International Journal of Pharmaceutics 498 (2016) 40–48

TKD peptide as a ligand targeting drug delivery systems tomemHsp70-positive breast cancer

Ying Menga,b, Shanshan Wanga, Chengyi Lia, Min Qiana, Yufan Zhenga, Xueying Yanb,Rongqin Huanga,*aDepartment of Pharmaceutics, School of Pharmacy, Key Laboratory of Smart Drug Delivery, Ministry of Education, Fudan University, 826 Zhangheng Road,Shanghai 201203, Chinab School of Pharmacy, Heilongjiang University of Chinese Medicine, Harbin 150040, China

A R T I C L E I N F O

Article history:Received 12 September 2015Received in revised form 14 November 2015Accepted 6 December 2015Available online 8 December 2015

Keywords:TKD peptidememHsp70Active targetingMicellesBreast cancer

A B S T R A C T

Breast cancer has been considered as a serious threat to females’ life. Active targeting drug delivery is apotential strategy in cancer therapy, which however is hindered by the targeting efficiency. Herein, a 14-mer peptide (TKD) derived from the oligomerization domain of membrane heat-shock protein 70(memHsp70), for the first time, was exploited as a tumor-targeting ligand to modify polymeric micelles.NMR results demonstrated the successful synthesis of TKD-PEG-PLGA polymer. No difference wasobserved in the drug release between TKD-modified doxorubicin (DOX)-loaded micelles (TKD-D-M) andunmodified counterparts. The modification of TKD mediated apparently higher cellular uptake withinmemHsp70-positive MCF-7 cells, compared to normal MCF-10A cells. Excessive TKD pretreatmentsignificantly inhibited the cellular uptake of TKD-D-M, indicating the receptor-mediated mechanism.Enhanced accumulation of TKD-D-M within the tumor of MCF-7 bearing mice further demonstrated thetargeting ability of TKD in vivo. CCK-8 assay showed that the modification of TKD significantly increasethe anti-proliferation effect against MCF-7 cells. The findings demonstrated that TKD peptide is apotential ligand which can target drug delivery systems to memHsp70-positive breast cancer.

ã 2015 Elsevier B.V. All rights reserved.

Contents lists available at ScienceDirect

International Journal of Pharmaceutics

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1. Introduction

Breast cancer is the most frequently diagnosed cancer amongwomen, with the incidence of 29%, followed by lung (13%) andcolon & rectum (8%) cancers (Siegel et al., 2015). In clinicaltreatment, women with triple-negative breast cancers, had poorersurvival than those with other types of breast cancers (Sledge et al.,2014). Triple-negative breast cancers with the absence expressionof estrogen receptor (ER), progesterone receptor (PR) and humanepidermal growth factor receptor-2 (HER2) are not amenable toany form of endocrine therapy yet have high incidence ofmetastasis and poor prognosis. In spite of the treatment withcombination of surgery, radiotherapy and systemic chemotherapy,the therapeutic efficiency is still modest because of high risk ofrelapse, insufficient intracellular drug accumulation in thepathological sites and severe side effects due to unspecific cellularuptake by normal tissues (Darby et al., 2011; Liu et al., 2011;Yabbarov et al., 2013; Zhu et al., 2012). Thus, how to enhance the

* Corresponding author.E-mail address: rqhuang@fudan.edu.cn (R. Huang).

http://dx.doi.org/10.1016/j.ijpharm.2015.12.0130378-5173/ã 2015 Elsevier B.V. All rights reserved.

tumor-specific accumulation of drugs and at the same time reduceunwanted side effects has been one of principle hindrances ofbreast cancer therapy.

Tumor-targeted drug delivery is one of potential strategies tosolve these problems. Active targeting nanoparticulate drugdelivery systems which can selectively accumulate in cancer cellswithout apparent cellular uptake by normal cells, especiallybiodegradable nanoparticles, have great potential for cancertherapy with high specificity, favored tumor accumulation andreduced side effects. Due to overexpression of some receptors suchas HER2 and ER on the surface of mammary cancer cells, a series ofmultifunctional drug delivery systems have been designed foractive tumor targeting, and enhanced drug accumulations havebeen achieved after systemic administration (Ngamcherdtrakulet al., 2015; Wang et al., 2015). However, more targets besides ER,PR or HER2 should be exploited for the design of drug deliverysystems due to the refractory property of triple-negative breastcancers.

Among these receptors, membrane heat-shock protein 70(memHsp70) has attracted more and more attentions. First,memHsp70 is present on the surface of human tumor cell linesincluding breast, leukemic cells, lung and colorectal cancer cells,

Scheme 1. Illustration of the targeting behavior of TKD-D-M to memHsp70-positive cancer.

Y. Meng et al. / International Journal of Pharmaceutics 498 (2016) 40–48 41

which is also up-regulated in the metastatic tumors (Stangl et al.,2011; Sun et al., 2008). Quite a number of researches aboutHsp70 have been focused on its roles in prognostic implications,chaperone machinery and cytokine production (Asea et al., 2000;Pfister et al., 2007; Tsan et al., 2004). Second, expression ofmemHsp70 is not detected in adjacent cells and other healthytissues (Gehrmann et al., 2014). Third, high expression ofmemHsp70 has been determined in triple-negative breast cancers.Therefore, memHsp70 has been considered to be a potential targetfor diagnostic and therapeutic applications.

Recently, a 14-mer tumor peptide (TKD), the sequence derivedfrom the oligomerization domain of Hsp70, has shown specificbinding and rapid internalization in a variety of cancer cells with amemHsp70-positive phenotype in vitro. It has been reported thatTKD peptide could stimulate the activity of human nature killercells (NK cells), and attract NK cells’ migration in a concentration-dependent and highly selective manner (Gastpar et al., 2004).However, no researches have been performed, to our bestknowledge, to explore TKD peptide as a targeting ligand in drugdelivery systems.

In this work, TKD peptide was employed as a specific ligand toconjugate polymeric micelle systems to target memHsp70-positivebreast cancer. Poly(D,L-lactide-co-glycolide)-b-poly(ethylene glycol)(PEG-b-PLGA), a widely used biodegradable amphiphilic polymer,was applied to self-assemble into micelles to load anti-tumor drugdoxorubicin (DOX). The synthesis, characterizations, targeting

Table 1Size distribution, LC% and EE% of different DOX-loaded micelles (mean � SD, n = 3).

Molar percentage of TKD-PEG-PLGA Size distribution (nm

0% 24.2 � 1.78

2.5% 26.8 � 3.16

5% 25.9 � 4.49

10% 32.0 � 2.41

20% 28.1 � 3.20

40% 34.8 � 4.14

ability and anti-breast cancer effects of TKD-modified DOX-loadedmicelles (TKD-D-M) were evaluated in vitro and in vivo, usingunmodified DOX-loaded micelles (D-M) as the positive control. Thepeptide-mediated selective targeting of TKD-D-M to memHsp70-positive cancer was shown in Scheme 1.

2. Materials and methods

2.1. Materials

TKD (CTKDN NLLGR FELSG) was synthesized by ZiYu BiotechCo., Ltd. (Shanghai, China). mPEG4K-b-PLGA2.2K and MAL-PEG5.5K-b-PLGA(75/25)2.2K were purchased from Advanced PolymerMaterials Inc (Montreal, Canada). Doxorubicin (DOX) was pur-chased from Huafeng United technology Co.,Ltd (Beijing, China).Cell Counting Kit-8 (CCK-8) was bought from Dojindo Laboratories(Japan). Centrifugal filters (MWCO = 3 K Da) were purchased fromMerck Millipore Ltd. 1,1-dioctadecyl-3,3,3,3-tetramethylindotri-carbocyanine iodide (Dir) was purchased from Biotium Co., Ltd.(Shanghai, China). Trimethylamine (TEA), acetonitrile and otherreagents, if not specified, were obtained from Sinopharm Chemicalreagent Co., Ltd. (Shanghai, China).

MCF-7 cells (memHsp70-positive human breast cancer cellline) and MCF-10A cells (non-cancerous breast epithelial cell line)were obtained from America Type Culture Collection (ATCC), andrespectively incubated in RPMI-1640 and DMEM medium supple-mented with 10% fetal bovine serum (FBS), 1% penicillin, 1%streptomycin and 1% L-glutamine at 37 �C under 5% CO2 humidifiedatmosphere. All the reagents used in cell culture were purchasedfrom Gibco (Tulsa, OK, U.S.A.).

2.2. Methods

2.2.1. Fabrication of TKD-PEG-PLGATKD peptide was conjugated to MAL-PEG-PLGA via the reaction

between the sulfhydryl groups at terminal cysteine of the peptideand the C¼C double bond of maleimide groups at one end of thepolymer chain to form thioether bonds. Briefly, MAL-PEG-PLGAand TKD peptide were dissolved in PBS (pH 7.0, 0.2 M) at a molarratio of 1:1.2 and stirred at room temperature overnight. Theredundant peptide was removed via dialysis against distilledwater. Finally, the resulting complex was freeze-dried undervacuum for characterizations and further usage.

2.2.2. Preparation of micellesDOX-loaded micelles were prepared by thin-film rehydration

method (Sun et al., 2014). Briefly, DOX � HCl was dispersed inacetonitrile under sonication for 1 h, and neutralized withexcessive TEA. To prepare DOX-loaded micelles, mPEG-PLGA andTKD-PEG-PLGA were mixed according to the molar percentage ofTKD-PEG-PLGA ranging from 2.5% to 40% (Table 1), then DOX wasadded at a molar ratio of 6.6:1 to total PEG-PLGA. The solvent wasremoved by rotary evaporation at 60 �C to obtain a thin film. The

) LC% EE%

26.70 � 2.21 73.00 � 3.5328.92 � 1.58 82.39 � 2.8930.06 � 2.43 88.04 � 1.7429.62 � 3.65 88.16 � 3.3228.59 � 2.81 87.58 � 3.2724.91 � 3.52 78.72 � 2.49

42 Y. Meng et al. / International Journal of Pharmaceutics 498 (2016) 40–48

resultant thin film was hydrated with physiological saline.Unloaded DOX was discarded by ultrafiltration and measured bya fluorescence spectrophotometer (Cary Eclipse, Agilent, USA). Thedrug loading content (LC%) and encapsulation efficiency (EE%)were calculated by the following equations, respectively.

LC %ð Þ Weight of drugin the micellesWeight of drugin corporated micelles

� 100%

EE %ð Þ ¼ Weight of the drugin micellesWeight of the feeding drug

� 100%

The blank micelles were prepared as described above withoutthe addition of DOX.

2.2.3. CharacterizationsIn order to confirm the successful conjugation of TKD peptide to

PEG-PLGA polymer, both TKD-PEG-PLGA and MAL-PEG-PLGA weredissolved in deuterochloroform and 1H nuclear magnetic reso-nance (NMR) spectra were recorded utilizing a 400 MHz NMRspectrometer (Varian, USA). The sizes of different micelles freshlyprepared were determined using Zeta Potential/Particle Sizer

Fig. 1. 1H NMR spectra of MAL-PEG-PLGA and TKD-PEG-PLGA (A). TEM image ofblank TKD-modified micelles (B).

(Malvin, U.S.A.). The morphology of blank TKD-modified micelleswas characterized by transmission electron microscopy (TEM)using a negative staining method with phosphotungstic acid.

2.2.4. Drug release analysisThe in vitro release of DOX from different micelles were

investigated. Briefly, 1 mL DOX (0.5 mg) loading micelles weresubjected to dialysis (MWCO = 3500 Da) and incubated in PBS(0.01 M, pH 7.4) for 48 h at 37 �C. At predetermined time points,10 mL external release medium was withdrawn and equal amountof fresh PBS was replenished. The concentration of DOX wasdetermined by a fluorescence spectrophotometer with excitationwavelength at 488 nm. The in vitro release experiments werecarried out in triplicate and the release profiles were plotted withcumulative drug release as a function of time.

2.2.5. Optimization of the formulationsFor qualitative experiment, TKD-D-M with different TKD molar

percentage were incubated with MCF-7 cells at the sameconcentration of DOX. Then the cellular uptake was visualizedunder a fluorescent microscope. Briefly, MCF-7 cells were seeded in96-well plates at the density of 1 �104 cells per well, allowing forattachment for 24 h. Afterwards, cell culture media were removedand cells were washed with PBS. After that, cells were treated withTKD-D-M with the TKD-PEG-PLGA molar percentage ranging from2.5% to 40% at a same concentration of DOX (20 mg/mL) for 2 h.After the incubation, the drugs were removed and cells werewashed three times with PBS, fixed with 4% formaldehyde for15 min and were examined under a fluorescent microscope (LeicaDMI4000D, Germany).

For quantitative studies, MCF-7 cells were seeded in 24-wellplates at the density of 5 �104 cells per well. After attaching for24 h, cell culture media were removed and cells were washed withPBS. Then cells were incubated with DOX-loaded micelles withdifferent molar percentage of TKD-PEG-PLGA at the DOXconcentration of 100 mg/ml for 4 h. After that, cells were furtherwashed with PBS for three times, lysed, and the mean protein levelwas detected using the Bradford method (Bradford, 1976). Inaddition, DOX amounts within cells were measured using afluorescence spectrophotometer with the excitation wavelength at488 nm. Finally, the values of cellular DOX to cell proteins per unitwas calculated.

Fig. 2. In vitro cumulative DOX release from TKD-D-M and D-M in PBS (pH = 7.4) for48 h.

Y. Meng et al. / International Journal of Pharmaceutics 498 (2016) 40–48 43

2.2.6. Evaluation of targeting capability and cellular uptakemechanism

To evaluate cellular selectivity of TKD-D-M, memHsp70-positive MCF-7 cells and memHsp70-negative non-cancerousMCF-10A cells were seeded in 96-well plates at the density of8 � 103 cells/well, respectively. After attachment for 24 h, cellculture media were removed and both kinds of cells were treated

Fig. 3. Cellular uptake of TKD-D-M with different molar percentage of TKD-PEG-PLGA asignal. Bar = 200 mm. (For interpretation of the references to color in this figure legend

with 100 mL TKD-D-M and D-M with concentration of DOX at50 mg/mL. After incubation for 1 h, cells were washed three timeswith PBS, fixed with 4% formaldehyde for 15 min and wereexamined under a fluorescent microscope.

In addition, competitive inhibition assay was performed toexplore the cellular uptake mechanism. MCF-7 cells were pre-treated with TKD peptide at the concentration of 1 mg/mL for

fter incubation with MCF-7 cells for 2 h at the same DOX concentration. Red: DOX, the reader is referred to the web version of this article.)

44 Y. Meng et al. / International Journal of Pharmaceutics 498 (2016) 40–48

30 min, following treated with TKD-D-M and D-M with DOXconcentration at 100 mg/mL, respectively. After incubation for 1 h,cells were washed three times with PBS, fixed with 4% formalde-hyde for 15 min and were examined under a fluorescentmicroscope.

To further verify the specificity of the active targeting micelles,both cell lines were seeded in a glass bottom cell culture dishes atthe density of 4 �104 cells/dish, allowing attachment for 24 h. Aftercell culture media were removed, 800 mL TKD-D-M and D-M withDOX concentration at 50 mg/mL were added, respectively. Thefollowing steps were the same as above-mentioned. After fixedwith 4% formaldehyde, confocal laser scanning microscopy (CarlZeiss LSM710, Germany) was used to examine the uptake by bothkinds of cells.

2.2.7. Biodistribution in vivoFemale Balb/c nude mice with body weight around 20 g were

maintained under specific pathogen free conditions at theDepartment of Experimental Animals, Fudan University (Shanghai,China). To obtain MCF-7 tumor-bearing mice models, 2 � 106 MCF-7 cells suspended in 100 mL PBS, were injected under the rightshoulder subcutaneously. Dir-loaded micelles (TKD-Dir-M and Dir-M) were prepared in the same way with preparation of loadingDOX. When the tumor reached about 150 mm3 in volume, TKD-Dir-M and Dir-M in 150 mL physiological saline were injected via tailvein with Dir dose of 1 mg per nude mice. The fluorescent imageswere acquired at predetermined time points (1, 4, 8, 24 h) via anIVIS in vivo imaging system (IVIS Spectrum, Cailper PerkinE-lemmer, U.S.A.). Twenty-four hours after administration, the nudemice were sacrificed, and the major organs (heart, liver, spleen,lung and kidney) and tumor were harvested for fluorescentimaging. All animal experiments were performed in accordancewith guidelines evaluated and approved by the ethics committee ofFudan University.

2.2.8. Anti-proliferation assay in vitroTo verify the pharmacological activity of TKD-D-M and DOX-M,

the in vitro cytotoxicity tests against MCF-7 cells were conductedby CCK-8 assay. Briefly, MCF-7 cells were seeded in 96-well platesat the density of 8 � 103 cells/well, allowed for attachment of 24 h.The cells were incubated with DOX-loaded formulations atindicated concentrations (from 0.1 to 100 mg/mL). After exposurefor 24 h, cells were washed three times with PBS, replaced with

Fig. 4. Intracellular DOX level of TKD-D-M with different molar percentage of TKD-PEG-PLGA in MCF-7 cells after incubation for 4 h (n = 3).

100 mL fresh media and added 10 mL CCK-8 solution in each wellfor 2 h at 37 �C. The absorbance of each well was measured by amicroplate reader (Synergy2, Bio-tek, U.S.A.) at 450 nm. Cellswithout exposure to the DOX-loaded formulations were used ascontrols. Cell viability (%) was calculated based on the followingequation:

Cell viability ¼ Asample

Acontrol

� �� 100%

where Asample and Acontrol represent the absorbance of the sampleand control wells, respectively.

2. .2.9 Statistical analysisAll data were expressed as mean � SD (standard deviations).

Statistical significance between control and test groups wasevaluated by paired student's t test. The differences wereconsidered statistically significant when p < 0.05.

3. Results and discussions

3.1. Characterizations of TKD-PEG-PLGA and TKD-modified micelles

TKD-PEG-PLGA was synthesized via specific reaction betweenthe sulfhydryl groups from the cysteine at the terminal end of TKDpeptide and maleimide groups at the end of the polymer chain toform thioether bonds. Similar reaction has been demonstratedpreviously in our lab (Yao et al., 2015). The characterization of thesynthesized TKD-PEG-PLGA and non-conjugated PEG-PLGA wereconducted via 1H NMR spectra after lyophilization (Fig. 1A). Thepeak around 3.6 ppm was attributed to the methylene protons ofPEG chain. The signals at about 1.5, 4.8 and 5.2 ppm were ascribedto lactide methyl doublets, glycolide protons and lactide methinequartets in PLGA, respectively (Hong et al., 2014; Wang et al., 2011).The maleimide group showed its characteristic peak (HC¼CH) at6.7 ppm (Fig. 1A) (Jiang et al., 2014). After TKD conjugation,nevertheless, the peak at 6.7 ppm disappeared, confirming thereaction between sulfhydryl and maleimide groups. The peak at7.3 ppm was attributed to the solvent peak of CDCl3. The NMRresult demonstrated the successful synthesis of TKD-PEG-PLGA. Inaddition, TEM image showed that TKD-modified micelles were inhomogeneously spherical shape with the size around 20 nm(Fig. 1B).

3.2. Characterizations of different micelles

Different DOX-loaded micelles with TKD-PEG-PLGA molarpercentage ranging from 2.5% to 40% were prepared by thin-filmrehydration method (Sun et al., 2014). The results of sizedistribution, LC% and EE% of different formulations were exhibitedin Table 1. No significant difference was observed between theprepared TKD-D-M in sizes with increased molar percentage ofTKD-PEG-PLGA. All the micelles had small diameters ranging from25 to 35 nm. LC% did not show any notable differences while EE%increased as the increasing molar percentage of TKD-PEG-PLGA.The possible reason might be that surface hydration layer canstabilize the micelles with even more hydrophobic segmentaggregated together (Cheng et al., 2015). However, this effectreached a platform and then in the contrast decreased the EE%when the percentage was 40%. The results indicated that propermodification of TKD might achieve suitable LC% and EE%.

3.3. Drug release

The release behavior of the DOX-loaded micelles wereevaluated in PBS using dialysis method. The cumulative release

Fig. 5. Fluorescent microscopy images of TKD-D-M and D-M after incubated 1 h at a DOX concentration of 50 mg/mL with cells. (A–D): MCF-7 cells, (E–H): MCF-10A cells, (I–L): MCF-7 cells pretreated with excessive TKD. Red: DOX signal. Bar = 200 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to theweb version of this article.)

Y. Meng et al. / International Journal of Pharmaceutics 498 (2016) 40–48 45

Fig. 6. CLSM images of MCF-7/MCF-10A co-cultured cells incubated with D-M (A–D) and TKD-D-M (E–H) at 37 �C for 30 min. D and H: the magnification for blue line-dottedparts in C and D, respectively. Red: DOX signal. Bar = 20 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of thisarticle.)

46 Y. Meng et al. / International Journal of Pharmaceutics 498 (2016) 40–48

of DOX from the micelles to 48 h were monitored (Fig. 2). About60% drug release was achieved after 48 h. No significant differencewas observed between targeting and non-targeting micelles.

3.4. Optimization of the formulations

To optimize the molar percentage of TKD-PEG-PLGA for cellularinternalization, different TKD-D-M with molar percentage rangingfrom 2.5% to 40% with the same concentration of DOX wereevaluated in MCF-7 cells. The fluorescent microscopy resultshowed that TKD-D-M with the molar percentage of TKD-PEG-PLGA at 2.5% exhibited the highest cellular internalization by MCF-7 cells (Fig. 3). This was verified with the quantitative result (Fig. 4),which showed that the intracellular DOX amount reached over3 mg/mg cell protein in 2.5% group. It is interesting that the cellularuptake decreased with the increased molar percentage of TKD-PEG-PLGA ranging from 2.5% to 20%. And when the TKD-PEG-PLGAmolar percentage reached 40%, the uptake of DOX increased up tothat of 5%, yet lower than that of 2.5%. These results indicated thatthe amount of targeting ligand makes sense in the targeting abilityof the final formulations. And, TKD-D-M with the molar percentageof TKD-PEG-PLGA at 2.5% were used in the following experiments.

3.5. Targeting ability and competitive assay

To verify the selectivity of TKD-modified micelles tomemHsp70-positive cancerous cells, cellular uptake experimentswere performed with independent and co-culture of MCF-7 andMCF-10A cells. After incubation with TKD-D-M and D-M for 1 h, thefluorescent images were obtained and shown in Fig. 5. On onehand, the results showed that TKD-D-M had more cellular uptakethan D-M in MCF-7 cells (Fig. 5A-D), but not significant differencesin MCF-10A cells (Fig. 5E-H) at the same DOX concentration. On theother hand, more cellular uptake was observed in MCF-7 cells thanthat in MCF-10A cells when treated with TKD-D-M (Fig. 5B and F),verifying the active targeting ability with the TKD modification.The confocal laser scanning microscopy further confirmed the

results. As shown in Fig. 6, the cellular uptake was performed in co-culturing model of MCF-7 and MCF-10A cells. Although the two celllines had been miscellaneous, it was not difficult to identify themfrom each other distinctly via the different morphology. MCF-7 cells are generally with rounder shape while MCF-10A cells arefusiform (Trejo-Vargas et al., 2015). As shown in Fig. 6, when cellswere treated with D-M, it seemed that the non-cancerous cells hadmore accumulation of DOX than cancerous cells (Fig. 6A–D),properly because MCF-7 cells had drug resistance. In TKD-D-Mtreated dish, MCF-7 cells exhibited higher cellular uptake thanMCF-10A cells (Fig. 6E–H). The notable difference might bepossibly attributed to the specific binding of TKD-modifiedmicelles to MCF-7 cell membranes, leading to rapid and massivecellular uptake within MCF-7 cells.

Furthermore, competitive assay was performed to preliminarilyexplore the cellular uptake mechanism. Pretreating MCF-7 cellswith excessive TKD peptide mediated apparent decrease in thecellular uptake of TKD-D-M but not remarkable influence in thecellular uptake of D-M (Fig. 5A–D and I–L). This result demon-strated the involvement of receptor-mediated mechanism in thecellular uptake of TKD-modified micelles, which was in accordancewith results demonstrated by other groups (Liao et al., 2015; Parket al., 2015).

3.6. In vivo biodistribution

The real-time in vivo biodistribution was performed in MCF-7 tumor-bearing female nude mice. After intravenous administra-tion of TKD-Dir-M and Dir-M, IVIS spectra were gained at theestablished time points. As shown in Fig. 7A, after injection, theaccumulation of micelles in the tumor sites was apparentlyenhanced after TKD modification (Fig. 7A and B). The mice weresacrificed after 24 h and major organs and tumors were excisedsubsequently. The ex vivo results showed that significantly higheramount of TKD-Dir-M were accumulated within the tumor thanthat of Dir-M (Fig. 7D). In contrast, less accumulation of TKD-Dir-Mwas found within other main organs including heart, lung and

Fig. 7. (A and B): In vivo imaging of tumor-bearing mice injected with Dir-M (A) and TKD-Dir-M (B) for 1 h, 4 h, 8 h and 24 h, respectively. (C): ex vivo imaging of main organsafter 24 h. D: ex vivo imaging of tumors.

Y. Meng et al. / International Journal of Pharmaceutics 498 (2016) 40–48 47

kidney, compared to that of unmodified micelles (Fig. 7C). Theseresults demonstrated the excellent ability of TKD peptide to targetdrug delivery systems to memHsp70-positive tumor in vivo.

3.7. Anti-proliferation effects

The cell viability of different micelles to MCF-7 cells wasassessed using CCK-8 assay. As shown in Fig. 8, the cell viabilitydecreased while increasing the concentration of DOX-loadedmicelles. The cell inhibition rate enhanced rapidly over 50% whenthe DOX concentration reached 5 mg/mL treated with TKD-D-M.The IC50 value of different micelles were calculated with theimproved Karber's method (Du et al., 2012). The obtained IC50

value was 5.4 mg/mL for TKD-D-M and 9.1 mg/mL for unmodifiedD-M, respectively, indicating about 1.7-fold higher cytotoxicityafter TKD modification. Similar results have been obtained in othergroups who prepared different DOX-loaded micelles to treatdifferent cells for 24 h. For example, Chun Wang and co-workersdesigned PEG-b-PEYM micelles as the drug carrier to encapsulate

DOX against T98G cells, obtaining IC50 value with 2.5 mg/mL(Zhong et al., 2015). Moreover, Zhiyuan Zhong and colleagues gotan IC50 value with 3.4 mg/mL treating MCF-7 cells with for 48 h(Tang et al., 2011). There was no apparent discrepancy betweenunmodified and TKD-modified micelles when the concentration ofDOX less than 1 mg/mL.

4. Conclusions

In this work, TKD peptide was evaluated as a targeting ligand tomodify drug delivery systems to memHsp70-positive breastcancers for the first time. Apparently higher cellular uptake ofTKD-D-M in MCF-7 cells in vitro and biodistribution within tumorsin MCF-7 bearing mice in vivo, compared to unmodified D-M,demonstrated the targeting ability of the peptide. Simultaneously,TKD-modified micelles mediated higher anti-proliferation effectagainst MCF-7 cells than that of non-targeting counterparts.Conclusively, TKD could be applied as a ligand to target drugdelivery systems to memHsp70-positive cancers, which is

Fig. 8. Histogram (A) and profile (B) of cell viability for MCF-7 cells treated withTKD-D-M and DOX-M at a series of DOX concentrations from 0.1 to 100 mg/mL(n = 4). **: p < 0.01; ***: p < 0.001.

48 Y. Meng et al. / International Journal of Pharmaceutics 498 (2016) 40–48

extremely potential in the diagnosis and therapy of refractorycancers including triple-negative breast cancers.

Conflict of interest

The authors declare no conflict of interest.

Acknowledgements

This work was supported by the grants from National Key BasicResearch Program (2013CB932502) of China (973 Program),National Natural Science Foundation of China (81573002), Sino-German Research Project (GZ995) and “Zhuo Xue” Talent Plan ofFudan University.

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