.2. Synthesis of Poly(ethylene glycol)-block-Poly(2-methyl-2-benzoxycarbonyl propylene carbonate) (PEG-PBC). PEG-PBC polymer was synthesized as previouslydescribed.20,36 Briefly, DBU (40 μL) was added to the mixtureof given amount of mPEG and MBC in 10 mL of anhydrousCH2Cl2 and reacted under stirring for 3 h. The reaction mixturewas then dissolved in CH2Cl2, precipitated with a large amountof ice-cold diethyl ether, and dried under vacuum at roomtemperature. Polymers were characterized by 1H nuclearmagnetic resonance (NMR) with a 400 MHz JEOL ECSNMR and deuterated chloroform (CDCl3) as a solvent. Themolecular weight and polydispersity index (PDI) of PEG-PBCpolymers were determined by gel permeation chromatography(GPC) using a Waters GPC system equipped with a GPCcolumn (Styragel HR 4E) and a differential refractive indexdetector. Tetrahydrofuran was used as an eluent at a flow rate

Scheme 1. Schematic Illustration of the Doxorubicin and Lapatinib Combination Nanomedicine for Treating Resistant BreastCancer

Molecular Pharmaceutics Article

dx.doi.org/10.1021/mp400687w | Mol. Pharmaceutics 2014, 11, 2600−26112601

of 0.35 mL/min. A series of narrow polystyrene standards (50−100 000 Da) were used for calibration. The critical micelleconcentration (CMC) was determined using pyrene as ahydrophobic fluorescent probe.19 Briefly, 40 μL of pyrene stocksolution (2.4 × 10−3 M) in acetone was added to 40 mL waterto prepare a saturated pyrene aqueous solution. Polymersamples were dispersed in water with concentration rangesfrom 0.5 to 4.8 × 10−7 mg/mL and mixed thoroughly withabove pyrene solution at a volume ratio of 1:1. The fluorescentintensity was recorded with a fluorescence spectrometer with Ex= 338 nm (I3) and 333 (I1) and Em = 390 nm. The intensityratio (I338/I333) was plotted against the logarithm of polymerconcentration. The CMC value was obtained as the point ofintersection of two tangents drawn to the curve at high and lowconcentrations, respectively.2.3. Preparation and Characterization of Micelles.

2.3.1. Preparation of Micelles. Polymeric micelles wereprepared with a film dispersion method as previously reportedwith some modifications.18−20 Briefly, 15 mg of polymer and agiven amount of doxorubicin or lapatinib were dissolved in 0.5mL of CHCl3, and then the solvent was removed underreduced pressure. The resulting film was hydrated in 3 mL ofPBS (pH 7.4) and sonicated for 1 min. Then, the residue freedrug was removed by centrifugation at 10 000 rpm for 5 min.The supernatant was filtered using a 0.22 μm filter. For thelyophiliztion, micelles were frozen overnight at −80 °C andlyophilized with a freeze-dry system (LABCONCO, USA).2.3.2. Drug Loading and Encapsulation Efficiency. To

determine doxorubicin loading in micelles, freeze-dried micellesamples were dissolved in dimethyl sulfoxide (DMSO), anddrug concentrations were determined with a fluorescencespectrometer (Ex: 485 nm, Em: 590 nm). Various concen-trations of doxorubicin dissolved in DMSO (0.39, 0.78, 1.56,3.125, and 6.25 μg/mL) were used to prepare the standardcurve of doxorubicin based on their fluorescent intensities. Todetermine lapatinib loading in micelles, freeze-dried sampleswere dissolved with methanol and drug concentrations weredetermined with a HPLC method (Agilent Technologies 1200series HPLC; DIKMA Suprsil-C 18 column; temperature 25°C; mobile phase: methanol/H2O 85:15, 1 mL/min). Variousconcentrations of lapatinib dissolved in methanol (3.125,6.25,12.5, 25, 50, and 100 μg/mL) were used to prepare thestandard curve. Drug loading and encapsulation efficiency werethen calculated using the following equations, respectively.

=

×

drug loading(%) (amount of loaded drug

/amount of polymer) 100%

=

×

encapsulation efficiency(%) (amount of loaded drug

/amount of drug added) 100%

=

×

theoretical loading(%) (amount of drug added

/amount of polymer) 100%

2.3.3. Morphology and Particle Size. The particle sizedistribution of micelles was determined by dynamic lightscattering (Malvern Nano ZS). The morphology of micelleswas determined with a transmission electron microscope usingan acceleration voltage of 100 kV. Micelle samples were loadedon a copper grid and stained with 0.2% (w/v) phosphotungsticacid solution before test using transmission electron micros-copy (TEM).

2.4. In Vitro Cytotoxicity. MCF-7 breast cancer cells anddrug resistant MCF-7/ADR breast cancer cells were used forthe in vitro cytotoxicitiy studies. MCF-7 and MCF-7/ADR cellswere obtained from the American Type Culture Collection(ATCC, Manassas, VA) and Keygen Biotech. Co., Ltd.(Nanjing, China), respectively. Cells were cultured in RPMI1640 media supplemented with 10% fetal bovine serum (FBS),100 U/mL penicillin G sodium, and 100 μg/mL streptomycinsulfate at 37 °C in humidified environment of 5% CO2.Doxorubicin (1 μg/mL) was added into the medium for MCF-7/ADR cells to maintain the drug resistance property. Cellswere seeded in 96-well plates at a density of 6000 cells per wellone day before treatment. Then, cells were treated withdifferent formulations for additional 48 h and followed by the3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT) assay or the lactate dehydrogenase (LDH) releasestudy. Multiple doxorubicin concentrations were tested in eachgroup including 0, 0.5, 1, 2, 4, 8, and 12 μM, respectively.During the optimization process, lapatinib at a dose of 10 μMor 15 μM was found to be able to effectively sensitize drugresistant MCF-7/ADR cells to DOX treatment, while notshowing significant cytotoxicity by lapatinib itself. Therefore,these two lapatinib concentrations were selected for combina-tion therapy.For MTT assay, cell culture medium was replaced with 200

μL of fresh medium at the end of treatment. Then, 20 μL ofMTT (5 mg/mL) solution was added and incubated for 4 h.The medium was carefully removed, and 200 μL of DMSO wasadded into each well to dissolve the formazan crystals. Theabsorbance was measured in a microplate reader (Thermo,USA) at a wavelength of 570 nm. The relative cell viability wascalculated with the following equation:

= −

− ×

cell viability(%) (OD OD )

/(OD OD ) 100%test DMSO

control DMSO

For the LDH release study, cell culture supernatant washarvested at the end of treatment and was analyzed with a LDHrelease assay kit (Beyotime, Shanghai, China). The releasedLDH were expressed as the percentage of LDH released in cellstreated with 1% Triton X-100.

2.5. Cellular Uptake Studies. Fluorescence Microscopy.The ability of micelles to transport doxorubicin into cells wasdetermined using a fluorescence inverted microscope (Olym-pus IX81, Ex: 488 nm, Em: 570 nm). Cells were seeded at adensity of 1 × 104 cells/well and cultured for 24 h. Aftertreating cells with different formulations for 4 h at 37 °C, mediawere removed, and PBS was added to rinse cells for three timesbefore observation with the microscope.

Flow Cytometry. Cells were plated at a density of 1 × 105

cells/well into 6-well microplates and cultured for 24 h. Then,cells were incubated at 37 °C for 4 h with different test articlesdiluted with fresh culture medium. At the end of treatment,cells were washed with PBS and digested with 0.25% trypsin.Then, cells resuspended in PBS were analyzed with a flowcytometer (BD Biosciences FACS Calibur, Germany). Thedoxorubicin inherent fluorescence was detected as FL-2 (488/585 nm).

2.6. In Vivo Anticancer Efficacy Studies. Animalexperiments were performed in accordance with the protocolapproved by Institutional Animal Care and Use Committee(IACUC) of Shanghai Institute of Materia Medica, ChineseAcademy of Sciences. Female BALB/c-nu nude mice (4−5



weeks old, 18−22 g) were housed under specific pathogen-freeconditions. Animals possessed continuous access to sterilizedfood pellets and distilled water in a 12 h light/dark cycle. All ofthe animals were in quarantine for a week before treatment.Tumor models were created by subcutaneously injecting

MCF-7/ADR cells (1 × 106 cells/mouse) into the back ofBALB/c-nu nude mice with a 25 gauge needle. When tumorsreached approximately 100 mm3, mice were randomly assignedto different treatment groups (n = 3). Different treatmentformulations were injected in the tail vain of these miceaccording the following groups: Group 1, PBS (control);Group 2, DOX solution (7.5 mg/kg/3 d); Group 3, DOXmicelles (7.5 mg/kg/3 d); Group 4, DOX micelles (7.5 mg/kg/3 d) + LAPA micelles (7.5 mg/kg/3 d). The tumor volume wasmeasured and calculated using the following formula:

= ×V W L( )/22

where W and L are the shortest and longest diameters,respectively.The body weight of mice was also monitored during the

course of the study. At day 21, mice were killed, and tumorswere immediately harvested and weighed. In addition, majororgans including heart, spleen, and kidney were harvested andsubjected to a histological examination after H&E staining.

3. RESULTS

3.1. Synthesis and Characterization of Polymers. PEG-PBC copolymer was synthesized using DBU as a catalyst whichgives a high conversion rate of monomer.20,37 The PEG-PBCpolymer chemical structure was confirmed by 1H NMRspectrum (Figure 1A), with the following characteristic peaks:δ 1.2 (CH3 in BC unit), δ 3.6 (CH2 in PEG), δ 4.3 (CH2 in BCmain chain), δ 5.2 (CH2 in BC side group), and δ 7.3 (phenylring). All signals are assigned to methoxy poly(ethylene glycol)(mPEG) and polymerized BC units. The molecular weight ofthe PBC block of the polymer was 7000, as estimated based onthe peak areas of PEG CH2 groups at δ 3.6 and those of CH2 inBC main chain at δ 4.3. We also characterized PEG-PBCpolymer with GPC, which indicated a narrow molecular weightdistribution with a PDI of 1.39 (Figure 1B). The molecularweights determined by GPC were Mp 10812, Mw 8686, and Mn6268, respectively. The low CMC value (1.5 × 10−3 g/L)indicated the good stability of PEG-PBC micelles (Figure 1C).3.2. Preparation and Characterization of Micelles.

3.2.1. Drug Loading Efficiency. To determine the ability ofPEG-PBC to load doxorubicin (DOX) and lapatinib (LAPA),the loading efficiencies of these two drugs were calculated usingthe equations described in the experimental methods. Forlapatinib, when we increase the theoretical loading from 5% to25% (w/w), the drug solubility increased accordingly from 0.5to 2.1 mg/mL, with loading efficiency decreased slightly from98% to 84%. The solubility of lapatinib increased from 1.9 to5.1 mg/mL, when the polymer concentration was increasedfrom 5 to 25 mg/mL (Figure 2). Similar studies were alsoperformed for doxorubicin loaded PEG-PBC micelles. Doxor-ubicin solubility increased from 0.3 to 0.85 mg/mL when thetheoretical loading increased from 3% to 10%, with drugloading efficiency decreased from 97.4% to 94.8%. Whenpolymer concentration increased from 10 to 30 mg/mL, theconcentration of doxorubicin was increased accordingly from0.7 to 2.1 mg/mL with a loading efficiency above 95% in allgroups (Figure 3).

3.2.2. Morphology and Particle Size. The mean particle sizeof various micelle formulations determined with dynamic light

Figure 1. Characterization of PEG-PBC polymer. (A) 1H NMRspectrum and chemical structure of PEG-PBC polymer. (B) GPCchromatogram. (C) Critical micelle concentration.

Figure 2. Optimization of lapatinib-loaded PEG-PBC micelleformulations. (A) Effect of theoretical drug loading. The polymerconcentration was 10 mg/mL. (B) Effect of polymer concentration.The theoretical loading was 20%.



scattering (DLS) was around 100 nm, and there was littlechange in particle size due to the drug loading (data notshown). The particle size and morphology of these micelleswere also determined using a transmission electron microscopy(TEM); TEM results showed that these micelles were sphericaland had a narrow size distribution with a mean number averagediameter less than 50 nm (Figure 4), which was lower than theZ-average diameters obtained from DLS. This was consistentwith the previous report that the smaller particle size wasobserved with TEM than with DLS.35 The loading ofdoxorubicin or lapatinib did not cause any significant changein morphology and particle size (Figure 4). In addition, we alsomeasured the effects lyophilization/reconstitution process onthe change of particle sizes. Results demonstrated that thelyophilization/reconstitution process did not lead to significantchange in particle size of blank micelles as well as drug-loadedmicelles (data not shown).

3.3. In Vitro Cytotoxicity. The cytotoxicity of various drugformulations was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. For MCF-7cells, there was no significant difference in cytotoxicity amongfree doxorubicin, doxorubicin micelle, doxorubicin + lapatinib(10 μM) micelle, and doxorubicin + lapatinib (15 μM) micelle(Figure 5A). The IC50 was 1.2 μM doxorubicin for all four

tested groups. The results indicated no enhanced internal-ization effect and no cytotoxic effect of lapatinib on cells underthe test concentration. MCF-7/ADR cells displayed strong

Figure 3. Optimization of doxorubicin-loaded PEG-PBC micelleformulations. (A) Effect of theoretical drug loading. The polymerconcentration was 10 mg/mL. (B) Effect of polymer concentration.The theoretical loading is 7.5%.

Figure 4. TEM images of PEG-PBC micelles.

Figure 5. MTT assay. Viability of MCF-7 cells (A) and MCF-7/ADRcells (B) after treatment with free DOX, DOX micelles, and DOX +LAPA micelles for 48 h. (C) The effects of blank PEG-PBC micelletreatment on the viability of MCF-7 and MCF-7/ADR cells. Nosignificant difference was demonstrated between DOX + LAPA (10μM) and DOX + LAPA (15 μM) groups.



resistance to free doxorubicin, leaving it virtually ineffectiveeven with a high dose up to 12 μM. However, PEG-PBCmicelles formulated doxorubicin exhibited improved activitycompared to free doxorubicin, indicating its ability to overcomeMDR. Notably, micelle-formulated doxorubicin plus 10 μM or15 μM of lapatinib combination greatly increased thecytotoxicity. IC50 was 2 μM doxorubicin for these twocombination therapy groups. The anticancer efficacy wascomparable to that of sensitive MCF-7 cells, indicating theability of lapatinib to sensitize drug resistant MCF-7/ADR cellsand reverse drug resistance. Since lapatinib alone at 10 μM or15 μM had no cytotoxicity on MCF-7/ADR cells, the observedanticancer efficacy in combination therapy might be attributedto the inhibition of MDR transporters rather than the inhibitionof HER-2 or EGFR kinases (Figure 5B). There was nosignificant difference between the two LAPA + DOX groupswith LAPA concentrations of 10 and 15 μM. No significantcytotoxicity was observed at PEG-PBC polymer concentrationup to 1 mg/mL on both MCF-7 and MCF-7/ADR cells,suggesting its good biocompatibility (Figure 5C).Furthermore, LDH release study was used to verify the in

vitro anticancer activities. The results correlated well with theMTT assay data. In MCF-7 cells, the treatment of doxorubicininduced a concentration-dependent increase of LDH release,displaying no significant difference among different groups(Figure 6A). As expected, the free doxorubicin induced LDH

release was significantly deceased in MCF-7/ADR cells.Doxorubicin micelles also showed a higher level of LDHrelease than free drugs. The combination of doxorubicin andlapatinib micelles showed highest activity among these threegroups. No significant difference was observed between twocombination therapy groups with different lapatinib concen-trations (10 μM vs 15 μM, Figure 6B), indicating 10 μM was a

sufficient dose for overcoming MDR. These in vitro cytotoxicityresults revealed that micelle formulated doxorubicin andlapatinib when used as a combination therapy can effectivelyovercome MDR in breast cancer cells.

3.4. In Vitro Cellular Uptake Studies. To elucidate themechanism that doxorubicin and lapatinib loaded micellesovercomes MDR, the cellular uptake of doxorubicin by MCF-7and MCF-7/ADR was determined. Cells were treated with freedoxorubicin, doxorubicin micelle (8 μM), and doxorubicin (8μM) + lapatinib (10 μM) micelles. In MCF-7 cells, high levelsof cellular uptake of doxorubicin were achieved in all of thethree treatment groups with 91.8% positive cells in freedoxorubicin group, 99.3% in doxorubicin micelle group, and99.8% in doxorubicin (8 μM) + lapatinib (10 μM) micellegroup, respectively (Figure 7A,B). In MCF-7/ADR cells, due tothe overexpression of Pgp, almost no intracellular accumulationof doxorubicin was observed in the free doxorubicin treatedgroup (1.6% positive cells). The micelle formulation signifi-cantly enhanced intracellular accumulation of doxorubicin asdemonstrated by higher percentage of positive cells than that offree drug treated group. The addition of lapatinib (10 μM)further increased the percentage of positive cells in MCF-7/ADR cells (Figure 7A,B).

3.5. In Vivo Anticancer Studies. After characterizing theanticancer activities of the lapatinib and doxorubicin micellecombination therapy with in vitro studies, we moved forward totest their anticancer efficacies with an in vivo animal tumormodel. Drug-resistant MCF-7/ADR cells were used to establishthe tumor model. Though inhibition of tumor growth wasachieved by both doxorubicin free drug and micelleformulations compared to the PBS control group, combinationtherapy with doxorubicin and lapatinib micelles showed themost potent anticancer efficacy. The inhibition of tumor growthwas significantly enhanced in combination therapy group incomparison to the doxorubicin monotherapy groups. Thetumor growth in the combination therapy group wascompletely arrested (Figure 8A). Tumor samples werecollected at the end of study and weighted (Figure 8B,C). Allof the groups that received chemotherapeutic agent showedsignificantly less tumor weight than the PBS control group. Thetumor weight was the smallest in the doxorubicin plus lapatinibmicelle combination therapy group, which was significantly lessthan those in doxorubicin monotherapy groups. The change inbody weight was also measured for preliminarily evaluation ofthe in vivo safety and tolerance (Figure 8D). The treatmentwith doxorubicin free drug showed most severe toxicity asindicated by the significant loss of body weight during thecourse of study. By contrast, neither micelle formulateddoxorubicin nor doxorubicin plus lapatinib combinationinduced body weight loss. Moreover, the histologicalexamination was found that the micelles did not cause obviouschanges in the major organs (spleen, kidney, and heart),whereas the free DOX induced severe damage in spleen andkidney (Figure 9), suggesting the reduced toxicity and bettertolerance of the micelle-based combination therapy.

4. DISCUSSIONDue to the significance of MDR in cancer therapy, many effortshave been made in developing different strategies to reverseMDR in cancers, which include the use of MDR transporterinhibitors as the adjuvants to sensitize resistant cancer cells tochemotherapy,18,38 the development of anticancer drugs whichare not substrates for known MDR transporters,19,24 and the

Figure 6. LDH release study. The release of LDH from (A) MCF-7cells and (B) MCF-7/ADR cells treated with different formulations for48 h. No significant difference was demonstrated between DOX +LAPA (10 μM) and DOX + LAPA (15 μM) groups.



inhibition of MDR transporters with gene silencing ap-proach.39,40 The use of MDR transporter inhibitors to reverseMDR in cancer therapy has attracted great interest. Pgp arewidely distributed in normal human tissues and cells, and theyare responsible for protection of vital tissue or cells (e.g., centralnervous system, bone marrow stem cells). The inhibition ofPgp will result in high chemotherapeutic agent concentration inthese vulnerable tissues or cells and cause toxicities. Therefore,many treatment protocols using MDR inhibitors, thoughdelayed the growth of tumors, failed to improve the overallsurvival rate of patients due to the severe toxicities.In the current study, a combination therapy of lapatinib and

doxorubicin was developed for the treatment of MDR breastcancers. Our results demonstrated that lapatinib significantlyincreased anticancer activities of doxorubicin on MCF-7/ADRMDR breast cancer cells. In contrast, lapatinib did not changethe cytotoxicity of doxorubicin on MCF-7 cells (Figure 5). Thetreatment with lapatinib alone showed no effects on both MCF-7 and MCF-7/ADR cells at the tested concentrations. Thisobservation is consistent with our previous studies wherenontoxic concentrations of lapatinib enhanced cytotoxicty of

paclitaxel on MDR prostate cancer cells.18 Several other studieshave also identified lapatinib as an inhibitor for MDRtransporters.13,14,16,18,41 Lapatinib as well as many othertyrosine kinase inhibitors (e.g., nilotinib, apatinib, and erlotinib)are modulators of ATP-binding cassette MDR transport-ers.42,14,43 Lapatinib can inhibit both Pgp and BCRP by directbinding to their ATP-binding site and minimize the efflux ofchemotherapeutic agents which are the substrates of MDRtransporters.13 Due to the inhibitory activity of lapatinib onPgp, lapatinib can enhance the intracellular accumulation ofdoxorubicin in MDR cancer cells and thus reverse drugresistance and enhance cytotoxicity. To further confirm themechanism that lapatinib overcomes MDR, we determined theeffect of lapatinib treatment on the intracellular accumulation ofdoxorubicin in drug-resistant MCF-7/ADR cells. The intra-cellular accumulation of doxorubicin was determined with thefluorescence microscopy and the flow cytometry (Figure 7). Alow level of intracellular accumulation of doxorubicin wasobserved in MCF-7/ADR cells when cells were treated withfree doxorubicin alone. The low intracellular drug concen-tration was a major reason that MCF-7/ADR cells were

Figure 7. Cellular uptake of DOX on MCF-7 and MCF-7/ADR cells. (A) Fluorescence microscopy; (B) flow cytometry analysis of MCF-7 cellstreated with free DOX (8 μM); DOX micelles (8 μM); DOX micelles (8 μM) + LAPA micelles (10 μM); and (C) flow cytometry analysis of MCF-7/ADR cells treated with free DOX (8 μM); DOX micelles (8 μM); DOX micelles (8 μM) + LAPA micelles (10 μM).



resistant to doxorubicin monotherapy (Figures 5 and 7). Theuse of lapatinib significantly increased the intracellularaccumulation of doxorubicin in MCF-7/ADR cells. This isconsistent with our previous studies, which showed thatlapatinib inhibited Pgp mediated drug efflux but had no effecton Pgp mRNA or protein levels.18 In summary, lapatinibovercomes MDR by directly inhibiting the activities of MDRtransporters, thus reducing drug efflux and increasing theintracellular accumulation of doxorubicin in resistant cancercells.Currently, lapatinib has been used in combination therapy

for treating HER-2 positive breast cancer cells.11,12 Manycombination therapy protocols have been developed withlapatinib plus other therapeutic agents to treat refractory breastcancers.44,45 Lapatinib and other chemotherapeutic agents workon different mechanisms or targets which may enhance the

anticancer activities through either synergistic or additiveeffects. The overexpression of EGFR and HER-2 are associatedwith a higher risk of breast cancer recurrence and with poorclinical outcomes. Lapatinib can inhibit these receptor mediatedbreast cancer growth and significantly improve the clinicaloutcomes.46−48 It was noted that the combination of lapatiniband doxorubicin in our study did not further enhance thecytotoxcicity in nonresistant MCF-7 cells. The use of lapatinibalone did not have any anticancer effects on nonresistant MCF-7 or resistant MCF-7/ADR cells. These results are expectedbecause MCF-7 cells are HER-2 negative breast cancer cells.However, we expect to have additional anticancer effects inHER-2 positive breast cancers by using lapatinib as an adjuvantin chemotherapy. Lapatinib will enhance the anticancer efficacythrough two distinctive mechanisms: (1) inhibit HER-2receptor mediated tumor growth; and (2) reverse MDR

Figure 8. In vivo anticancer efficacy study. (A) Change of tumor sizes in MCF-7/ADR tumor-bearing nude mice received intravenous injection ofdifferent formulations; ** P < 0.01, * P < 0.05, compared with the PBS control; + P < 0.05, compared with DOX monotherapy (free drugs or micelleformulations); (B) tumor size and (C) tumor weight measured at the end of study (21 days post the initiation of treatment). ** P < 0.01, comparedwith the PBS control; ++ P < 0.01, compared with DOX + LAPA micelle combination. (D) Change of body weight in MCF-7/ADR tumor-bearingmice during the course of study. ** P < 0.01, * P < 0.05, compared with the PBS control; # P < 0.05, compared with DOX micelles; + P < 0.05,compared with DOX + LAPA micelle combination. Points are presented as mean ± SD (n = 3).



transporter mediated drug resistance. Due to the highprevalence of MDR and HER-2 overexpression in advancedbreast cancers as well as their association with the aggressivecancer phenotype, the use of lapatinib as an adjuvant inchemotherapy will be a promising approach for treatingrefractory breast cancers.Doxorubicin was selected in this study as a chemotherapeutic

agent in combination with lapatinib. Doxorubicin is a first linedrug for monotherapy or combination therapy to treat breastcancers in the clinics. The clinical use of doxorubicin isassociated with toxic side effects. The situation becomes worsein patients with resistant breast cancers because a higher dose isneeded to control the tumor growth, which may lead to moresevere toxicities and side effects. A recent research reported thatlapatinib strongly potentiated the cardiac toxicity of doxor-ubicin due to the inhibition of doxorubicin efflux in myocytes.49

Therefore, it is important to specifically deliver anticancer drugsand MDR inhibitors into tumor and minimize their exposure innormal tissues or organs. Nanocarriers can efficiently deliverdrugs into tumors and thus have a great potential to be used asdelivery systems for MDR inhibitor and chemotherapy drugcombination therapy. In this study, PEG-PBC polymericmicelles were used for the delivery of lapatinib and doxorubicin.This polymer formed a stable micelle with a low critical micelleconcentration (Figure 1). The stability of micelles is critical fordrug retention and in vivo drug delivery. The benzyl moietieslocated at the hydrophobic block of PEG-PBC polymer arebelieved to stabilize the micelles by enhancing hydrophobicinteraction. The benzyl ring may also interact with doxorubicinthrough π−π interaction which will stabilize micelle as well asprotect the encapsulated drugs from degradation.50 Theprepared micelle formulations had a mean particle size of 100nm, which is suitable for enhancing drug delivery into tumorsvia passive tumor targeting. Doxorubicin micelle monotherapy

and lapatinib plus doxorubicin micelle combination therapywere well-tolerated and did not show significant systemictoxicity (Figure 8). This result is consistent with previousclinical studies using pegylated liposomal formulation ofdoxorubicin (PLD) plus oral lapatinib. Patients received thattreatment showed low toxicities and no treatment-relatedcardiac toxicity.51

We observed that doxorubicin loaded PEG-PBC micellesshowed greater anticancer activities than free doxorubicin indrug resistant MCF-7/ADR cells (Figure 5). Our results alsodemonstrated that doxorubicin micelles induced more LDHrelease in MCF-7/ADR cells (Figure 6). The cellular uptakestudies showed that MCF-7/ADR cells treated with doxor-ubicin micelles had a higher intracellular drug concentrationthan cells treated with doxorubicin free drugs (Figure 7), whichmight be a reason that PEG-PBC micelles enhanced anticancereffects of doxorubicin in MCF-7/ADR cells. The enhancedintracellular accumulation of doxorubicin is possibly due to theinhibition of Pgp by the PEG-PBC polymer or due to thealteration of cellular uptake route.52 The PEG-PBC polymerused in our study is an amphiphilic molecule. Many amphiphilicmolecules have been reported to be able to sensitize resistantcancer cells to chemotherapy. These amphiphilic molecules(e.g., pluronics, polyoxyl 15 hydroxylstearate <solutol HS15>,polyoxyethylene 20 stearyl ether <Brij 78>, and vitamin ETPGS) can deplete intracellular ATPs and inhibit MDR drugefflux transporters.53−56 These molecules may induce thechange of mitochondrial membrane permeability, cause the lossof the membrane polarity, and lead to the depletion of ATPs.The enhanced drug accumulation and reversal of MDR mayalso due to the alteration in the route of cellular uptake and thechange of subcellular localization of drugs encapsulated inmicelles. Free drugs enter cells through simple diffusion and aresensitive to the effects of drug efflux transporters. In contrast,

Figure 9. Histological examination of heart, spleen, and kidney after treatment. The micelles caused little changes in the organs, but the free DOXinduced severe toxicity (shrinking white pulp in the spleen and renal tubular necrosis).



micelle encapsulated drugs enter cells through endocytosiswhich transports drugs into perinuclear regions where drugs areless susceptible to the effects of efflux transporters which arelocated in the cell membranes.52 This hypothesis has beeninvestigated in several research papers, which have demon-strated the abilities of nanocarriers to reverse MDR incancers.57−59

PEG-PBC micelles could efficiently load both lapatinib anddoxorubicin. The solubility of these two drugs was significantlyimproved when PEG-PBC micelles were used to formulatethem. Lapatinib showed higher drug loading in PEG-PBCmicelles than doxorubicin (Figures 3 and 4). This was due tothe difference in the chemical structures of lapatinib anddoxorubicin, which leads to different compatibility of these twodrugs with PEG-PBC polymers. The compatibility between adrug and a polymer (i.e., the miscibility or interaction betweena drug and a polymer) is a key factor that determines the drugloading efficiency, stability, and drug release. Our previousstudies have demonstrated the influence of polymer/drugcompatibility on drug loading efficiency and release pro-files.20,35 The drug/polymer compatibility and its relevancewith drug loading could be studied by various methodsincluding Flory−Huggins interaction parameters and moleculardynamics.60−62 Because of the difference in drug chemicalstructures, it is almost impossible to design a single polymer asa universal delivery carrier which will have high drug loading forall drugs. For combination therapy, the polymer used toformulate multiple drugs will not have the identical loadingefficiency for different drugs. However, the selected polymershould have satisfactory loading efficiency for all the drugs inthe combination therapy.In conclusion, PEG-PBC polymeric micelle formulations

have been developed for the delivery of lapatinib anddoxorubicin as a combination therapy to treat resistant breastcancers. The use of lapatinib as an adjuvant significantlyincreased anticancer effects of doxorubicin in resistant breastcancer cells. The reversal of MDR was mainly due to theinhibition of MDR transporters by lapatinib. The combinationtherapy formulation was also evaluated with an in vivo animaltumor model and showed an inhibition of resistant breastcancer cells as well as a good tolerance in animals. These novelformulations have a great potential for treating resistant breastcancers.

■ AUTHOR INFORMATIONCorresponding Authors*Hampton University, School of Pharmacy, Kittrell Hall RM216, Hampton, VA 23668. E-mail: [email protected]. Tel.:(757) 727-5585.*Shanghai Institute of Materia Medica, Chinese Academy ofSciences, 501 Hai-ke Rd, Shanghai 201203, China. E-mail:[email protected]. Tel.: + 86 21 20231000, ext 1401.

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis work was supported by the following grants: NationalBasic Research Program of China (973 Program2013CB932503, 2014CB931900), NSFC, China (81172996,81373357), and Hampton University Faculty Research Award(F.L.). This work was also supported in part by the ChinesePostdoctoral Science Foundation (2012M510097,

2013T60478) and Shanghai Pu-jiang Scholar Program(11PJ1411800). We would also thank Dr. Godman NwoKoguat Hampton University for help with the NMR studies; Mrs.Dundee Schettino and Dr. Mike Eitcher at Waters for help withthe GPC characterization of polymers.

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mailto:[email protected]

http://www.cancer.gov/cancertopics/druginfo/fda-lapatinib#Anchor-HER6789

http://www.cancer.gov/cancertopics/druginfo/fda-lapatinib#Anchor-HER6789

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