Rationally Designed Aberrant Kinase-Targeted Endogenous ProteinNanomedicine against Oncogene Mutated/Amplified RefractoryChronic Myeloid LeukemiaArchana P. Retnakumari,† Prasanna Lakshmi Hanumanthu,† Giridharan L. Malarvizhi,†

Raghuveer Prabhu,‡ Neeraj Sidharthan,‡ Madhavan V. Thampi,§ Deepthy Menon,† Ullas Mony,†

Krishnakumar Menon,† Pavithran Keechilat,‡ Shantikumar Nair,† and Manzoor Koyakutty*,†

†Amrita Center for Nanosciences and Molecular Medicine, ‡Department of Medical Oncology, and §Department of HumanCytogenetics, Amrita Institute of Medical Science and Research Center, Amrita Vishwavidyapeetham University, Cochin, India

*S Supporting Information

ABSTRACT: Deregulated protein kinases play a very critical role in tumorigenesis,metastasis, and drug resistance of cancer. Although molecularly targeted smallmolecule kinase inhibitors (SMI) are effective against many types of cancer, pointmutations in the kinase domain impart drug resistance, a major challenge in the clinic.A classic example is chronic myeloid leukemia (CML) caused by BCR-ABL fusionprotein, wherein a BCR-ABL kinase inhibitor, imatinib (IM), was highly successful inthe early chronic phase of the disease, but failed in the advanced stages due toamplification of oncogene or point mutations in the drug-binding site of kinasedomain. Here, by identifying critical molecular pathways responsible for the drug-resistance in refractory CML patient samples and a model cell line, we have rationallydesigned an endogenous protein nanomedicine targeted to both cell surface receptorsand aberrantly activated secondary kinase in the oncogenic network. Moleculardiagnosis revealed that, in addition to point mutations and amplification of oncogenicBCR-ABL kinase, relapsed/refractory patients exhibited significant activation ofSTAT5 signaling with correlative overexpression of transferrin receptors (TfR) on the cell membrane. Accordingly, we havedeveloped a human serum albumin (HSA) based nanomedicine, loaded with STAT5 inhibitor (sorafenib), and surfaceconjugated the same with holo-transferrin (Tf) ligands for TfR specific delivery. This dual-targeted “transferrin conjugatedalbumin bound sorafenib” nanomedicine (Tf−nAlb-Soraf), prepared using aqueous nanoprecipitation method, displayed uniformspherical morphology with average size of ∼150 nm and drug encapsulation efficiency of ∼74%. TfR specific uptake andenhanced antileukemic activity of the nanomedicine was found maximum in the most drug resistant patient sample having thehighest level of STAT5 and TfR expression, thereby confirming the accuracy of our rational design and potential of dual-targetingapproach. The nanomedicine induced downregulation of key survival pathways such as pSTAT5 and antiapoptotic protein MCL-1 was demonstrated using immunoblotting. This study reveals that, by implementing molecular diagnosis, personalizednanomedicines can be rationally designed and nanoengineered by imparting therapeutic functionality to endogenous proteins toovercome clinically important challenges like molecular drug resistance.

KEYWORDS: chronic myeloid leukemia, drug resistance, targeted nanomedicine, rational design, albumin, transferrin

1. INTRODUCTION

Genomic and proteomic approaches have identified many levelsof intimately cross-linked and complex aberrant protein kinasesresponsible for the uncontrolled proliferation, metastasis,invasion, and drug resistance of cancer.1,2 In the past decade,one of the major breakthroughs in the area of cancer therapywas the invention of small molecule drugs that can specificallyinhibit aberrant protein kinases through various mechanisms.These molecularly targeted tyrosine kinase inhibitors (TKIs)were greatly beneficial compared to the conventional DNAintercalating agents because of their high specificity to aparticular kinase and thus remain nontoxic to healthy cells.2

Among many clinically approved TKIs, imatinib (IM) targetingBCR-ABL fusion protein in chronic myeloid leukemia (CML),

erlotinib and gefitinib targeting epidermal growth factorreceptors (EGFR), everolimus, and rapamycin targetingmammalian target of rapamycin (mTOR) are some of themost studied and clinically used small molecule inhibitors.However, despite promising initial response, one of the criticalchallenges faced by these molecules was the development ofpoint mutations in the kinase domain where the drug issupposed to bind, leading to drug resistance and relapse as seenin CML.3,4 CML is a hematological malignancy caused by

Received: April 2, 2012Revised: August 23, 2012Accepted: September 12, 2012

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Philadelphia chromosome (Ph) translocation t (9:22).5 Thistranslocation generates a constitutively active fusion tyrosinekinase BCR-ABL, leading to uncontrolled proliferation ofmyeloid progenitor cells.6 IM was the first TKI approvedagainst BCR-ABL fusion and was found highly effective againstchronic phase CML, but later failed in the advanced stages ofblast crisis due to amplification of BCR-ABL oncogene oracquired mutations in the tyrosine kinase domain.7,8 The mostcritical mutation was T315I (tyrosine → isoleucine at 315position), a “gate-keeper” mutation that offers steric hindranceto the drug binding at the BCR-ABL kinase.9 In certain cases,even the second-generation small molecule drugs such asdasatinib (DS) and nilotinib (NL) were also found ineffectivedue to a multitude of reasons.10

In addition to the mutations or amplification of BCR-ABLgene, activation of alternate survival kinases was also foundresponsible for TKI resistance.11 Among the prominent survivalpathways, signal transducer and activator of transcription(STAT5), mitogen-activated protein kinase-1/2 (MEK1/2),and extracellular signal-regulated kinase-1/2 (ERK1/2) collec-tively confer CML cells a survival advantage.12 Hence, theclinical condition of IM/DS/NL resistance in CML demandsurgent need for the development of alternative therapies forthis disease.13 One of the possible approaches is to identify thecritical secondary survival kinase in the oncogenic network andtarget the same using personalized therapeutic interven-tions.14,15 However, such pathways may also be important forthe functioning of normal healthy cells. Therefore, thechallenge is to specifically inhibit these kinases mainly in therefractory cancer cells without causing serious damage tohealthy cells. This demands the cell-specific targeted delivery ofmolecularly targeted kinase inhibitors. Effectively, this neces-sitates “dual-targeting” of both specific receptors in cancer cellsand their intracellular aberrant kinases.Nanotechnology offers versatile platforms for the targeted

delivery of chemotherapeutic agents, particularly for cancertherapy. However, most of the current translational cancernanomedicine approaches were limited to the theme ofimproving the bioavailability of cytotoxic chemo drugs andreducing their toxic side effects. However, the potential use ofnanomedicines to rationally target aberrant cancer kinome bycell-specific delivery of nanoencapsulated small molecule drugsremains less explored. One of the most clinically successfulexamples of the first category is “albumin-bound-paclitaxel”(Abraxane), which is used for treating metastatic breast cancerand currently under clinical trial for many other solid tumors.16

A key aspect of this formulation was the use of human serumalbumin as the drug-delivery system. Endogenous proteins likealbumin possess great advantages over polymeric nanoparticlesdue to their excellent biocompatibility, degradability, non-immunogenicity, and ease of conjugation with other targetingligands. Considering this, we have selected serum albumin as anideal nanocarrier for implementing our “dual targetingapproach” against oncogene mutated/amplified refractoryleukemia. Since albumin is an integral constituent of bloodserum, it is highly nonimmunogenic and biocompatible, andhence best suitable for drug delivery to leukemia cells.17

In this paper, based on the extensive molecular character-ization of IM refractory/relapsed CML patient samples and amodel cell line, we have identified that, in addition to theacquired point mutations in the BCR-ABL kinase domain andamplification of the oncogene, resistant phenotype possessespreferentially activated secondary survival kinase, STAT5, in the

downstream of BCR-ABL. In addition, the resistant phenotypealso showed an interesting correlative overexpression of a cell-surface receptor, transferrin (TfR), which is a transcriptionaltarget of STAT5. Based on this lead information, we haverationally designed an endogenous protein (HSA) nano-medicine to inhibit phosphorylation of STAT5 by deliveringanti-STAT5 small molecules through the overexpressed TfRpresent on the surface of refractory CML cells. This “cellreceptor plus kinase targeted” (dual-targeted) protein nano-medicine efficiently induced apoptosis in most resistant patientsamples and refractory cell line, without causing any major sideeffects such as hemolysis, inflammatory response, or toxicity toprimary blood cells.

2. EXPERIMENTAL SECTION

2.1. Cell Culture. K562 CML cell line was purchased fromNCCS. Cells were cultured and maintained in RPMI 1640(Sigma Aldrich, USA) supplemented with 10% FBS (Gibco,USA), 1% L-glutamine (Sigma Aldrich, USA), and 1%penicillin/streptomycin (Gibco, USA). Cells were maintainedat 37 °C, under 85% relative humidity and 5% CO2.

2.2. Development of IM/DS Resistant CML Cells. IMand DS were purchased from LC Laboratories (Woburn, MA).The drug stocks were prepared in DMSO, and aliquots werepreserved at −20 °C. Cells maintained in liquid culture, asmentioned above, were gradually exposed to increasingconcentrations of IM and DS at a rate of 0.1 μmol/Lincrements every 10 days of culture. After three months, livecells were subcultured and maintained under culture con-ditions. The cells were further characterized for IM and DSresistance. The drug sensitive K562 cells are represented asK562S and the resistant cells as K562R.

2.3. Characterization of IM/DS Resistant K562R Cells.2.3.1. MTT Cell Viability Assay. Cell viability assay wasperformed using MTT, (Sigma Aldrich, USA), which measuresthe number of viable cells. 1 × 104 cells seeded in a 96-welltissue culture plate, incubated overnight under cultureconditions, were exposed to respective concentrations of IMand DS (0−10 μM) for 48 h. Cell viability was determined bytetrazolium conversion to formazan. The solubilization offormazan crystals was carried out using solubilization buffer,containing 10% Triton X-100 (Sigma Aldrich, USA), 0.1 MHCl (Qualigens, India) in isopropanol (Qualigens, India) andabsorbance of solubilized formazan at 560 nm was measuredusing an automated microplate reader (Biotex power Wave XSmodel, USA).

2.3.2. Flow Cytometric Analysis of Drug Efflux Protein.FITC conjugated mAb against human P-gp (BD Biosciences,USA) was used to determine the expression of P-gp drug effluxprotein. Briefly 1 × 105 cells were collected, washed withphosphate buffered saline (PBS, pH 7.4), and stained withantibody according to the manufacturer’s instructions. After theincubation period, cells were washed to remove the unboundantibody and flow cytometric analysis was performed (FACSAria II, USA).

2.3.3. FISH Analysis for BCR-ABL Amplification. FISH wasperformed on interphase nuclei using LSI BCR probe labeledwith Spectrum Green (22q11.2) and LSI ABL probe labeledwith Spectrum Orange (9q34) Dual Color, Dual Fusion Probe(Vysis, Downers Grove, IL), according to the manufacturer’sinstructions. Two hundred interphase nuclei were analyzed, andthe number of fusion signals/cell was enumerated.

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2.3.4. Gene Expression Studies Using RT-PCR. Theactivation of alternative signaling pathways of cell survival wasanalyzed by gene-expression studies using qRT-PCR. TotalRNA from ∼5 × 105 cells was isolated using RNAqueous MicroRNA isolation kit (AMBION, USA). The concentration andpurity of resulting RNA were estimated at 260 and 280 nmusing the Nanodrop spectrophotometer Nd-1000 (ThermoScientific, USA), and only those samples with A260/A280 ratiosbetween 1.9 and 2.1 were considered further. The isolated RNAwas reversely transcribed into cDNA with random hexamersusing high capacity cDNA Reverse Transcription kit (AppliedBiosystems, Fostercity, CA, USA). The relative expression ofthe above-mentioned genes was evaluated by qRT-PCR in ABIPrism 7900 HT qRT-PCR system (Applied Biosystems, FosterCity, CA, USA). The reaction mixtures were prepared intriplicate in a final volume of 20 μL using TaqMan UniversalPCR Master Mix and TaqMan gene expression Assays-on-Demand Products (Applied Biosystems Foster City, CA, USA).50 ng of cDNA was used for the real time reaction. The geneexpression profile of BCR-ABL, STAT5A, PI3K, and Akt wasanalyzed using GAPDH as the endogenous control. cDNA fromK562S cells was used as the control.2.3.5. Immunostaining for TfR Expression. Cell surface

expression of TfR on K562R cells was analyzed using flowcytometry and confocal microscopy. Human TfR mAbconjugated to APC from R&D systems, USA, was used forTfR immunostaining. 1 × 105 cells were counted, washed, andincubated with 20 μL of antibody for 30−45 min at 2−8 °C.Later, the cells were washed and resuspended in PBS pH 7.4supplemented with 0.5% bovine serum albumin (BSA) for flowcytometric reading (FACS Aria II, USA). The analysis wasperformed using FACS Diva software. The fluorescence wasdetected using 633 nm excitation and emission was collectedusing 660/20 nm band-pass filter. For confocal microscopy, thecells were stained with the human TfR-APC mAb as describedearlier. The cells were washed, resuspended and plated on polyL-lysine (PLL, Sigma Aldrich USA) coated coverslips, andobserved under microscope. The fluorescence was detectedusing 633 nm laser excitation.2.4. Isolation and Characterization of Patient Derived

Leukemic Mononuclear Cells. Blood/bone marrow samplesfrom three CML patients (P1, P2, and P3) were obtained afterinformed consent and approval from institutional ethicalcommittee. All three patients displayed resistance to eitherIM or DS during the course of the treatment. The mononuclearcells were isolated using Ficoll-Histopaque density gradientcentrifugation. The cells were washed and diluted in RPMI-1640 medium supplemented with 10% FBS, 1% L-glutamine,and 1% penicillin/streptomycin and used for further studies.The in vitro sensitivity of the patient derived cells to IM andDS, TfR and STAT5 expression were tested in the same way asthat of the cell lines. In addition to these, we performed IMresistance mutation analysis (IRMA) in patient samples toanalyze the possibility of point mutations in the BCR-ABLkinase domain, which confer resistance to TKIs. The analysiswas performed using qRT-PCR at Oncquest diagnostics, NewDelhi, India.2.5. Synthesis of HSA Nanoparticles Loaded with

Sorafenib Tosylate (nAlb-Soraf). All the chemicals werepurchased from Sigma Aldrich, USA, unless otherwise specified.HSA stock solution (fraction V, ≥98% purity) was prepared inPBS, pH 7.4. Sorafenib was obtained as a tosylate salt fromSelleck chemicals (USA). Sorafenib stock was prepared in

DMSO, and aliquots were stored at −20 °C. In a typicalsynthesis, 15.7 mM sorafenib tosylate in DMSO was addeddropwise to 5 mg/mL HSA solution predoped with fluorescentnanoclusters of Au28. The solution was kept under continuousstirring, where the final concentration of sorafenib was adjustedto 500 μM. The stirring was continued for ∼30 min at roomtemperature. The individual HSA molecules were cross-linkedusing 2 mg of 1-ethyl-3-[3-dimethylaminopropyl] carbodiimidehydrochloride (EDC), a zero-length cross-linker for effectiveentrapment of the drug in the protein shell. The reaction wascontinued for ∼2 h at room temperature. Sorafenib embeddedalbumin nanoparticles (nAlb-Soraf) were subjected to dialysisusing Slide-A-Lyzer dialysis cassettes with 2 kDa molecularweight cutoff (Thermo Scientific, USA), and lyophilized for 48h in Martin Christ Lyon Chamber Guard (USA). Thepercentage entrapment of the drug was determined fromstandard graph of sorafenib prepared in DMSO.

2.6. Bioconjugation of nAlb-Soraf with Tf. Thebioconjugation of nAlb-Soraf with iron-saturated Tf wasperformed using EDC-Sulfo NHS coupling chemistry. 5 mg/mL Tf was prepared in conjugation buffer [100 mM 2-(N-morpholino)ethanesulfonic acid (MES), 500 mM sodiumchloride (NaCl), pH 6.0]. Tf was activated by the addition of2 mg of EDC and 6 mg of sulfo-NHS. The reaction mixture wasgently vortexed for the complete dissolution of all thecomponents. The activation was allowed to proceed for 15min at room temperature in the dark. Later, activated Tf waspurified in the desalting column for the removal of unreactedEDC and sulfo-NHS. A colloidal solution of the lyophilizednAlb-Soraf was prepared in the conjugation buffer at aconcentration of 2 mg/mL. The activated Tf was added tothe colloidal suspension of nAlb-Soraf. The solution mixturewas stirred continuously at room temperature for 2 h. Later theunreacted components were removed by dialysis with amolecular weight cutoff of 100 kDa. The Tf conjugated nAlb-Soraf is represented as Tf−nAlb-Soraf.

2.7. Characterization of Tf−nAlb-Soraf. Molecularmodeling of the interactions between albumin and sorafenibwas performed using Autodock Version 4.0. The morphologyand size characterization of Tf−nAlb-Soraf was done usingscanning electron microscopy (SEM, JEOL JSM-6490LA,Japan). The hydrodynamic diameter and size distributionanalysis was performed using dynamic light scattering (DLS-ZP/particle sizer Nicomp 380 ZLS, USA). Photoluminescencespectra of free sorafenib and Tf−nAlb-Soraf were recordedusing a spectrofluorimeter (HORIBA JOBIN-VYON Fluoro-max 4, Japan).

2.8. Hemocompatibility Analysis. 2.8.1. HemolysisAssay. Whole blood from healthy donors was used for thestudy after the approval from the institutional ethicalcommittee and donor’s informed consent. 3.8% aqueoussolution of trisodium citrate (Qualigens, India) was used asthe anticoagulant. 20 μM free sorafenib and Tf−nAlb-Sorafwere incubated with 900 μL of whole blood at a volume ratio of1:10 for 3 h at 37 °C in a shaking incubator (Rivotek, India).1% Triton X-100 was used as the positive control whereasuntreated blood served as the negative control. Duplicates wereset up for the sample as well as the controls. Plasma wascollected from the treated blood by centrifugation at 4000 rpmfor 15 min at 20 °C in a temperature-controlled centrifuge(Thermo Scientific, USA). The absorption of plasma optimallydiluted with 0.01% sodium citrate was recorded at 380 nm, 415nm, and 450 nm in a UV−vis spectrophotometer (UV-1700

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Pharma Spec Japan). The percentage hemolysis was calculatedusing the Soret band method.2.8.2. Cell Viability Analysis of PBMCs Treated with Tf−

nAlb-Soraf. Whole blood from healthy donors was collected inheparinized vacutainers (BD Biosciences, USA). PBMCs wereisolated using Ficoll histopaque density gradient centrifugationas per the established protocol. The isolated cells were washedusing RPMI 1640 medium and resuspended in the samesupplemented with 10% FBS, 1% L-glutamine, and 1%penicillin/streptomycin. 1 × 104 cells were seeded per well ina 96 well-tissue culture plate and were treated with differentconcentrations of free sorafenib and Tf−nAlb-Soraf for 48 h.The cell viability was assessed using MTT conversion toformazan. 1% Triton X-100 was added to the positive control,and untreated cells served as the negative control. Theabsorption of solubilized formazan crystals was measured asdescribed before.2.8.3. Inflammatory Response Analysis of Tf−Alb NPs by

Cytokine Induction Assay. Inflammatory response of thecarrier vehicle, i.e., nAlb and Tf−nAlb NPs, was analyzed bycytokine induction assay on human PBMCs using flowcytometry. PBMCs from healthy donors were isolated asdescribed earlier. 1 × 106 cells/mL were seeded in a 24 welltissue-culture plate and incubated with 50 μg/mL nAlb andTf−nAlb NPs for 24 h under culture conditions. Later, the cellswere harvested and supernatant was collected. The supernatantwas incubated with CBA bead array (BD Biosciences, USA)specific for the cytokines IL8, IL1β, IL6, IL10, TNF, and IL12P70. The beads were washed, incubated with the detectionreagent, and analyzed using flow cytometry. Untreated cellsserved as the negative control whereas 1 μg/mL bacteriallipopolysaccharide treated cells were used as the positivecontrol. All the samples including the controls were set asduplicates for the study. Cytokine induction for the negativecontrol was set as 0% and for the positive control as 100%.2.9. Targeted Uptake Studies of Tf−nAlb-Soraf Nano-

medicine. K562R cells maintained under culture conditionswere seeded at a density of 1 × 105 cells per well in a 24 welltissue culture plate and incubated for 24 h under cultureconditions. After 24 h of incubation, the cells were exposed to500 μg/mL Tf−nAlb-Soraf. nAlb-Soraf was kept as the control.In order to confirm the TfR mediated uptake, another set ofcells with same seeding density was pretreated with 50 μg offree Tf 1 h prior to the addition of Tf−nAlb-Soraf. Albumin waspredoped with red-NIR emitting Au28 nanoclusters. The Au28fluorescence was recorded in a flow cytometer as well as aconfocal microscope to assess the uptake of nAlb-Soraf andTf−nAlb-Soraf.2.10. Cytotoxicity of Tf−Alb-Sora-NPs. 2.10.1. MTT Cell

Viability Assay. The cytotoxicity of free sorafenib, nAlb-Soraf,and Tf−nAlb-Soraf on K562R cells was assessed for 48 h.Typically 1 × 104 cells were seeded per well in a 96 well tissueculture plate and incubated for 24 h under culture conditions.The cells were treated with 2.5−20 μM free sorafenib, nAlb-Soraf, and Tf−nAlb-Soraf for 48 h. 1% Triton X-100 was addedto the positive control, and the negative control was untreatedcells. Triplicates were set up for each sample concentration aswell as the controls. MTT conversion to formazan was used asa measure of cell viability. The formazan absorption wasmeasured as described earlier.2.10.2. Apoptosis Assay. The drug-induced apoptosis on

K562R cells was assessed using flow cytometry as well asconfocal microscopy. Apoptosis assay was performed on K562R

cells using Annexin V-FITC Apoptosis Detection Kit (BDPharmingen, USA) according to the manufacturer’s instruc-tions. Propidium iodide (PI) was used as a nuclear stain foridentifying late apoptotic and necrotic cells. Briefly 1 × 105 cellsseeded in a 24 well tissue culture plate were incubated in thepresence of 20 μM free sorafenib, nAlb-Soraf, and Tf−nAlb-Soraf for 48 h under culture conditions. Controls were cellscultured in the absence of the drug. Later the cells wereharvested, washed in 1× binding buffer containing 0.01 MHEPES (pH 7.4), 0.14 M NaCl, 2.5 mM CaCl2 provided alongwith the kit and stained with Annexin V-FITC and PI accordingto the manufacturer’s instructions. The cells were furtheranalyzed using flow cytometry. Staining the samples withAnnexin V and PI individually performed the compensation fordouble fluorescence. For confocal microscopic imaging, thecells were treated with the drug as described before. Later thecells were harvested, washed in 1× binding buffer, and stainedwith Annexin V-FITC and PI. After the incubation period, thecells were washed again to remove the unconjugated dye, platedon PLL coated glass Petri dishes, and observed under amicroscope. Annexin V-FITC fluorescence was detected using488 nm laser excitation, and PI fluorescence was detected using543 nm laser excitation.

2.10.3. Cytotoxicity of Tf−Alb-Soraf on IM RefractoryPatient Samples. MTT cell viability assay was performed toanalyze the cytotoxicity of Tf−nAlb-Soraf in IM refractorypatient samples. The mononuclear cells from three patientsrepresented as P1, P2, and P3 were treated with Tf−nAlb-Soraffor 48 h. 1 × 104 cells seeded per well in a 96 well tissue cultureplate were treated with 10−20 μM Tf−nAlb-Soraf for 48 hunder culture conditions. The viability of the cells treated withthe drug was assessed using MTT conversion to formazan asdescribed earlier. The apoptotic cells were further stained withAnnexin V-FITC Apoptosis Detection Kit as per themanufacturer’s instructions. The cells were collected, washedin 1× binding buffer [0.01 M HEPES (pH 7.4), 0.14 M NaCl,and 2.5 mM CaCl2], stained with Annexin V-FITC and PI, andobserved under a confocal laser scanning microscope for theqaulitative evaluation of apoptosis. PI was used as a nuclearstain for identifying late apoptotic cells and necrotic cells.

2.11. Immunoblot Analysis. Immunoblotting of therelevant proteins was performed on whole cell lysates ofK562R cells. 2.5 × 105 cells/well were incubated in a 24 welltissue culture plate in the presence of 10 μM free sorafenib andTf−nAlb-Soraf for 48 h. Untreated cells were kept as thecontrol for the study. After the incubation period, the cells werecollected and washed in ice cold PBS (pH 7.4). The cells werelysed in 1 mL of lysis buffer [50 mM Tris-HCl (pH 6.8), 0.1%SDS, 1% Triton X-100, 0.5% deoxycholic acid, 150 mM NaCl,0.01 M EDTA] all purchased from Sigma Aldrich, USA,supplemented with complete protease inhibitor and phospha-tase inhibitor cocktail (Sigma Aldrich, USA). The proteinconcentration was estimated using bicinchoninic acid assay(BCA assay, Sigma Aldrich, USA) according to themanufacturer’s instructions. 25 μg of the whole cell lysatewas boiled in 1× Lamelli buffer (63 mM Tris HCl, 10%glycerol, 2% SDS, 0.0025% Bromophenol blue, pH 6.8) for 5min, resolved on 12% SDS−PAGE gel, and later transferredonto a polyvinylidene difluoride (PVDF) membrane (Millipore,USA). The proteins detected were BCR-ABL, MCL-1, STAT5,and pSTAT5. α-Tubulin was used as the loading control. Anti-BCR antibody (Cell Signaling Technology, USA), anti-MCL-1antibody (Cell Signaling Technology, USA), anti-STAT5

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antibody (Cell Signaling Technology, USA), anti-pSTAT5antibody (Cell Signaling Technology, USA), and anti α-tubulinantibody (Santa Cruz Biotechnology, USA) were used forimmunoblot analysis. After overnight incubation at 4 °C withprimary antibody, the membranes were washed and incubatedwith horseradish peroxidase (HRP) conjugated secondaryantibody (Cell Signaling Technology, USA) at room temper-ature for 1 h. The membranes were washed, and boundantibodies were detected with Immobilon Western reagent(Millipore, USA) as specified by the manufacturer.

3. RESULTS AND DISCUSSION

3.1. Molecular Characterization of IM/DS ResistantK562 Cell Line (K562R). In order to identify the uniquemolecular characteristics of refractory CML, first, we havedeveloped an IM/DS resistant CML cell line K562R andextensively characterized it with reference to its sensitivecounterpart K562S, before proceeding to IM refractory patientsamples. K562 cell line carries a typical b3a2 fusion transcriptencoding a p210 kDa BCR-ABL chimeric oncoprotein. Theresistant variant K562R was generated according to a previouslyreported method.18 Cells maintained under culture conditionswere progressively exposed to higher concentrations (up to 1μM) of IM and DS for three months until an outgrowth ofresistant phenotype was observed. After three months, viablecells, which could survive in the presence of 1 μM drug, wereisolated and cultured. These cells did not present a reversion ofthe resistant phenotype even after removal of the drugs fromthe culture medium. The sensitivity of these cells to both IM

and DS was quantified using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric reduc-tion method, where the viable cells reduce MTT to formazancrystals. Typically, 1 × 104 cells were treated with 0−10 μM IMor DS for 48 h under culture conditions. The optical density(OD) values at 560 nm of the dissolved formazan crystals wereconverted to percentage cell viability. As shown in Figure 1A,IM or DS caused dose dependent inhibition of proliferation inthe sensitive cell line K562S, whereas the resistant phenotypeK562R remained unaffected over the same or higher dosage.This indicates the development of clinically relevant TKIresistance in the K562R cell line, and we have selected thispopulation for further studies.To understand the mechanisms of resistance, we have probed

various possible molecular pathways. This includes over-expression of multidrug resistance gene (MDR1) encoding P-glycoprotein (P-gp), which was reported in many drug resistanttumor types.19 P-gp is an intricate molecular pump, which canefficiently efflux drugs from intracellular regions renderinglower toxicity.20 Whether P-gp can mediate resistance to IM orDS is a clinically relevant question because the expression of P-gp was noted in certain cases of blast crisis CML patientsinsensitive to IM.21 We have studied P-gp expression in bothK562S and K562R cell lines by flow cytometry using FITCconjugated anti-P-gp mAb. However, the flow data (Figure 1B)shows that both the cell lines were P-gp−ve, indicating that therole of P-gp efflux pumps in the observed IM/DS resistancewas insignificant.In the case of oncogene-addicted cancer, the amplification of

oncogene was another major cause of resistance.22 Amplifica-

Figure 1. Characterization of CML cells. (A) Cell viability of K562S and K562R cells upon exposure to increasing concentrations of IM and DS for48 h. (B) P-gp expression studies in K562S and K562R. (C) FISH analysis of K562RS and K562R cells. (D) 304bp BCR-ABL cDNA transcriptsfrom K562R and K562S run in 1% agarose gel. (E) Gene expression profile showing upregulation of BCR-ABL and STAT5 in K562R cells comparedto K562S, taken as baseline.

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tion or duplication of BCR-ABL fusion gene was foundsignificant in the case of IM/DS refractory CML patients.23 Wehave used the fluorescence in situ hybridization (FISH)technique to investigate BCR-ABL gene amplification inK562R cells compared to K562S. FISH analysis was performedusing interphase nuclei of K562S and K562R cells with greenand red fluorescent probes targeted against BCR and ABLgenes, respectively. This renders yellow fluorescence from thefusion regions of BCR-ABL gene. Figure 1C shows therepresentative FISH patterns of K562S, which shows a fewregions of BCR-ABL fusion, indicated by yellow fluorescence,whereas, in the case of K562R, multiple copies of yellow signalscould be seen, suggesting significant amplification of BCR-ABLfusion gene. The inset shows an enlarged image of the fusionsignals, which substantiates that K562R cells harbored multiplecopies of BCR-ABL gene compared to that of K562S, indicatingchimeric gene amplification in the former. We have furtherverified this by polymerase chain reaction (PCR) where reversetranscription and amplification revealed a highly intense 304bpBCR-ABL signal in K562R compared to K562S as depicted inFigure 1D. Enhanced expression of the BCR-ABL fusiontranscript in K562R is clear from the figure. This confirms thatthe IM/DS resistance conferred by the K562R cell line isassociated with BCR-ABL gene amplification, which correlateswith the clinical case of refractory/relapsed CML.23

3.2. Activated Survival Pathways in Resistant CMLCell Line. The fact that even therapeutically very high doses ofBCR-ABL TKIs (IM/DS) could not induce significant toxicityto K562R cells indicates that, in addition to BCR-ABLamplification, other pathways in this oncogenic network mayalso be activated to support the uncontrolled proliferation.Recent reports also suggest that the resistance to secondgeneration TKIs such as DS and NL is attributed to theconstitutive activation of many secondary pathways including

PI3K-mTOR, Akt, RAS/RAF/MEK/ERK, and JAK/STAT.24−26

In such cases, these pathways could be considered as potentialtargets for therapeutic intervention for IM/DS/NL refractorycases.27 In view of this, we have investigated the involvement ofsuch alternative survival pathways in the K562R cell linecompared to its drug sensitive counterpart. The relative geneexpression of BCR-ABL, PI3K, AkT, and STAT5 was studiedusing quantitative real-time polymerase chain reaction (qRT-PCR) with GAPDH as endogenous control. Interestingly, thedata (Figure 1E) shows that, compared to the drug sensitiveK562S cells, the resistant phenotype shows ∼2.5-fold increasedexpression of STAT5, which is as high as that of the oncogene(BCR-ABL) itself. This means, in addition to the amplificationof oncogene, its immediate downstream pathway, STAT5, wasalso upregulated in the resistant phenotype. We haveinvestigated the critical role of this STAT5 upregulation inIM resistance by inhibiting STAT5 phosphorylation usingsorafenib, pretreated before IM. K562R cells treated with a lowconcentration of sorafenib (2.5 μM) for 24 h were exposed togradually increasing concentrations of IM and incubated for 48h. The results show that downregulation of STAT5 couldsignificantly increase the sensitivity of the resistant phenotype(K562R) toward IM (Supporting Information, Figure S1).These findings are in conjunction with the very recent

reports of Warsch et al.28 and Rahmani et al.29 that showedintimate association of STAT5 upregulation with TKI resistancein CML. Moreover, irrespective of the downregulation of BCR-ABL kinase by IM, the STAT5 upregulated cells exhibiteduninterrupted capacity to survive and proliferate. STAT5 hasbeen reported as a critical protein kinase in the BCR-ABLoncogenic network, behaving as an important signal for cellulartransformation and growth factor independent proliferationleading to leukemiogenesis.30 Scherr et al.31 found that RNAinterference mediated silencing of STAT5 can lead to

Figure 2. Transferrin receptor expression studies. (A) Flow cytometry analysis of TfR expression in PBMC, K562S, and K562R cells. (B) Graphicalrepresentation of mean fluorescence intensity (MFI) of TfR mAb stained cells. (C) Confocal microscopic image of TfR-mAb stained PBMC and(D) K562R cells. (E) Magnified image of TfR-mAb stained K562R single cell showing TfR expression on the cell membrane.

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diminished capacity of primary CD34+ve CML cells topropagate colonies in cytokine containing soft agar assay,demonstrating an important role of STAT5 in CML primarycells. In addition, STAT5 activation was also found correlatingwith the enhanced expression of antiapoptotic protein MCL-1belonging to the BCL-2 family, which enables the leukemiccells to evade the growth inhibitory effects of TKIs.32 Inanother recent report, Wang et al.33 revealed a correlationbetween elevated STAT5 phosphorylation and IM resistance inpatient samples. Further, Hoelbl et al.34 also confirmed theindispensable role of STAT5 for the maintenance of BCR-ABL+ve leukemia. Collectively, our observations and all theabove reports clearly suggest that STAT5 is a potentialintracellular protein kinase target for developing newtherapeutic approaches against drug resistant CML.3.3. Immunophenotyping of Transferrin Receptors

(TfR) in Drug Resistant Cell Line. After identifying STAT5as the aberrant kinase to be targeted in refractory CML, ournext question was how to deliver small molecule nanomedicineagainst STAT5, in a cell-targeted fashion, specifically to theresistant population. This led us to investigate the potentialtrans-membrane receptors that may be overexpressed in theresistant population. There was no prior report on any uniquecell surface marker for TKI refractory CML cells. However,studies done by Barabas et al.35 found that adriamycin resistantK562 CML cell line overexpresses transferrin receptors (TfR)compared to their sensitive counterpart and TfR was found to

play a remarkable role in the resistance mechanism. TfRmediated endocytosis is a major pathway for cellular ironuptake, and more importantly, high levels of TfR have beenfound in malignant cells including leukemia.36 Transferrin is aniron transport glycoprotein, which induces growth-promotingactivities through TfR (CD71). TfR mRNA level was foundsignificantly upregulated in primary CD34+/CD38− CML cellsas well.37 Considering this, we have analyzed the possibility ofTfR overexpression in drug resistant CML cells.TfR immunostaining was performed on K562S and K562R

using flow cytometry as well as confocal microscopy. PBMCsisolated from healthy individuals were taken as control. Theflow cytogram in Figure 2A shows an insignificant level of TfRexpression in the PBMC control, when stained withallophycocyanin (APC) conjugated TfR monoclonal antibody(mAb), whereas ∼99.0% of both K562S and K562R cellsshowed TfR expression. More importantly, although both thecell types showed similar TfR expression in most of the cells,the mean fluorescence intensity (MFI), which is a measure ofthe number of receptors per cell, showed ∼3-fold increase inK562R compared to K562S (Figure 2B). This suggests theoverexpression of TfR in resistant population compared to thesensitive case. Representative confocal microscopic images(Figure 2C) also confirmed this data where PBMC showedmuch less staining by mAb targeted to TfR, whereas K562Rregistered high TfR staining on the cell membrane (Figures 2Dand 2E). The cell membrane was found completely decorated

Figure 3. Characterization of imatinib (IM) refractory patient samples. (A) FISH analysis of drug resistant CML patients. (B) IM resistancemutation (IRMA) analysis of patient samples. (C) Cell viability of mononuclear cells derived from drug resistant CML patients upon exposure toincreasing concentrations of IM and DS for 48 h. (D) qRT-PCR analysis of STAT5 expression of patient 1−3 (P1, P2, and P3), relative to that of adrug sensitive chronic phase patient, taken as baseline.

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with fluorescently stained TfR antibody. These results correlatewith the reports of Barabas et al.35 that the refractory CML cellsexpress high levels of TfR and indicate the possibility oftargeting TfR for cell specific delivery of anti-STAT5nanomedicines.3.4. Isolation and Characterization of IM Refractory/

Relapsed CML Patient Cells. To determine the clinicalvalidity and relevance of the above findings, we have studied themolecular characteristics of cells isolated from IM resistantCML patients who were under relapse after the treatment for atleast 2 years with TKI. Peripheral blood/bone-marrow sampleswere collected from three clinically confirmed refractorypatients, after their informed consent and approval from theinstitutional ethical committee of Amrita Institute of MedicalSciences, Cochin. From the clinical investigation, patient 1 (P1)was on IM, developed blast crisis, and was started on DS, butthere was no response to the same. Patient 2 (P2) was on IM,developed blast crisis, and was on DS for 2 years; he was inclinical and hematologic remission. Patient 3 (P3) was achronic phase CML patient who was losing response to IM asevidenced by increasing level of BCR-ABL. We performed dualcolor FISH analysis to confirm the presence of BCR-ABL genein these patients. All three cases showed typical BCR-ABLfusion patterns. In all three cases, multiple copies of BCR-ABLfusion signals were observed as shown in Figure 3A. Moreimportantly, PCR based IM resistance mutation analysis(IRMA) identified different point mutations in the BCR-ABLkinase domain (Figure 3B) in two of these patients, P1 atG250E, and P2 at F311I. P3 did not show any TKD mutation.The presence of the above mutations in P1 and P2 (G250E andF311I) was associated with poor prognosis for IM refractory

patients. Our clinical data indicated that P1 harboring G250Emutation was the most resistant compared to the other twocases.38 G250E is a highly relevant mutation in the ATPbinding loop (P-loop) of BCR-ABL, and reports suggest that P-loop mutations are more oncogenic compared to mutations inthe other regions of kinase domain. F311I mutation detected inP2 resides in the IM binding domain of BCR-ABL, which wasalso found to be clinically significant and responsible for IMresistance.39

To quantify the level of IM/DS resistance of these patientsamples, under in vitro conditions, we have tested thecytotoxicity of leukemic cells isolated from blood/bone marrowof patients by treating with a 0−10 μM concentration of thesedrugs over ∼48 h. As expected, among the three samples, P1displayed an extreme insensitivity to both the BCR-ABL TKIsup to relatively high concentration of 10 μM as depicted inFigure 3C. P2 and P3 also showed insensitivity toward IM andDS, which correlates with their respective clinical data. Eventherapeutically higher concentrations of IM or DS could notinduce much toxicity to these cells, indicating the functioning ofa strong drug resistant mechanism. We have studied the role ofP-gp, if any, on IM refractory patient samples using flowcytometry. The results (see Supporting Information Figure S2)showed little expression of P-gp on all refractory samples,suggesting that IM resistance was not contributed by P-gpefflux proteins.In the next step, we have evaluated the level of STAT5 in

these patient samples by qRT-PCR. The relative mRNAexpression was compared with that of a patient (P0) in thechronic phase showing clinical response to IM. Interestingly, itcan be seen from Figure 3D that the most drug resistant patient

Figure 4. TfR expression in IM refractory CML patient cells. (A) Flow cytometry analysis of TfR expression in IM refractory patients P1, P2, and P3.(B) Confocal microscopic images of patient derived leukemic cells stained with APC conjugated TfR mAb, showing increasing level of TfRexpression as P1 > P2 > P3. (C) Percentage expression of TfR expression in P1, P2, and P3.

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sample P1 showed the highest expression of STAT5 mRNA(∼6.54-fold) compared to P2 (∼5.41-fold) and P3 (∼1.2-fold).This data correlated well with that obtained using the K562Rcell line (Figure 1E) and other reports on the preferentialactivation of STAT5 and its role in the mechanism of drugresistance in CML. Thus, with these results, we have confirmedour rationale to select aberrant STAT5 kinase as the target fordesigning nanomedicine against refractory/relapsed CML.We have further quantified the level of TfR expression in IM

refractory patient samples (P1, P2, and P3). Mononuclear cellsisolated from the peripheral blood of these patients werestained with APC conjugated TfR mAb and analyzed using flowcytometry as well as confocal microscopy. The flow datarevealed that P1 had the highest level of TfR expression of∼59.4%, whereas P2 and P3 showed ∼16.7% and ∼6.7%expression as indicated by Figure 4A. The flow result wasfurther reinforced by confocal imaging, which also showedsimilar gradual increase in the expression level of TfR from P3to P1 (Figure 4B). The percentage TfR expression in the threeIM refractory patients is graphically represented in Figure 4C.These results unambiguously correlate the TfR expression levelwith STAT5 expression and the severity of drug resistance.This throws more light onto a unique correlation between

the TKI resistance of CML and the activation of STAT5 withoverexpression TfR, which was not well studied earlier. Ourresults on cell lines as well as patient samples clearly show that,with increasing levels of drug resistance, both STAT5 activationand TfR expression increase in a correlative fashion. This result

has great significance considering the recent reports on theregulatory role of STAT5 in the Tf-mediated iron uptake inerythroid cells. It was shown that STAT5 null mice display∼50% reduction in the expression of TfR genes on theerythrocytes leading to microcytic hypochromic anemia.40 Itwas also shown that STAT5 binds to the first intron on theTf R1 gene and constitutively activated STAT5 increases theexpression of TfR1. Kerenyi et al.41 also showed ∼2-foldlowering of TfR expression on the erythroid cells in micelacking STAT5. This suggests that STAT5 possesses transcrip-tional regulatory control over TfR and the overexpression ofthe former leads to similar effects on the latter (See SupportingInformation Figure S3). From the perspective of drug resistantCML, this means constitutive activation of STAT5 kinasethrough its aberrant upstream BCR-ABL oncogene willeventually increase the expression of trans-membrane proteinreceptor TfR, as we have seen in both resistant cell line andpatient samples. We consider this as a “window of opportunity”to specifically deliver our proposed anti-STAT5 nanomedicineby conjugating them with Tf ligand for targeting its receptorTfR. In effect, the aberrant tyrosine kinase (STAT5)responsible for the survival of drug resistant CML cells canbe inhibited by stealthily delivering nanoencapsulated STAT5inhibitors through its own translationally controlled membranereceptor TfR.

3.5. In Silico Design and Synthesis of TfR and STAT5Targeted Endogenous Nanomedicine. Based on the aboveinformation derived from cell lines and patient samples, we

Figure 5. Development of Tf−nAlb-Soraf. (A) In silico molecular modeling of the interactions between human serum albumin (HSA) and sorafenib.(B) Schematic representation of Tf conjugated albumin bound sorafenib (Tf−nAlb-Soraf) nanomedicine. (C) SEM image of Tf−nAlb-Sorafnanoparticles showing size ∼150 nm. Inset: Photograph of colloidal Tf−nAlb-Soraf nanomedicine. (D) DLS data showing the hydrodynamicdiameter of Tf−nAlb-Soraf, ∼84 ± 20 nm. (E) Photoluminescence characteristics of free sorafenib and Tf−nAlb-Soraf nanomedicine.

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have selected preferentially activated STAT5 as the aberrantkinase to be inhibited in IM/DS refractory CML and TfR(CD71) as the cell receptor target for specific delivery ofnanomedicine against STAT5. Accordingly, an FDA approved,small molecule drug, sorafenib, that showed potential activityagainst STAT5 (IC50−10 μM) was selected as the drug ofchoice. Considering the abundance of albumin protein in thehuman serum and its clinically demonstrated ability toencapsulate hydrophobic drugs such as paclitaxel (Abraxane),we chose human serum albumin (HSA) as the nanocarrier forsorafenib delivery to refractory CML cells.16 Being anendogenous protein, albumin is highly biocompatible, non-immunogenic, and easy to conjugate with targeting ligands suchas antibodies, peptides, or other proteins.42 To target TfR, inthe present case, we have selected transferrin (Tf), which isanother endogenous protein, as the ligand to be conjugatedwith albumin−sorafenib nanoparticles. To track the cell specificuptake using fluorescence techniques, we have predoped HSA(nanocarrier) with ∼28 atom clusters of fluorescent gold(Au28), as reported in our earlier work.43

Prior to wet-chemical synthesis, we have performed ligand-protein docking simulations using Auto Dock 4.2 to understandthe possible chemical interactions and associated bindingenergy between sorafenib and HSA. After docking sorafenibwith the experimentally determined six possible drug bindingpockets of HSA,44 we have identified favorable amino acidresidues that are interacting with sorafenib (within 4 Å space)and explored the type of interactions in each of the HSAbinding sites. The docking results revealed that sorafenibdifferentially binds with ∼6 drug-binding pockets of albuminwith binding energies varying from −8.17 to −4.71 kcal/mol(see Supporting Information Figure S4). The best docking posewith binding energy −8.17 kcal/mol indicates that sorafenibinteracts with albumin through a combination of van der Waalsforces, hydrogen bonds, and intermolecular interactions. Thebest binding energy conformation suggested that nitrogen andoxygen atoms of sorafenib could make hydrogen bonds withArg 117, Glu 518, Ala 176, Cys 177, and Leu 115 residues.Furthermore, intermolecular energy offered by hydrophobicinteractions also well stabilizes the binding of sorafenib withalbumin (residues involved in the hydrophobic interactions aregiven in the Supporting Information Table S1). Similarly, wealso explored the other possible sorafenib binding sites inalbumin and obtained the least binding energy ∼−4.71 kcal/mol in one of the pockets which may also be significant enoughto provide the necessary hydrophobic interactions betweensorafenib and albumin. In effect, these docking simulationssuggested that at least 6 molecules of sorafenib can effectivelybe loaded into a single albumin molecule without anysignificant cross talk, thus indicating the possibility of improvedsolubility of this otherwise hydrophobic drug (Figure 5A).Computationally evaluated model of sorafenib loaded

albumin nanoparticles (nAlb-Soraf) was prepared by a wet-chemical nanoprecipitation method under optimized con-ditions. Typically, ∼15.7 mM (10 mg/mL) solution ofsorafenib dissolved in DMSO was added dropwise to ∼5mg/mL aqueous solution of HSA, predoped with Au28, underprobe sonication. The final concentration of sorafenib wasadjusted to 500 μM. After ∼5 min, moderately turbidprecipitate appeared, indicating the formation of albuminbound sorafenib nanoparticles. The extent of precipitation,particle size, and aggregation properties were controlled byoptimizing the concentration of albumin, sorafenib, reaction pH

(7.4), and rate of addition (2 μL/min). After the precipitation,we used 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hy-drochloride (EDC) mediated cross-linking of albumin nano-particles to effectively encapsulate the drug within the proteinmatrix thus limiting their degradation and release. Unboundfree drug from the reaction medium was removed by dialysis,and the UV−vis absorption studies of washed precipitateindicate excellent encapsulation efficiency of ∼74%. Thissuggests that sorafenib and albumin had very strong bindingaffinity as also predicted by the docking experiments. nAlb-Soraf was lyophilized to obtain fine powder which was very wellredispersible in the aqueous medium without losing the stabilityof sorafenib as monitored using UV−vis and photolumines-cence spectroscopy.For the bioconjugation of nAlb-Soraf with the targeting

ligand Tf, EDC-sulfo NHS coupling chemistry was employed.Typically, 5 mg of Tf dissolved in conjugation buffer was madeamine reactive using EDC, and the resulting semistable O-acylisourea ester was stabilized by sulfo-NHS. nAlb-Sorafnanoparticles dispersed in PBS were added to the activatedTf and reacted for ∼3 h at room temperature. Afterbioconjugation reaction, free Tf was removed by dialysis andlyophilized to make a fine powder. Thus obtained Tfconjugated nAlb-Soraf showed excellent redispersion propertiesin aqueous medium. Moreover, the bioconjugation reaction didnot affect the chemical stability of sorafenib as evaluated usingUV−vis spectroscopy studies (data not shown). The Tfconjugated nAlb-Soraf is hereafter referred to as “Tf−nAlb-Soraf” nanomedicine, which is schematically depicted in Figure5B. The morphological characterization of Tf−nAlb-Soraf wasperformed using SEM, which revealed formation of sphericalparticles of size ∼150 nm (Figure 5C). Dynamic light scattering(DLS) analysis showed average particle size of ∼84 ± 20 nm(Figure 5D) and zeta potential ∼−36.37 mV rendering a stablecolloidal suspension of nanomedicine as shown in the opticalphotograph, Figure 5C (inset). The dissociation of sorafenibfrom Tf−nAlb-Soraf nanoparticles at the lysosomal pH (4.5)follows a slow and sustained release from Tf−nAlb-Soraf, with apeak at 72−192 h time frame. This is a promising trend as thenanoparticles will have sufficient time to get into the leukemiccells before releasing the drug (See Supporting Information,Figure S5).The molecular interactions between sorafenib and albumin

carrier were characterized using fluorescence spectroscopy. Theexcitation and emission spectra of free and albumin boundsorafenib are shown in Figure 5E. Free sorafenib shows thefluorescence emission spectrum with doublet peaks at 405 and450 nm, and the corresponding excitation spectrum has a peakat 380 nm. However, after encapsulation in albumin nano-particles, the excitation spectrum of sorafenib was foundsignificantly modified due to the appearance of new peaks inthe high-energy region (260−280 nm), which correlated withthe absorption band of tryptophan residues in the albumin.This suggests that radiative transitions in albumin boundsorafenib were contributed not only by the molecular orbitals ofsorafenib but by that of albumin as well. This is possible byForster type fluorescence resonance energy-transfer (FRET)where the excited state electrons within the lowest unoccupiedmolecular orbitals (LUMO) of albumin transfer its excited stateenergy to electrons in the highest occupied molecular orbitals(HOMO) of sorafenib rendering radiative transitions in thelatter. This is possible due to strong molecular orbitalinteractions through intimate proximity between the two

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entities, confirming that sorafenib is very well encapsulatedwithin the protein carrier.3.6. Hemocompatibility Analysis of Tf−nAlb-Soraf. It

is important for Tf−nAlb-Soraf nanomedicine, being a leukemiatargeted nanomedicine, to have minimum toxicity to normalblood components other than leukemic cells. To evaluate this,we have subjected Tf−nAlb-Soraf to three important bloodtoxicity tests such as hemolysis, proinflammatory cytokineresponse, and compatibility with healthy mononuclear cellsincluding immune cells. Hemolytic potential was tested usingwhole blood derived from healthy volunteers, after theirinformed consent and approval by the institutional ethicscommittee. The optical absorption of plasma derived from thewhole blood treated with 20 μM Tf−nAlb-Soraf and the sameconcentration of free sorafenib was recorded at 380, 415, and450 nm for the detection of hemoglobin in the plasma using theSoret band analysis method.45 Untreated whole blood was usedas the control for the study. Figure 6A shows that both freesorafenib and Tf−nAlb-Soraf did not induce any leakage ofheme into the plasma and thus remained non-hemolytic. Thescanning electron microscopy (SEM) image of nanomedicinetreated red blood cells also revealed their intact biconcavemorphology, confirming no adverse hemolytic effects by Tf−nAlb-Soraf nanomedicine (Figure 6B). Figure 6C shows thephotograph of treated whole blood after centrifugation.The influence of Tf−nAlb-Soraf on the viability of peripheral

blood mononuclear cells (PBMCs) isolated from healthyvolunteers was studied by MTT assay. PBMCs are crucialcomponents of the immune system to fight infections andevoke immune response to foreign intruders. The interaction of

nanomedicine with PBMCs and subsequent immune activationor suppression is a serious toxicity concern. We have analyzedPBMC viability upon interaction with Tf−Alb-Soraf, and thesame concentration of free sorafenib was used as control.Figure 6D shows the percentage viability of free sorafenib andTf−nAlb-Soraf treated PBMCs. There was no significanttoxicity shown by either free sorafenib or Tf−nAlb-Sorafnanomedicine over the 1−15 μM range, and slight toxicity wasobserved only at a higher concentration (20 μM). Consideringthis, 20 μM was taken as the cutoff limit for further experimentswith leukemic cells.In the next step, inflammatory and immunogenic potential of

nanomedicine were studied using flow cytometry basedquantitative cytokine induction assay. Immune cells, inresponse to interaction with the nanoparticles, may releaseproinflammatory cytokines, which marks the onset ofinflammatory response against the nanoparticles.46 Cytokinescan evoke various downstream signaling pathways during acuteor chronic inflammation, which may sometimes be fatal.Though protein nanoparticles are generally noncytotoxic, itmay cause inflammatory or immunogenic response leading topremature clearance of nanomedicine from the circulation.PBMCs isolated from the healthy individuals serve as the bestmodel to study the inflammatory response of nanoparticles invitro.47 Accordingly, we have studied the proinflammatoryresponse of Tf−nAlb-Soraf nanomedicine by evaluating thecytokine expression of PBMCs. The cells were treated with 50μg/mL nAlb and Tf−nAlb under culture conditions for 24 h.Later, the cells were centrifuged and the culture supernatantwas analyzed for the presence of cytokines released using CBA

Figure 6. Hemocompatibility studies of Tf−nAlb-Soraf. (A) Hemolysis analysis of free sorafenib and Tf−nAlb-Soraf. (B) SEM image of RBCstreated with Tf−nAlb-Soraf. (C) Photograph of whole blood treated with (i) 1% Triton X-100 (+ve control), (ii) untreated whole blood (−vecontrol), (iii) 20 μM free sorafenib, (iv) 20 μM Tf−nAlb-Soraf. (D) Cell viability of PBMC treated with sorafenib and Tf−nAlb-Soraf. (E) Graphicalrepresentation of inflammatory response analysis of 50 μg/mL nAlb and Tf−nAlb by cytokine induction assay using flow cytometry. (F)Representative scatter plots of flow analysis of cytokine induction assay.

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bead array (BD Biosciences USA) for the presence ofproinflammatory cytokines IL8, IL1β, IL6, IL10, TNF, andIL12 P70. Flow-cytometric evaluation (Figure 6E) has revealedthat nAlb or Tf−nAlb did not cause induction of proin-flammatory cytokines and their levels remain comparable tothat of untreated control. The representative scatter plot(Figure 6F), drawn by taking 100% cytokine expression bypositive control (LPS) and 0% by PBS, also showed that bothnAlb and Tf−nAlb did not evoke any cytokine induction, andthe results were comparable to that of negative control. Thisindicates that Tf−nAlb nanomedicine possesses no inflamma-tory potential and may not trigger the cascade of events leadingto premature immune response. Collectively, the above testssuggest insignificant nonspecific adverse side effects by Tf−nAlb-Soraf nanomedicine, and we believe that the formulationhas the potential to be well tolerated by healthy blood cells.3.7. Targeted Uptake Studies. In order to confirm

whether TfR mediated the intracellular accumulation of Tf−nAlb-Soraf, cell targeted uptake studies were carried out usingTf−nAlb-Soraf with unconjugated nAlb-Soraf as control. Thefluorescent emission in the red−NIR region of Au28 nano-clusters doped within albumin was utilized for trackingintracellular uptake of Tf−nAlb-Soraf in the cells. ∼1 × 105

cells were treated with 500 μg/mL Tf−nAlb-Soraf and nAlb-Soraf (carrying ∼12 μM sorafenib) for 4 h under cultureconditions. In order to clearly demarcate the TfR specificuptake, in another set of experiments, TfR downregulation incells was obtained by pretreating with an excess dose of free Tf(50 μg) for 1 h, prior to incubation with Tf−nAlb-Soraf. Bothflow cytometry and confocal microscopy were used to analyzethe results. For the flow analysis, treated cells were collected,

washed, and excited using 535 nm laser excitation and emissionwas collected using a 610 nm long pass filter. The flow datashows that (Figure 7A) while ∼14.5% K562R cells have takenup unconjugated nAlb-Soraf within 4 h, ∼60% cells have takenup Tf−nAlb-Soraf during the same period of incubation,showing enhanced uptake in the latter sample. In the case of Tfpretreated cells, uptake was reduced to ∼19.3%, which clearlyindicates that the observed enhancement of 60% in TfRexpressing cells was due to receptor specific endocytosis. Toconfirm the flow data, we conducted confocal microscopicimaging where the cells treated as before were collected,washed, plated onto glass Petri dishes, and observed under amicroscope. The fluorescence from Au28 atomic clusters wascollected using 543 nm He−Ne laser excitation. As shown inthe representative microscopic images (Figure 7B), both nAlb-Soraf treated cells and Tf pretreated cells showed reducedfluorescence staining whereas Tf−nAlb-Soraf treated cellsshowed bright red fluorescence from the internalized nano-medicine. Figure 7C shows the excitation−emission spectra ofAu28 nanoclusters doped into albumin, with an excitation peak∼510 nm and emission peak ∼656 nm. This confirms the TfRmediated enhanced endocytosis of Tf−nAlb-Soraf nano-medicine in drug resistant CML cells.

3.8. Cytotoxicity of Tf−nAlb-Soraf on K562R Cells.Targeted cytotoxic effects of Tf−nAlb-Soraf were first tested onthe drug resistant cell line K562R having amplified BCR-ABLand STAT5, using the cell viability assay (MTT). 1 × 104 cellsmaintained under culture conditions were treated with 2.5−20μM concentrations of free sorafenib, nAlb-Soraf, and Tf−nAlb-Soraf for 48 h. Untreated cells were used as the negativecontrol. Figure 8A shows that, compared to free sorafenib or

Figure 7. Targeted nanomedicine uptake studies. (A) Flow cytogram showing differential uptake of 500 μg/mL nAlb-Soraf, or 500 μg/mL Tf−nAlb-Soraf in TfR overexpressing CML cells (i and ii) and (iii) Tf pretreated (TfR downregulated) cells. (B) Corresponding confocal microscopic imagesshowing TfR specific enhanced uptake. Imaging was done using the fluorescence signal (C) from Au28 clusters embedded within albuminnanoparticles.

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nAlb-Soraf, the Tf conjugated nAlb-Soraf exerted significantlyhigher toxicity to IM/DS resistant K562R cells. Interestingly,nAlb-Soraf also showed higher toxicity compared to free drug,probably due to the improved uptake and stability of proteinbound sorafenib.48 However, Tf−nAlb-Soraf exhibited greaterdose-dependent cytotoxicity than that of its unconjugatedcounterpart, nAlb-Soraf. While the IC50 of free drug and nAlb-Soraf was ∼15 and 5 μM, respectively, Tf−nAlb-Soraf couldachieve the same at 2.5 μM, which is ∼6 times lower aconcentration compared to the free drug. It may be recalledthat, within 2.5−10 μM, Tf−nAlb-Soraf did not induce anyserious toxicity to normal healthy PBMCs (Figure 6D). Clearly,Tf−nAlb-Soraf nanomedicine showed enhanced antileukemicactivity without causing nonspecific toxicity effects to primaryhuman blood cells.In addition to the cell viability assay, quantitative

determination of viable versus apoptotic cells was carried outusing the Annexin V/propidium iodide (PI) assay. Briefly, 1 ×105 cells were treated with 10 μM concentration of freesorafenib, nAlb-Soraf, and Tf−nAlb-Soraf for 48 h. The flowcytogram (Figure 8B) revealed that while the free drug inducedearly apoptosis in ∼22% cells and nAlb-Soraf caused ∼50% celldeath, Tf−nAlb-Soraf caused late stage apoptosis in ∼92% ofcells. This is a very important result because K562R was highlyresistant to IM/DS therapy and holds clinically relevantfeatures of refractory CML such as BCR-ABL amplificationand activation of STAT5 signaling. This result was furtherconfirmed by confocal microscopy as shown in Figure 8C. It

can be seen that, while the morphology of free-sorafenib treatedcell remains largely unchanged with intact cell membrane andnuclear contents within 48 h of incubation, the morphology ofTf−nAlb-Soraf treated K562R was significantly altered. Thecytoplasm contents including the nucleus underwent con-densation showing typical features of apoptosis (Figure 8C).Annexin V/PI staining showed that most of the Tf−nAlb-Soraftreated cells were in the late phase of apoptosis with significantnuclear staining by PI compared to that of free drug or nAlb-Soraf. Though there was some difference between the MTTand FACS results, regarding the extent of toxicity, flow databased on the Annexin V/PI staining assay is more reliable injudging the cytotoxicity compared to MTT assay, whichmeasures only the metabolic activity of the cells. This confirmsthat our nanomedicine approach to target both cell surfacereceptor and aberrant STAT5 kinase has great potential toinduce apoptosis in IM/DS resistant CML cells.

3.9. Targeted Toxicity of Tf−nAlb-Soraf Nanomedi-cine in IM Refractory Patient Samples. Final testing of Tf−nAlb-Soraf nanomedicine was performed on IM/DS refractorypatient samples P1, P2, and P3. As mentioned earlier, P1carried G250E mutation in P-loop with highest resistance toIM/DS, and maximum expression of TfR (∼ 59.4%) and∼6.54-fold increase of STAT5, while P2 had F311I mutationwith 16.7% TfR expression and ∼5.41-fold increase of STAT5,and P3 had no mutation with ∼6.7% TfR expression and ∼1.2-fold increase of STAT5. For the toxicity evaluation, ∼1 × 104

cells were exposed to 10−20 μM Tf−nAlb-Soraf nanomedicine

Figure 8. Cytotoxicity of free sorafenib and Tf−nAlb-Soraf in K562R cells. (A) Cell viability analysis of K562R cells treated with free sorafenib, nAlb-Soraf, and Tf−nAlb-Soraf. (B) Flow cytogram showing variation in the level of apoptosis in K562R cells treated with 10 μM free sorafenib, nAlb-Soraf, and Tf−nAlb-Soraf. (C) Confocal microscopic images showing morphological changes and apoptosis markers (Annexin V and PI) in K562Rcells treated with 10 μM free sorafenib versus Tf−nAlb-Soraf.

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for 48 h. MTT assay was used for assessing the viability of thecells after exposure to free sorafenib, nAlb-Soraf, and Tf−nAlb-Soraf. Figure 9A shows the Tf−nAlb-Soraf dose−response

curves for P1, P2, and P3. From the figure, it is evident that allthree IM/DS refractory patient samples exhibited profoundtoxicity with increasing concentrations of the nanomedicine

Figure 9. (A) Cell viability of patient derived leukemic cells treated with different concentrations of Tf−nAlb-Soraf nanomedicine showing toxicity inall patient samples in the order P1 > P2 > P3. (B) Confocal microscopic images of patient cells treated with Tf−nAlb-Soraf and stained with AnnexinV-FITC and PI, showing apoptosis in all samples. (C) Immunoblot analysis of MCL-1, pSTAT5, and STAT5 with α-tubulin as loading control innanomedicine treated sample.

Figure 10. Schematic representation of molecular mechanism of drug resistance in refractory CML cells and the mechanism of action of Tf−nAlb-Soraf.

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and, more importantly, the most resistant sample P1 showedmaximum toxicity. This was again confirmed by apoptosis assayusing confocal microscopy (Figure 9B), which showedinduction of apoptosis in all three patient samples with P1registering maximum number of apoptotic cells. It may berecalled that both STAT5 and TfR expression levels of thesethree patients were in the order P1 > P2 > P3, indicating thatthe sample with highest expression of TfR and STAT5registered maximum cytotoxicity with the nanomedicine. Asmentioned earlier, this sample also harbored P-loop mutationG250E, a diagnosis with very poor prognosis.37 It is alsoimportant to note that, at 10 μM concentration, where allresistant samples showed significant toxicity, the nanomedicinecaused practically no toxicity to healthy RBC or immune cells,as shown in Figure 6. This clearly indicates the molecularspecificity of our nanomedicine and the accuracy of the rationaldesign to target both STAT5 and TfR in drug resistant CMLcells.3.10. Preferential Downregulation of Survival Kinases

by Tf−nAlb-Soraf. In the next step, we have evaluated theanti-CML mechanism of Tf−nAlb-Soraf nanomedicine bystudying the effect on targeted kinases of STAT5. Recently,Rahmani et al.28 reported that sorafenib can potentially induceapoptosis in refractory CML through a mechanism involvingdownregulation of STAT5 and MCL-1. MCL-1 is a well-characterized antiapoptotic protein of BCL-2 family that isregulated at the transcriptional level by STAT5. Rahmani etal.29 revealed that sorafenib adopts a MEK1/2/ERK1/2-independent mechanism for the inhibition of STAT5phosphorylation and downregulation of MCL-1. By immuno-blotting of these proteins, we have investigated whether Tf−nAlb-Soraf evokes a similar mechanism of cell death inrefractory leukemia through the downregulation of pSTAT5and MCL-1. α-Tubulin was used as the loading control for theimmunoblotting experiments.As shown in Figure 9C, exposure of K562R cells to 10 μM

free sorafenib for 48 h induced significant downregulation ofpSTAT5. At this exposure level, the percentage of cell death,obtained from the MTT assay, was ∼50% (Figure 8A).Interestingly, the same concentration of Tf−nAlb-Soraf causedenhanced downregulation of pSTAT5 and correspondinglybetter cell death ∼75%. However, both free drug and Tf−nAlb-Soraf did not induce downregulation of BCR-ABL, indicatingsuccessful molecular targeting. MCL-1 was also found betterdownregulated at the translational level by Tf−nAlb-Soraf.Clearly, the mechanism of cell death by Tf−nAlb-Soraf can beattributed to the inhibition of pSTAT5 and downregulation ofMCL-1 in refractory CML having BCR-ABL amplification andSTAT5 upregulation. The successful systematic induction ofapoptosis in drug resistant patient samples by TfR targetednanomedicine and its correlative effects with the level of TfRexpression and STAT5 phosphorylation status clearly suggestthe success of our rationally designed nanomedicine for treatingrefractory CML. Figure 10 schematically depicts the drugresistance mechanism of refractory CML and the molecularactivity of our nanomedicine that targets the key features ofrefractory cells leading to apoptosis. By presenting this modelof rationally designed personalized nanomedicine, we havedemonstrated that molecular diagnosis at the individual patientlevel and custom-made design and development of targetednanomedicine formulation has great potential for managingdrug resistance in cancer.

5. CONCLUSION

Drug resistance caused by various factors such as pointmutations in the drug-binding domain of the aberrant cancerkinome, amplification of oncogene, and/or preferentialactivation of alternative survival pathways is one of the majorchallenges faced by the current molecularly targeted chemo-therapeutic regime. The best clinical example is the case ofCML, where, despite having amazing initial success of smallmolecule inhibitor IM against BCR-ABL kinase of CML, drugresistance developed in ∼25% of patients leads to treatmentfailure and relapse. This demands urgent development of newtherapeutic strategies to treat refractory patients who do notrespond to existing drugs. This requires extensive molecularcharacterization of patient samples to identify the cause of drugresistance, newer targets for therapeutic intervention, andrational design of novel drug formulations. Here, we haveemployed the concept of personalized molecular diagnosis-based design and development of targeted nanomedicines toovercome drug resistance in CML. Evaluation of patientsamples and a model resistance cell line (K562R) revealed that,in addition to the amplification and mutations of BCR-ABLoncoprotein, a prominent downstream kinase, STAT5, waspreferentially activated in the refractory patient samples as wellas resistant cell line, and the extent of its activation wasproportional to the level of resistance. Additionally, we havealso found overexpression of a TfR cell membrane proteinreceptor, which was directly under transcriptional regulation ofthe aberrant STAT5 kinase. Thus, we have identified these twokey features of refractory cells and designed a “dual targeted”nanomedicine, where the aberrant STAT5 kinase was taken asthe first target of intracellular survival pathway and the trans-membrane protein TfR as the second target for the cell specificdelivery of anti-STAT5 nanomedicine. Accordingly, a multi-kinase inhibitor, sorafenib, which effectively inhibits STAT5phosphorylation, was loaded into an endogenous proteinnanoparticle based on HSA using optimized wet chemicalnanoprecipitation and conjugated with Tf ligand. The use ofprotein nanoparticles as the drug carrier provided exceptionaladvantages of improving the aqueous solubility of the otherwisehydrophobic sorafenib and ease in bioconjugation with thetargeting ligand. Thus formed Tf conjugated, sorafenib loadedalbumin nanoparticles (Tf−nAlb-Soraf) with ∼150 nm size andspherical morphology showed remarkable intracellular uptakeand enhanced toxicity to TfR/STAT5 overexpressing IM/DSresistant cells, while retaining compatibility with primary bloodcells such as RBC and WBC. More importantly, the extent ofcellular uptake and toxicity correlated well with the level ofSTAT5 and TfR overexpression, which were the key features ofthe resistant phenotype. The molecular accuracy of nano-medicine in downregulating phosphorylated STAT5 andantiapoptotic protein MCL-1 was confirmed by immunoblot-ting. In effect, our nanomedicine showed maximum toxicity tothe most resistant clinical samples, thereby emphasizing thesuccess of our rational design. In conclusion, the present studydemonstrates the potential of molecular diagnosis based designof futuristic nanomedicines to tackle critical challenges likecancer drug resistance by engineering therapeutic functionalityto endogenous proteins abundant in our own body.

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■ ASSOCIATED CONTENT*S Supporting InformationIM sensitivity on sorafenib treated cells, P-gp expression on IMrefractory patients, transcriptional regulation of TfR by STAT5,in silico analysis of sorafenib binding sites in HSA, andsorafenib dissociation studies. This material is available free ofcharge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*Amrita Centre for Nanosciences and Molecular Medicine,Amrita Institute of Medical Sciences and Research Centre,Kochi, PIN 682 041, India. E-mail: manzoork@aims.amrita.edu; Phone: (0) 484 4001324 (Extn: 8768), Fax: (0) 4842802020.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThe authors thank Department of Biotechnology (DBT),Government of India, for financial support under RNAiprogram Grant No. BT/PR10716/AGR/36/603/2008 andNanotoxicology and Nanomedicine Grant No. BT/PR9357/NNT/28/1042007. A.P.R. is thankful to CSIR for seniorresearch fellowship. M.K. thanks Prof. T. R. N. Kutty, IISc,Bangalore, for valuable suggestions and critical reading of themanuscript.

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