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BiomedicalNanotechnologyVol. 9, 1–10, 2013www.aspbs.com/jbn

Doxorubicin-Loaded Cyclic Peptide Nanotube BundlesOvercome Chemoresistance in Breast Cancer Cells

Yongzhong Wang, Sijia Yi, Leming Sun, Yujian Huang, Scott C. Lenaghan, and Mingjun Zhang∗

Nano Bio-Systems and Bio-Mimetics Lab, Department of Mechanical, Aerospace and Biomedical Engineering,The University of Tennessee, Knoxville, TN 37996, USA

The purpose of this study was to design and fabricate a new cyclic peptide-based nanotube (CPNT) and to explore itspotential application in cancer therapy. For such a purpose, the CPNT bundles with a diameter of ∼10 nm and a lengthof ∼50–80 nm, self-assembled in a micro-scaled aggregate, were first prepared using a glutamic acid and a cysteineresidue-containing cyclic octapeptide. In order to explore the potential application of these supramolecular structures, theCPNTs were loaded with doxorubicin (DOX), and further modified using polyethylene glycol (PEG). The PEG-modifiedDOX-loaded CPNTs, showing high drug encapsulation ratio, were nano-scaled dispersions with a diameter of ∼50 nmand a length of ∼200–300 nm. More importantly, compared to free DOX, the PEG-modified DOX-loaded CPNT bundlesdemonstrated higher cytotoxicity, increased DOX uptake and altered intracellular distribution of DOX in human breastcancer MCF-7/ADR cells in vitro. In addition, an enhanced inhibition of P -gp activity was observed in MCF-7/ADR cells bythe PEG-modified DOX-loaded CPNT bundles, which shows their potential to overcome the multidrug resistance in tumortherapy. These findings indicate that using cyclic peptide-based supramolecular structures as nanocarriers is a feasibleand a potential solution for drug delivery to resistant tumor cells.

KEYWORDS: Cyclic Peptide, Nanotube, Doxorubicin, Multidrug Resistance, Breast Cancer.

INTRODUCTIONNanotubes are generally defined as an elongated nano-object with a definite inner hole,1 which can be fabricatedfrom a variety of materials, such as inorganic, carbon,amyloid proteins, synthetic polymers, and other organicsystems.1 Molecular self-assembly is a main bottom-upapproach for the production of this type of supramolec-ular structures.1 Despite considerable research in differ-ent types of nanotubes in recent years, the design andfabrication of multifunctional nanotubes remains a sig-nificant challenge to the scientific community. Creatingand developing new nanotubes with different supramolec-ular structures for medicine, diagnostics, drug delivery,and tissue engineering, has been a fundamental strategyto meet this challenge.2 In the past several decades, themost extensively studied nanotube structures are carbonnanotubes (CNTs). Considering the highly desirable prop-erties of CNTs, they have been designed as nanocarriers

∗Author to whom correspondence should be addressed.Email: mjzhang@utk.eduReceived: 29 January 2013Accepted: 20 May 2013

for drug and gene delivery, scaffolds for tissue engineering,and smart devices for disease diagnosis.3 However, numer-ous concerns exist related to the toxicity, solubility andbiodegradability of CNTs,3–7 which presents a significantobstacle for the widespread use of CNTs in biomedicalapplications.Self-assembled cyclic peptide-based nanotubes (CPNTs),

formed from the stacking of individual cyclic peptides(CPs),8 has garnered significant attention since the pio-neering work in 1993.9 Extensive studies have beenconducted about the design, self-assembly, and charac-terization of CPNTs formed from CPs containing alter-nating D,L-�-amino acids, alternating D,L-�, �-aminoacids, �-amino acids, �-amino acids, and many otherarrangements.8–15 It has been demonstrated that CP unitswith an even number of alternating D,L-�-amino acidscan adopt a flat conformation with all the amino acidside chains pointing outwards and the carbonyl andamino groups of the amide backbone groups oriented per-pendicular to the ring.8�9 Alternating D,L-�-amino acidresidues with conformationally equivalent �-type dihedralangles is crucial to form closed rings by stacking theCP units through anti-parallel �-sheet hydrogen bonding.8

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For example, using the octapeptide cyclo-[(L-Gln-D-Ala-L-Glu-D-Ala)2-], hollow CPNTs, with internal diametersof 0.75 nm, were formed by stacking the CP units throughtheir amide backbone–backbone hydrogen bonding.8

In addition, the diameters of CPNTs could be easily tunedthrough variation in the number of amino acid residues inthe CP units. For instances, the decapeptide cyclo-[L-Gln-(D-Leu-L-Trp)4-D-Leu-] was reported to self-assemble theCPNTs with internal diameter of 1.0 nm,16–17 whereas thedodecapeptide cyclo-[(L-Gln-D-Ala-L-Glu-D-Ala)3-] cangenerate the CPNTs with the internal diameter of 1.3 nm.18

In addition to the tunable properties of the CPNTs, dueto the superior biocompatibility, CPNTs are believed tobe a desirable candidate for biomedical applications.8 Fur-ther, modification and functionalization of the surface ofCPNTs can be achieved through reactions with the variousside chains of the amino acids present on the CP subunits.2

Therefore, by carefully designing the repeating CP unitand optimizing the self-assembly conditions, CPNTs couldbe tailored to meet needs of specific applications.8

Currently, CPNTs have been proposed for applica-tions in various fields, including biosensing,19–21 optics/electronics,22–26 and antimicrobials.27–29 However, usingthese supramolecular structures as drug delivery systemsfor cancer therapy, to the best of the authors’ knowledge,has not been done. The purpose of this study is to designand fabricate a new eight-residue CPNT and to exploreits potential application in cancer therapy. To achieve thisgoal, the PEG-modified doxorubicin (DOX)-loaded CPNTbundles were fabricated using a glutamic acid and a cys-teine residue-containing cyclic octapeptide. In previousreports of the antimicrobial activity of cyclic peptides,it is notable that some amphipathic D,L-�-CPs have beenreported to exhibit significant antibacterial activity againsta broad spectrum of bacteria that include methicillin-resistant Staphylococcus aureus (MRSA).8�28 Inspired bythese discoveries, we further investigated the cytotoxicityof the PEG-modified DOX-loaded CPNTs against DOXsensitive and resistant tumor cell lines to explore the poten-tial of CPNT bundles to function as drug carriers for can-cer therapy.

MATERIALS AND METHODSChemicals and Cell LinesThe cyclic peptide, cyclo-(-L-Gln-D-Ala-L-Glu-D-Ala-L-Gln-D-Ala-L-Cys-D-Ala-), was synthesized by Peptide2.0 Inc (Chantilly, VA). Trifluoroacetic acid, acetonitrile,doxorubicin, and methoxypolyethylene glycol maleimide(Maleimide-mPEG5000� were purchased from Sigma-Aldrich (St. Louis, MO). LysoTracker Green DND-26and Hoechst 33342 were obtained from InvitrogenLife Technologies (Grand Island, NY). Fetal bovineserum and RPMI 1640 medium were purchased fromMediatech (Manassas, VA). Penicillin (10000 units/ml)-streptomycin (10000 �g/ml) solution was procured from

MP biomedicals (Solon, OH). The human breast tumorcell line MCF-7 and its resistant cell line MCF-7/ADRwere obtained from the Frederick National Laboratory forCancer Research (Frederick, Maryland).

Preparation and Characterization of CyclicPeptide Nanotubes (CPNTs)10 mg of cyclic peptide unit, cyclo-(-L-Gln-D-Ala-L-Glu-D-Ala-L-Gln-D-Ala-L-Cys-D-Ala-), was suspendedin 12.1 ml of distilled water. The suspension (∼1 mM) ofpeptide subunit was dissolved by adding NaOH drop-wiseto a pH of 12.0, and the resulting peptide solution wascentrifuged to remove any traces of solid matter. In orderto observe the effect of pH on the formation of CPNTs,0.5 mM of the cyclic peptide stock solution at pH 12.0 wastitrated with 2% trifluoroacetic acid in acetonitrile.9�13�30

The particle sizes and zeta potentials of the resulting nano-tubes at different pHs ranging from 12.0 to 2.0 weredetected at 25 �C using a Malvern Zetasizer, NANO ZS(Malvern Instruments Limited, UK), with a He–Ne laser(wavelength of 633 nm) and a detector angle of 173�. Allsamples were measured in triplicate.The morphology of the CPNTs was analyzed using

transmission electron microscopy (TEM). Briefly, theCPNT particles were gradually formed at pH 2.0 as awhite suspension over a period of hours after titration with2% trifluoroacetic acid in acetonitrile. Nanotubes werethen collected by centrifugation and washed repeatedlywith distilled water to remove excess acids and salts.9�13�30

A drop of the CPNT suspension was then dried on a car-bon film-coated copper grid, then negative stained with0.5% (w/v) phosphotungstic acids, and observed usingTEM (Zeiss LIBRA 200 FE, Germany) at 80 kV.

Preparation and Characterization of DOX-LoadedCPNT Bundles (CPNT/DOX) and PEG-ModifiedCPNT/DOX Bundles0.5 mM of CP aqueous solution at pH 12.0 was diluted to0.25 mM by the addition of same volume of HEPES buffer(20 mM, pH= 7.0). The resulting CP solution was adjustedusing 6 N HCl to a pH of 8.0, 7.0 and 6.0, respectively.100 �l of 3 mM DOX in HEPES buffer was then addedto 900 �l of 0.25 mM CP solution with different pH, asprepared above. The mixtures of CP and DOX in HEPESbuffer (pH 8.0, 7.0 and 6.0) were placed overnight at roomtemperature. The nanotube/drug complexes between CPNTand DOX were centrifuged at 5000 rpm for 10 min, andthe precipitates were washed three times with distilledwater to remove any excess acids and salts. The mor-phology of the precipitated CPNT/DOX aggregates wasexamined using SEM (LEO 1525, high resolution FE-SEMsystem, Germany). In order to prepare the PEG-modifiedDOX-loaded CPNTs (PEG-modified CPNT/DOX), 14 mgof maleimide-mPEG5000 was dissolved in 1 ml of dis-tilled water (pH 7.0). The precipitates of the CPNT/DOX,

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prepared using the above procedure at pH 7.0, were imme-diately suspended in 1 ml of maleimide-mPEG5000 aque-ous solution, and the maleimide-thiol reaction betweenthe CP and PEG ([PEG]/[CP]= ∼ 12.4, molar ratio) wasperformed overnight at room temperature. The resultingPEG-modified CPNT/DOX was purified by column sep-aration using Sephadex G75 to remove any unreactedPEG and salts. The purified PEG-modified CPNT/DOXwas then imaged using SEM. The PEG concentrationin the purified PEG-modified CPNT/DOX was quanti-fied using a colorimetric PEG assay based on the forma-tion of PEG-barium iodide complexes31�32 after DOX wasremoved from the PEG-modified CPNT/DOX by dialysis(MWCO= 12�000 Da) against distilled water (pH 5.5) forthree days.

Cytotoxicity of PEG-Modified CPNT/DOXBundles on Tumor CellsThe cytotoxicity of the PEG-modified CPNT/DOX wasassessed by the MTT (3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay as previouslyreported.33�34 Briefly, 1× 104 tumor cells (human breastcancer cell lines, MCF-7 and MCF-7/ADR) were seededin 96-well plates in 100 �l of RPMI 1640. Serial dilutionsof the PEG-modified CPNT/DOX were added to the plateand incubated at 37 �C in 5% CO2 for 48 hr. 10 �l ofthe MTT stock solution (5 mg/mL in PBS, pH 7.4) wasthen added to the wells, and the plates were incubated at37 �C for another 4 hours. The medium was then com-pletely removed and 100 �l of DMSO was added to eachwell to solubilize the dye. The absorbance was measuredusing a microplate reader (Bio-Tek �Quant) at 570 nm,and the concentration of drug that inhibited cell survival by50% (IC50� was determined from cell survival plots usingthe “DoseResp” function of OriginPro 8.0.

Cellular Uptake AssayThe cellular uptake of the PEG-modified CPNT/DOX wasquantified according to the reported method.35 Briefly,tumor cells were seeded onto 6-well plates at densities of2× 106 cells/ml and incubated at 37 �C until 70% con-fluence was reached. Free DOX solution or PEG-modifiedCPNT/DOX at a DOX concentration of 10 �M was thenadded and incubated for 4 h at 37 �C. The medium wasremoved and cell monolayers were washed with cold PBSthree times. After trypsinization, the cell associated fluo-rescence was measured immediately using an Epics XLAnalyzer (Beckman Coulter Inc., Brea, CA) by collect-ing 20000 events for each sample. Each experiment wasperformed in triplicate.

Confocal Laser Scanning MicroscopyBoth cells (MCF-7 and MCF-7/ADR) were grown oncover slips to 50% confluence and incubated with freeDOX solution or PEG-modified CPNT/DOX bundles at

DOX concentrations of 10 �M at 37 �C for 4 h. Thecells were then incubated with 100 nM LysoTracker greenDND-26 and 4 �M Hoechst 33342 for 30 min prior tovisualization for endolysosome and nuclear labeling.35 Thecell monolayers were then washed three times with PBS,and immediately imaged with a FluoView FV1000 Confo-cal Microscope (Olympus, Japan).

P -gp Function AnalysisThe intracellular accumulation of Hoechst 33342, a spe-cific substrate of P-gp, was used as an index of Pgpactivity.36–38 Both tumor cell lines were grown in 12-wellplates to 80% confluence and treated with free DOX solu-tion or PEG-modified CPNT/DOX bundles at DOX con-centrations of 10 �M at 37 �C for 4 h. 4 �M Hoechst33342 was then added to each well and incubated at 37 �Cfor another 30 min. After the incubation, the cell mono-layers were washed three times with PBS and lysed with1 mL of 0.1% v/v Triton-X 100 dissolved in 0.3% NaOH.A 0.5 ml aliquot was then used for the determination of theHoechst content, using a Fluorescence Microplate Reader(BioTek, Winooski, VT). Excitation and emission wave-lengths were 370 and 450 nm, respectively. Another 0.5 mlaliquot was also used to quantify the protein concentra-tion using the BCA assay (Pierce, Rockford, IL) followingthe manufacturer’s protocol. The intracellular fluorescencewas standardized to nmol of Hoechst/mg of cell proteinsusing a calibration curve.

Statistical AnalysisThe data were expressed as mean ± S.D. Statistical signif-icance was determined using a one-way ANOVA followedby a Student’s t test for multiple comparison tests. A pvalue of < 0�05 was considered statistically significant.

RESULTS AND DISCUSSIONpH-Driven Self-Assembly of CyclicPeptide Nanotube (CPNT) BundlesIn this study, a new eight-residue cyclic peptide that con-tains a cysteine residue and a glutamic acid residue atthe symmetric positions on the peptide unit (Scheme 1(a))was used to form the CPNTs. According to the previ-ous reports, cyclic peptide units with an even numberof alternating D,L-�-amino acids can self-assemble to asupramolecular nanotube structures through an extensivenetwork of hydrogen bonds.8�9 The glutamic acid sidechain functions as a triggering mechanism for initiationof the self-assembly process, and protonation of the car-boxylate groups of glutamic acid in an acidic environ-ment should lead to the formation of the CPNTs. As such,we predicted that the self-assembly of the CPNTs was apH-dependant process (Scheme 1(b)). First, we examinedthe pH-dependant profile of self-assembly of the CPNTs.As shown in Figure 1(a), the initial formation of CPNTs,

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Scheme 1. Structure of the cyclic octapeptide (a) used in this study, pH-driven self-assembly of the blank cyclic peptide nano-tube (CPNT) bundles (b), and fabrication of PEG-modified DOX-loaded CPNT bundles (c).

as determined by dynamic light scattering (DLS), wasobserved at pH 6.0, with the micro-scaled nanotube aggre-gates appearing at lower pHs. Due to protonation of thecarboxyl group of the glutamic acid side chain, causing ahydrophobic property on the surface of the CPNT,8�9 thenanotube aggregates were formed in acidic pHs, which was

(a)

(b) (c)

Figure 1. pH-driven self-assembly of cyclic peptide nano-tube (CPNT) bundles. (a) Self-assembly profile of thecyclic octapeptide, cyclo-(-L-Gln-D-Ala-L-Glu-D-Ala-L-Gln-D-Ala-L-Cys-D-Ala-), over a pH range of 2.0–12.0; (b)–(c) The rep-resentative TEM images of the CPNT bundles prepared at pH2.0 by trifluoroacetic acid/acetonitrile method (see Materialsand Methods).

confirmed by the particle size (3–4 �m, Fig. 1(a)). At thesame time, due to the protonation, the zeta potentials of thenanotube aggregates increased from ∼−20 mV to ∼ 0 mVwhen the nanotubes spontaneously self-assembled at acidicpHs ranging from 6.0 to 2.0 (Fig. 1(a)). We also examinedthe morphology of the CPNTs prepared at the most acidicpH 2.0 using TEM. As shown in Figures 1(b)–(c), indi-vidual nanotubes found in the aggregates had lengths of∼ 50–80 nm and diameters of ∼ 10 nm. It has been previ-ously reported that the diameter of an un-aggregated CPNTformed from cyclic octapeptides was demonstrated to be< 1 nm.8�9 Due to their hydrophobic surface, the CPNTscan easily aggregate in acidic environments leading to anincrease in the particle size.9 From the TEM analysis,individual nanotubes with ∼10 nm in diameter and ∼ 50–100 nm in length were the organized bundles of tightlypacked individual CPNTs, which is consistent with previ-ous studies.9 In this study, we designate these individualnanotubes observed from the TEM images (Figs. 1(b)–(c))as the tightly packed CPNT bundles.

High DOX Loading in CPNT Bundlesand Site-Specific PEGylation ofDOX-Loaded CPNT BundlesThe purpose here is to fabricate a new nanocarrier usingCP for potential biomedical applications. We hypothe-sized that the individual CPNT bundles with the diame-ter of ∼ 10 nm and the length of ∼ 50–80 nm preparedby protonation of the carboxylate group would be effec-tive nanocarriers for drug delivery and cancer therapy if(1) drugs could be incorporated into the CPNT bundles,and (2) the nanotube aggregates could be effectively dis-persed using a proper hydrophilic polymer in aqueoussolution. First, we utilized the carboxyl group of the glu-tamic acid side chain of the CP subunit to form ion-pair

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(a)

(d) (e)

(f) (g)

(b) (c)

Figure 2. Doxorubicin was loaded into the CPNT bundles and PEG-modified CPNT/DOX bundles. (a)–(c) Ion-pair complex for-mation of CPNT/DOX at different pHs: (1) Cyclic peptide and DOX mixture; (2) DOX solution; (d)–(e) SEM images of CPNT/DOXbundles in aggregates at pH= 7�0; (f)–(g) SEM images of PEG-modified CPNT/DOX bundles.

complexes with a cationic antitumor drug, doxorubucin(DOX) (Scheme 1(c)).33 As shown in Figures 2(a)–(c),when the pH of the mixed solution of DOX and CP wasadjusted to 7.0 and 6.0, the DOX-associated CPNT bundleswere formed. The DOX-containing CPNT bundles rapidlyaggregated, due to hydrophobic surface of the CPNTs,leading to overnight precipitation. The encapsulation ratioof DOX within the CPNT bundles of the aggregates wasabout 58.3% and 66.7% at pH 7.0 and 6.0, respectively.However, no precipitates were observed in the mixed solu-tion of DOX and CP at pH 8.0. Presumably, there was avery small fraction of DOX protonated at pH 8.0 due tothe pKa of DOX (∼ 8.3–8.5),39 which resulted in failure toform the ion-pair complexes between the carboxyl groupand DOX. Such that, the nanotubes cannot be formed and

no precipitates were observed at this base pH value. We fur-ther examined the morphology of the DOX-loaded CPNTaggregates using SEM. As shown in Figures 2(d)–(e), theCPNT aggregates displayed scaffold-like structures, withparticle sizes of ∼ 1–2 �m measured by DLS method (datanot shown); however, individual CPNT bundles within theaggregates had diameters of∼ 50 nm and lengths of∼ 200–300 nm. Due to their large size, the micro-sized CPNTaggregates would not be an effective carrier for biomedicalapplications; however, we hypothesized that the individualCPNT bundles may be a potential drug delivery system ifthey could be properly dispersed.In order to disperse the CPNTs in solution, we utilized

the cysteine residue on the CP subunits for site-specificPEGylation using maleimid-mPEG5000 acting as the

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hydrophilic polymer (Scheme 1(c)). The cysteine residuewas designed so that its position was directly opposite ofthe glutamic acid residue (Scheme 1(a)). The thiol-groupin cysteine was used as a specific PEGylation site for amaleimide-modified PEG.40 We postulated that sulfhydrylspecific PEGylation of the DOX-loaded CPNTs wouldfacilitate the formation of a dispersed nanotube popu-lation. As shown in Figures 2(f)–(g), the PEG-modifiedCPNT/DOX bundles were well-dispersed, with an averagediameter of ∼ 50 nm and length of ∼ 200–300 nm. Aftermodification with PEG, the molar ratio of PEG to CPunits in the PEG-modified CPNT bundles was estimatedto be ∼ 0.21, and the encapsulation ratio of DOX withinthe CPNT bundles slightly decreased from 58.3% of theunmodified form to 54.2% at pH 7.0.

DOX-Loaded CPNT Bundles SensitizedResistant MCF-7/ADR Tumor Cells In VitroDue to high DOX loading within the PEG-modifiedCPNT/DOX bundles and the nano-scale dimensions of thewell-dispersed nanotubes, we hypothesized that they mighthave a potential as a drug delivery system for cancer ther-apy. To test the hypothesis, we first analyzed the cyto-toxicity of the PEG-modified CPNT/DOX bundles againstthe human breast tumor cell line MCF-7. As shown inFigure 3(a) and Table I, the PEG-modified CPNT/DOXbundles showed higher cytotoxicity against MCF-7 cellscompared to free DOX (0.53 �M vs. 0.16 �M), indicat-ing potential application of the PEG-modified CPNT/DOXbundles as a drug nanocarrier for cancer therapy. Apartfrom sensitive MCF-7 tumor cells, we further tested thecytotoxicity of the PEG-modified CPNT/DOX bundlesagainst a multidrug resistant cell line, MCF-7/ADR. Asshown in Figure 3(b) and Table I, the PEG-modifiedCPNT/DOX bundles showed higher cytotoxicity towardcultured resistant MCF-7/ADR cells than free DOX as well(0.84 �M vs. 4.21 �M). However, compared to the cyto-toxicity between the sensitive MCF-7 cells and resistantMCF-7/ADR cells, the PEG-modified CPNT/DOX bun-dles had much less effect on drug efficacy in the caseof sensitive MCF-7 cells than resistant MCF-7/ADR cells(3.3-fold decrease vs. 5.01-fold decrease in IC50�. Interest-ingly, the cytotoxicity of PEG-modified CPNT/DOX bun-dles in the resistant cells was decreased to the point wherethe sensitive cells responded to free DOX, resulting in thecomparable IC50 values of the same order of magnitude(0.84 �M vs. 0.53 �M, Table I). In order to rule out anypossible cytotoxicity from the unloaded carrier, we con-ducted the MTT assay in both tumor cell lines using theblank PEG-modified CPNT bundles as a control. As shownin Figure 3(c), there was no obvious cytotoxicity fromthe PEG-modified CPNT bundles at the cyclic octapep-tide concentration ranging from 0.01 �M to 25 �M. Theresult is consistent with the previous report that cyclic pep-tides had no significant cytotoxicity against rat hepatoma

Figure 3. Cytotoxicity of PEG-modified CPNT/DOX bundlesagainst MCF-7 (a) and MCF-7/ADR cell lines (b). Blank PEG-modified CPNT bundles (c) were used as a control.

cells below 100 �M.41 According to a 54.2% of DOXencapsulation ratio in the PEG-modified CPNT bundlesand the initial feeding amounts of cyclic peptide/DOX(0.225 �mol/0.3 �mol, see Section 2.3), 25 �M ofcyclic peptide corresponds to 18.1 �M of DOX equivalentconcentration in the PEG-modified CPNT/DOX bundles.From the Table I, 18.1 �M of a DOX equivalent concen-tration is far higher than the IC50 of the PEG-modifiedCPNT/DOX bundles against MCF-7 (∼ 0.16 �M) andMCF-7/ADR cells (∼ 0.84 �M), indicating that there isno obvious cytotoxicity from the CPNT bundles them-selves in the DOX concentration range used for MTT assayin this study. The cytotoxicity data in both sensitive and

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Table I. IC50 (mean± S.D.) and resistance reversion index(RRI) of PEG-modified CPNT/DOX bundles against humanbreast MCF-7 cells and multidrug resistant MCF-7/ADR cells.

MCF-7 MCF-7/ADR

Formulation IC50��M) IC50��M) RRI∗

Free DOX 0�53±0�05 4�21±0�65 –PEG-modified 0�16±0�03∗∗ 0�84±0�04∗∗ 5.01

CPNT/DOX bundles

Notes: ∗Resistance reversion index (RRI), i.e., ratio of IC50 of free DOX solu-tion to PEG-modified CPNT/DOX bundles. ∗∗P < 0�01, versus free DOX.

resistant cells indicate that the PEG-modified CPNT/DOXbundles have the potential for DOX delivery to the resis-tant cells, and are able to sensitize the multidrug resis-tant cells, exhibiting higher cytotoxicity compared to freedrug.

DOX-Loaded CPNT Bundles EnhancedIntracellular DOX Uptake and Altered IntracellularDistribution of DOX in Tumor CellsIt has been reported that the multidrug resistance incancer is a major cause for clinical failure of anti-cancer chemotherapy.34 Therefore, the sensitizing effectof the PEG-modified CPNT/DOX bundles against mul-tidrug resistant cells is a favorable property for cancertherapy. It has been demonstrated that increased uptake,different intracellular localization, inhibition of ABC pumpactivity, and enhanced apoptotic effect, play importantroles for overcoming drug resistance in cancer by differ-ent drug delivery carriers.42�43 In order to elucidate themechanism of the sensitizing effect of the CPNT bundlestoward the resistant MCF-7/ADR cells, we first tested theDOX uptake in both sensitive and resistant MCF-7 cellsusing flow cytometry by measuring cell-associated DOX

(a) (b)

Figure 4. Flow cytometry analysis for cellular uptake of PEG-modified CPNT/DOX bundles in human breast cancer MCF-7 cells (a)and the resistant counterpart MCF-7/ADR cells (b). Both cells were treated with the PEG-modified CPNT/DOX bundles or freeDOX at a DOX concentration of 10 �M for 4 h, and then the mean DOX fluorescence associated with the cells was measured bycollecting 20000 events for each sample.

fluorescence intensity. As shown in Figure 4, after a periodof 4 h, the uptake of DOX was significantly increased bythe PEG-modified CPNT/DOX bundles in both cells com-pared to free DOX at a DOX concentration of 10 �M.To further determine the sub-cellular distribution of PEG-modified CPNT/DOX bundles, the confocal imaging wasperformed, and the nuclei and endolysosome were labeledwith respective nucleus-selective dye (Hoechst 33342,blue) and acidic endolysosome-selective dye (LysoTrackergreen DND-26). As shown in Figure 5, a different intra-cellular distribution between PEG-modified CPNT/DOXbundles and free DOX was observed in both cells after a4-h treatment. MCF-7 cells and MCF-7/ADR cells treatedwith PEG-modified CPNT/DOX bundles showed a higherintracellular accumulation of DOX compared to the treat-ment with free DOX, which is consistent with the datafrom flow cytometry (Fig. 4). However, the majority ofDOX in both cells treated with PEG-modified CPNT/DOXbundles was predominantly located in the endolysosomalcompartment, whereas most of the free DOX was locatedoutside endolysosomes. It has been reported that nanopar-ticles internalized via endocytosis were typically foundmainly within endosomes or lysosomes.44 Therefore, wepresume that the PEG-modified CPNT/DOX bundles wereprobably taken up by the endocytic pathway in both tumorcell lines. In addition, due to a significant increase ofintracellular DOX in both cells treated with the PEG-modified CPNT/DOX bundles, the nuclear DOX localiza-tion accordingly increased compared to the treatment offree DOX. Enhanced nuclear localization of DOX is adesirable property in order to maximize DOX’s antitumoractivity.45 Overall, the increased uptake and altered intra-cellular distribution of DOX in both tumor cells probablyis one cause that led to higher cytotoxicity by the PEG-modified CPNT/DOX bundles.

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Figure 5. Intracellular distributions of PEG-modified CPNT/DOX bundles and free DOX at DOX concentration of 10 �M in humanbreast cancer MCF-7 cells (a) and the multidrug resistant cell line MCF-7/ADR (b). The cells were incubated with both samples at37 �C, 5% CO2 for 4 h, and then 100 nM Lysotracker Green DND-26 and 4 �M Hoechst 33342 were added for a 30-min incubationprior to visualization by confocal microscopy. Scale bars represent 20 �m.

DOX-Loaded CPNT Bundles Inhibited P -gpFunction in MCF-7/ADR Cell LineSince P -gp plays an important role in determining andconferring multidrug resistance in various tumors,46 wepostulated that the increased DOX uptake in the resis-tant MCF-7/ADR cells was attributable to the inhibitionof P -gp efflux activity by the PEG-modified CPNT/DOXbundles. To validate this hypothesis, we used Hoechst33342, a commonly used fluorescent substrate of P -gp, toanalyze the P-gp activity in multidrug resistant cells aftertreatment with the PEG-modified CPNT/DOX bundles.As shown in Figure 6, in the case of MCF-7/ADR cellstreated with the PEG-modified CPNT/DOX bundles and

free DOX for 4 h, both treatments significantly enhancedthe accumulation of Hoechst 33342 at a DOX concentra-tion of 10 �M. Compared to free DOX, more fluores-cent dye significantly accumulated in the resistant cellstreated with the PEG-modified CPNT/DOX bundles. How-ever, the treatments with both PEG-modified CPNT/DOXand free DOX didn’t alter the Hoechst accumulation inthe sensitive MCF-7 tumor cells. The quantitative dataof intracellular Hoechest accumulation in sensitive andresistant cells are consistent with the images from theconfocal analysis in Figure 5. However, considering thefact that enhanced DOX uptake in the sensitive cellstreated with the PEG-modified CPNT/DOX bundles was

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Wang et al. Doxorubicin-Loaded Cyclic Peptide Nanotube Bundles Overcome Chemoresistance in Breast Cancer Cells

Figure 6. P -gp activity in the presence of free DOX and PEG-modified CPNT/DOX bundles in the human multidrug resistantbreast tumor MCF-7/ADR cell line. The cells were incubatedfor 4 h in fresh medium (ctrl) or with 10 �M doxorubicin (DOX)or PEG-modified CPNT/DOX bundles, and 4 �M Hoechst 33342was added to the culture media for another 30-min incubationprior to quantitative analysis. The cultures were then washed,lysed and analyzed fluorimetrically for the intracellular contentof the dye. The dye concentration was standardized by theprotein concentration which was measured with BCA assay.The sensitive breast tumor MCF-7 cells were used as a con-trol. Measurements were performed in triplicate and data arepresented as mean±SD (n = 3). ∗∗p < 0�01, versus MCF-7/ADRcells ctrl; ∗P < 0�001, versus MCF-7/ADR cells ctrl; †P < 0�05,versus MCF-7/ADR cells treated with free DOX.

also observed (Fig. 4(a)), we propose that the inhibitiveeffect of P -gp exerted by the PEG-modified CPNT/DOXbundles is, at least partially, attributable to the enhancedDOX uptake in the resistant MCF-7/ADR tumor cells.

CONCLUSIONIn this study, cyclic peptide-based nanotube (CPNT) bun-dles with a diameter of ∼ 10 nm and a length of ∼ 50–80 nm were prepared using a glutamic acid and cysteineresidue-containing cyclic octapeptide. In order to explorethe potential application of these supramolecular struc-tures as a nanocarrier for cancer therapy, PEG-modifiedDOX-loaded CPNT bundles were fabricated, and showeda high drug encapsulation ratio and good dispersion witha diameter of ∼ 50 nm and a length of ∼ 200–300 nm.More importantly, the PEG-modified CPNT/DOX bundlesdemonstrated the activity to overcome the multidrug resis-tance in a human breast cancer cell line in vitro. Fur-ther, the PEG-modified CPNT/DOX bundles increased theuptake of DOX, altered the intracellular DOX distribution,and inhibited P -gp activity in MCF-7/ADR cells. The find-ings of this study highlight that the PEG-modified DOX-loaded CPNT bundles may be a useful nanocarrier for drugdelivery to resistant tumor cells.

Acknowledgments: This work was supported in partby the Office of Naval Research Young Investigator Pro-gram (award ONR-N00014-11-1-0622). The authors aregrateful for the support.

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