Micellization of antineoplastic agent to significantly upregulate efficacy and security

Full Paper

328

Micellization of Antineoplastic Agent toSignificantly Upregulate Efficacy and Securitya

Chen Li,y Weiguo Xu,y Jianxun Ding,* Ying Zhang, Jincheng Wang,*Xiuli Zhuang, Xuesi Chen

The amphiphilic diblock copolymer composed of methoxy poly(ethylene glycol) and racemicoligoleucine was synthesized which formed into micelle with uniform size in aqueousenvironment. Doxorubicin (DOX) was loaded into micelle aided by noncovalent interactionswith high drug loading efficiency. The DOX-loaded micelle (referred as M-DOX) demonstratedthe sustained drug release in vitro and excellent antiproliferative capability toward both
MG63 and Saos-2 cells. Fur-thermore, for both MG63 andSaos-2-xenografted BALB/cnude mouse models, M-DOXexhibited enhanced intratu-moral distribution, improvedantitumor efficacy, and re-duced side effects comparedwith free DOX. Therefore, thepolypeptide micelle showed abright prospect for controlleddelivery of antitumor drugs invivo.
Dr. C. Li, Prof. J. WangDepartment of Orthopedics, The Second HospiUniversity, Changchun 130041, P. R. ChinaE-mail: [email protected]. Xu, Dr. J. Ding, Y. Zhang, Prof. X. Zhuang, PKey Laboratory of Polymer Ecomaterials, ChangApplied Chemistry, Chinese Academy of Scienc130022, P. R. ChinaE-mail: [email protected] authors contributed equally to this wo

aSupporting Information is available from theWilefrom the author.

� 2014 WILEY-VCH Verlag GmbH & Co. KGaA, WeinheimMacromol. Biosci. 2015, 15, 328–341

tal of Jilin

rof. X. Chenchun Institute ofes, Changchun

rk.

y Online Library or

wileyonlinelibr

1. Introduction

Polymericmicelle belongs to one kind of nanoscale colloids,

which is self-assembled from amphiphilic copolymers in

aqueous solution, and consists of a hydrophilic shell and a

hydrophobic core.[1–4] Recently, in oncology therapeutic

areas, micellization has been an emerging valuable

technique to optimize treatments and reduce the unex-

pected systemic toxicities of conventional antitumor

drugs.[5–8] In the meantime, it is highly hoped to improve

the life quality of patients with malignancy and prolong

their survival time.[9,10] As a promising strategy, micelliza-

tion endows antineoplastic agents with: i) increased

DOI: 10.1002/mabi.201400356ary.com

Micellization for Upregulating Efficacy and Security

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solubility and stability in water environment by the core–

shellmicrostructure,[11,12] ii) extended half-life in the blood

circulation resulted from the hydrophilic ‘‘stealth’’ shell,[13]

iii) increased accumulation in lesion sites through the

enhanced permeability and retention (EPR) effect and/or

the mediation of targeting ligand–receptor interac-

tion,[14,15] and iv) directional and intelligent delivery

adjusted by various degradation kinetics of polymeric

matrices.[16] Given the above advantages and robust

demand, thousands of innovative formulations originated

from polymeric micelles are designed and prepared for

potential chemotherapy ofmalignancy.[17,18] Among them,

several formulations are underway various phases of

clinical trials (e.g., BIND-014[19] and NK105[20]) or even

already on the market (i.e., Genexol1-PM).[21]

Although the polymeric micelle-based drug delivery

platforms have been rapidly developed during the past few

decades,[22] several deficiencies, such asweak stability, low

drug loading efficiency (DLE), and almost inevitably burst

release, dramatically compress the space of their advance-

ments.[23,24] In recent years, the noncovalent interactions,

that is, strong hydrophobic,[25] electrostatic,[26] coordina-

tion,[27,28] and/or hydrogen bond interplays,[29,30] between

copolymer backbones and drugmolecules are employed to

make up for the above shortcomings. In these interactions,

hydrogen bonds are attracting more and more attention

resulting fromtheuniqueadvantagesofhighstrength (�5–

150 kJmol�1)[31] and less derived unfavorable properties

(e.g., variation of zeta potential accompanied with electro-

static interaction).[29,32] As a typical instance, Yang and

coworkers[33,34] synthesized a series of amphiphilic block

copolymers containing poly(ethylene glycol) (PEG) and

urea-modified polycarbonates. Doxorubicin (DOX) was

loaded into the urea-containing micelles with high DLEs

ascribed to the hydrogen bonds between DOX and the urea

groups in micellar cores, which simultaneously endowed

the DOX-loadedmicelles (noted asM-DOXs) with a gradual

release behavior.[34]

Synthetic polypeptides consisting of amino acids with

amide bond as a linkage are one kind of booming polymeric

biomaterials with excellent biocompatibility and appro-

priatebiodegradability.[35,36] Polypeptidescanbeappliedas

fascinating matrices in hydrogen bond-enhanced drug

delivery systems due to the imino (donor) and carbonyl

groups (receptor) in amide bond. For the levorotatory and

dextrorotatory homopolypeptides, the intrachain and/or

interchain hydrogen bonds are the original driving forces

for the formations of secondary structures (i.e., a-helices

and b-strands).[37,38] In contrast, the complete hydrogen

bonds in racemic polypeptides with an equivalent amount

of levorotatory and dextrorotatory amino acids are

destroyed by the staggered different chiralities. The

different structural features give the racemic polypeptides

some unique characteristics as potential materials for

Macromol. Biosci. 20

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controlled drug delivery, such as high water solubility and

drug loading capability.[39,40]

Inspired by the previous backgrounds, a noncovalent

interaction-assisted drug delivery platform based on

methoxy poly(ethylene glycol)-block-oligo(D,L-leucine)(mPEG-b-O(D,L-Leu)) was exploited for controlled DOX

delivery in vitro and in vivo. In detail, mPEG-b-O(D,L-Leu)was synthesized by the ring-opening polymerization (ROP)

of two equivalent D-leucine N-carboxyanhydride (D-Leu

NCA) and L-Leu NCAwith amino-terminated mPEG (mPEG-

NH2) as a macroinitiator. The chemical structure was

exactly confirmed, and the self-assembly properties were

thoroughly revealed. DOXwas loaded intomicelle through

a universal nanoprecipitation approach. Significantly, high

DLE anddurable release behaviorwere entrusted toM-DOX

by the noncovalent interactions, e.g., hydrogen bond and

hydrophobic interactions, between DOX and O(D,L-Leu)

backbone. Inaddition, the improvedcellular internalization

and proliferation inhibition, enhanced intratumoral accu-

mulation, and upregulated antitumor efficacy and security

in vivo of M-DOXwere further systematically revealed. All

the above properties confirmed that the noncovalent

interaction-aided drug delivery system showed highly

promising for encapsulation and controlled delivery of

antineoplastic agents.

2. Experimental Section

2.1. Materials

mPEG (number-average molecular weight (Mn)¼2 000gmol�1)

was purchased from Sigma–Aldrich (Shanghai, P. R. China), and

mPEG-NH2 was synthesized following the previously reported

approach.[41,42] D-LeuandL-LeuwereobtainedfromGLBiochem,Ltd.

(Shanghai, P. R. China), and D-Leu NCA and L-Leu NCA were

synthesized according to the previous literature.[43]N,N-Dimethyl-

formamide (DMF)waspretreatedwithcalciumhydridefor72 hand

subsequently distilled under reduced pressure. DOX hydrochloride

(DOX �HCl) was sourced in ZhejiangHisun Pharmaceutical Co., Ltd.

(Zhejiang, P. R. China). 40 ,6-Diamidino-2-phenylindole (DAPI), Alexa

Fluor1488phalloidin (Alexa488) andmethyl thiazolyl tetrazolium

(MTT) were purchased from Sigma–Aldrich. Terminal deoxynu-

cleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) kit

was purchased fromRoche Company (Mannheim, Germany). Clear

6- and 96-well tissue culture polystyrene (TCP) plates were

purchased from Corning Costar Co. (Cambridge, MA, USA). The

deionizedwaterwas prepared through aMilli-Qwater purification

equipment (Millipore Co., MA, USA).

2.2. Synthesis of mPEG-b-O(D,L-Leu) Copolymer

mPEG-b-O(D,L-Leu) was synthesized by the ROP of equivalent D-Leu

NCA and L-Leu NCA with mPEG-NH2 as a macroinitiator

(Scheme 1).[16] Typically, extremely little water in mPEG-NH2

(2.0 g, 1.0mmol)wasfirst removedbybeingazeotropically distilled

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Scheme 1. Synthesis of mPEG-b-O(D,L-Leu).

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C. Li et al.

330

with toluene at 120 8C. Subsequently, D-Leu NCA (471.5mg,

3.0mmol), L-Leu NCA (471.5mg, 3.0mmol) and dried DMF were

added together. The reactionwas carried out at 20 8C for 72h. Then,

the solutionwas precipitated in diethyl ether, and the productwas

filtered out and dried to constant weight in vacuum at room

temperature. The yields were �73%.

2.3. Preparation of Micelle

Micelle was prepared by a universal dialysis approach.[16] In brief,

10.0mg of copolymer was dissolved in 5.0mL of DMF, and stirred

for 3 h at room temperature. Subsequently, 5.0mL of phosphate-

buffered saline (PBS) was dropwisely added into above solution

with stirring. After being stirred for further 3 h at room

temperature, DMF was dialyzed out against PBS for 48h, and the

volume was set to 25.0mL to obtain the micelle solution.

2.4. Characterizations

Protonnuclearmagnetic resonance(1HNMR)spectraweredetected

on a Bruker AV 400 NMR spectrometer (Ettlingen, Germany) in

deuterated chloroform (CDCl3) or trifluoroacetic acid (TFA-d).Fourier transform infrared (FT IR) spectra were determined on a

Bio-Rad Win-IR instrument (Cambridge, MA, USA) through

potassium bromide approach. Mn, weight-average molecular

weight (Mw), and polydispersity index (PDI¼Mw=Mn) were

recorded by gel permeation chromatography (GPC) with DMF as

aneluent. The criticalmicelle concentration (CMC)wasdetermined

by pyrene-probe fluorescence spectroscopy on a Photon Technolo-

gy International Fluorescence Master System with software Felix

4.1.0 (Lawrenceville, NJ, USA, lem¼390nm). The micellar mor-

phology was revealed on a JEM-1011 transmission electron

microscope (TEM; JEOL, Tokyo, Japan). Thehydrodynamic diameter

(Dh) was detected by dynamic laser scattering (DLS) measurement

on a WyattQELS instrument (DAWN EOS, Wyatt Technology

Corporation, Santa Barbara, CA, USA) with scattering angle at 908.

2.5. Preparation of M-DOX

DOX was loaded into micelle through a usual nanoprecipitation

technique. Briefly, both copolymer (40.0mg) and DOX �HCl

(10.7mg) were dissolved in 20.0mL of DMF andmixed thoroughly

by stirring at room temperature for 3 h. And then, 18.0mL of

deionized water and 2.0mL of PBS were dropwisely added to the

mixture. The above solution was stirred at room temperature for

further 5 h and subsequently dialyzed against deionized water for

24 h to eliminate excess DOX and DMF (molecular weight cut-off


� 2014 WILEY-VCH Verlag Gmb

(MWCO)¼ 3 500 Da). Finally, M-DOX was obtained by lyophiliza-

tionafterdialysis andfiltration. Thedrug loadingcontent (DLC) and

DLE ofM-DOXwere calculated by Equation (1) and (2), respectively.

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H & Co

DLC ðwt%Þ ¼ amount of drug in micelle

amount of drug-loaded micelle� 100 ð1Þ

DLE ðwt%Þ ¼ amount of drug in micelle

total amount of feeding drug� 100 ð2Þ

2.6. In Vitro DOX Release

In vitro DOX release behavior of M-DOXwas assessed in PBS at pH

7.4, 37 8C. Typically, 1.0mg of freeze-driedM-DOXwas dissolved in

10.0mL of PBS and introduced into a dialysis bag (MWCO¼ 3500

Da). The extremity-sealed dialysis bag was subsequently placed in

100.0mL of PBS to start the release detection shocking at 75 rpm,

which mimicked the circulation circumstance in vivo. At the

predetermined points in time, 2.0mL of external medium was

taken out and the equivalent fresh PBS was replenished. In

addition, the accumulativeDOX releasewas tested byfluorescence

spectroscopy through a standard curve method (lex¼480nm).

The intracellular DOX release from M-DOX was further

determined via confocal laser scanning microscopy (CLSM) and

flow cytometry (FCM) toward both MG63 and Saos-2 cells (two

types of human osteosarcoma cells). For CLSM measurement, the

cellswere seeded in6-well TCPplatesat adensityof�2.0� 105 cells

in 2.0mL of complete high glucose Dulbecco’s modified Eagle’s

medium (HG-DMEM) per well. The medium was removed after

incubation for 24h, and then free DOX (F-DOX) or M-DOX with

10.0mgL�1 DOX equivalent in 2.0mL of complete HG-DMEMwas

added. After incubation for another 2 h and removal the culture

medium, the cellswerewashed four timeswith PBS and fixedwith

4% (w/v) PBS-buffered formaldehyde for 30min at room tempera-

ture, andsubsequently counterstainedwithDAPI for cellularnuclei

and Alexa 488 for F-actin following the manufacturer’s introduc-

tions. The cellular microimages were determined on a CLSM (LSM

780, Carl Zeiss, Jena, Germany). For FCM determination, the cells

were seeded and cultured similarly as CLSM tests. After incubation

with F-DOX orM-DOX (cDOX¼ 10.0mgL�1) for 2 h and removal the

culture medium, the obtained cells were subsequently washed

three times with PBS and digested with trypsin. Subsequently, the

cells in eachwell were suspended in 1.0mL of PBS and centrifuged

for 4min at 3 000 rpm. The harvest cells were then resuspended in

0.3mL of PBS, and the data for 10 000 gated events were collected.

The analyses were performed on a flow cytometer (Beckman,

California, USA).

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2.7. In Vitro Cytotoxicity Assays

In vitro cytotoxicities of micelle and M-DOX were evaluated by a

standard MTT assay toward MG63 and Saos-2 cells. The cells were

planted in 96-well TCP plates at�7 000 cells per well in 200.0mL of

complete HG-DMEM. After culture for 24h and removal the

incubation medium, the micelle (0–40.0mg L�1), F-DOX or M-DOX

(0–10.0mg L�1DOX) in 200.0mL of completeHG-DMEMwasadded.

The cells were assessed after being incubated for 72h. The

absorbances of above media were detected at 490nm on a Bio-

Rad 680 microplate reader (Bio-Rad Laboratories, Hercules, CA,

USA). Equation (3) was employed to calculate the cell viability.

www.M

Cell viabilityð%Þ ¼ Asample

Acontrol� 100 ð3Þ

In Equation (3), Asample and Acontrol corresponded to the

absorbances of sample and control wells, respectively.

Relativ

2.8. Tumor-Bearing Animal Procedure

The five-week-old female BALB/c nude mice were handled under

protocols approved by the School of Life Sciences Animal Care and

Use Committee of Northeast Normal University, and all efforts

were made to minimize suffering. The nude mice weighting from

20 to 22g were injected subcutaneously in the armpits of right

anterior limbs with 0.1mL of cell suspension containing 3.0�106

MG63 or Saos-2 cells in PBS.

2.9. Ex Vivo DOX Fluorescence Imaging

Thequalitativeor semi-quantitative tissuedistributionofDOXwas

determined by ex vivo fluorescence imaging. When tumors were

grown to about 200 mm3, F-DOX or M-DOX at a dosage of

4.0mgkg�1 DOX was injected into the tail vein of nude mice

bearing MG63 or Saos-2 tumor. The mice were sacrificed via the

dislocation of cervical vertebra at 6 or 12h post-injection. Then

major internalorgans (i.e., heart, liver, spleen, lung,andkidney)and

tumorswere separated, and the surfacewaswashedwithPBS three

times for ex vivo fluorescence imaging of DOXwith theMaestro in

vivo ImagingSystem(CambridgeResearch&Instrumentation, Inc.,

USA). The autofluorescence in analyzed images was identified,

separated, and removed by the commercial software (Maestro 2.4).

Moreover, the average fluorescence signalswere also quantitative-

ly analyzed using the software of Maestro 2.4.

2.10. In Vivo Antitumor Evaluations

In vivo antitumor efficacies of F-DOX and M-DOX were evaluated

using nude mice xenoimplanted with MG63 or Saos-2 tumor. PBS

was used as control. Treatmentswere performed at approximately

twoweeks after plantingwhen the tumor volumes reached to�80

mm3, and this day was defined as Day 1. For each kind of tumor-

bearingmousemodels, the nudemicewere randomly divided into

threegroups (sixmiceper group): PBS (control), F-DOX, andM-DOX,

according to the tumor volumes and body weights. The tail-vein

injections of 0.2mL of PBS, or PBS solution of F-DOXorM-DOXwith


� 2014 WILEY-VCH Verlag GmaterialsViews.com

an equivalent DOX dosage of 4.0mg per kg body weight were

implemented on Days 1, 4, 7, and 10. The chemotherapy efficacy

and security evaluationwere real-time assessed bymonitoring the

tumor volume and body weight, respectively. The tumor volume

was calculated by Equation (4).

15, 15,

bH & C

Vðnm3Þ ¼ L� S22

ð4Þ

In Equation (4), L and S (mm) were the largest and smallest

diameters of tumor, respectively.

2.11. Histopathological Evaluations

Hematoxylin and eosin (H&E) staining was employed for

histopathological evaluation. The mice were sacrificed via the

dislocation of cervical vertebra on 12 days. The MG63 and Saos-2

tumors, and major internal organs (i.e., heart, liver, spleen, lung,

and kidney) were isolated, fixed in 4% (w/v) PBS-buffered

paraformaldehyde overnight, and then embedded in paraffin.

�6mm thick transverse sections were cut and then stained with

H&E to evaluate the histological alterations by microscope. The

relative necrotic area (%) was calculated by Equation (5).

e necrotic area ð%Þ ¼ necrotic area in tumor section

total area in tumor section� 100

ð5Þ

2.12. In Situ Apoptosis Detections

The TUNEL apoptosis assays of MG63 and Saos-2 tumors were

carried out with a commercial kit in accordance with the

instruction of manufacturer (Roche, Basel, Switzerland). Approxi-

mately 6mm thick tumor sections were washed, and the nicked

DNA ends were labeled by the reaction mixture. The in situ cell

apoptosis of tumor tissues was observed by CLSM.

2.13. Statistical Analyses

All experiments were repeated at least three times, and the results

were represented as means� standard deviation (SD). Statistical

significances were analyzed using SPSS (Version 18.0, Chicago, IL,

USA). p< 0.01 and p<0.001 were considered highly statistically

significant.

3. Results and Discussion

3.1. Characterization of mPEG-b-O(D,L-Leu)

The amphiphilic block copolymers with polypeptides as

hydrophobic moieties can be facilely synthesized by the

ROP of amino acid NCAmonomerswith amino-terminated

PEG as a macroinitiator.[44–46] In this work, the diblock

mPEG-b-O(D,L-Leu) copolymer was synthesized through the

ROPofequivalentD-LeuNCAand L-LeuNCAwithmPEG-NH2

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as a macroinitiator (Scheme 1). 1H NMR and FT IR spectra,

and GPC chromatogram (Figure 1 and S1, Supporting

Information) were employed to demonstrate the chemical

structure of copolymer. As shown in 1H NMR spectra

(Figure 1A), all signals assigned to the protons in PEG and

PLeu blocks were well assigned, which confirmed the

successful preparation of copolymer. The degree of

polymerization (DP) of D,L-Leu unit in O(D,L-Leu) was

evaluated to be 4 based on the integrated area of signal

(f) at�0.9 ppmassignedto thesidemethylprotonsofD,L-Leu

(—CH2CH(CH3)2) andthatofpeak (b)at�3.8 ppmattributed

to themethylene proton in PEG (—CH2CH2—). According to

the DP of D,L-Leu,Mn of copolymer (Mn;NMR) was calculated

to be 2450 gmol�1. The resultant FT IR spectra also

confirmed the generation of O(D,L-Leu) block based on the

appearance of typical amide I and II bands at 1 651 (yC55O)

and 1 550 cm�1 (yC(O)—NH), respectively (Figure 1B). The low

Figure 1. 1H NMR (in CDCl3 (a) and TFA-d (b); A) and FT IR spectra(B) of mPEG-NH2 (a) and mPEG-b-O(D,L-Leu) (b).



PDI (i.e., 1.1) indicated the relatively controlled ROP of D,L-

Leu NCA, and the relative higher Mn from GPC (Mn;GPC ¼ 4

310 gmol�1) comparedwithMn;NMR should be attributed to

the different chemical structures of copolymer and

polystyrene standards in GPC measurement.

The amphiphilic block copolymers can spontaneously

self-assemble into various nanoparticles, such as vesicle

andmicelle, drivenby themicrophase separation.[47] In this

work, the formation of micelle was first confirmed by a

widely reported pyrene-probe-based fluorescence tech-

nique.[48] The excitation spectra variation of pyrene in

mPEG-b-O(D,L-Leu) aqueous solutions at different concen-

trationswas shown in Figure S2A, Supporting Information.

As the polymer concentration gradually elevated from

2.4� 10�5 to 0.4 g L�1, the absorptionbond shifted from335

to 338nm. The red shift resulted from the movement of

pyrene probe from the aqueous environment to the

hydrophobic micellar core, which displayed the informa-

tion about the location of pyrene molecule and the

formation of micelle.[49] In addition, the CMC was

calculated to be 47.8mg L�1 from the profile of excitation

intensity ratio (i.e., I338/I335) versus the logarithmic

concentration (lg c) of copolymer (Figure S2B, Supporting

Information). The micelle from mPEG-b-O(D,L-Leu) copoly-mer presented as spherical with an average diameter of

around 65nm, which were obtained from the TEM

micrograph (Figure 2). In addition, the Dh of micelle was

quantitatively detected to be 121� 4.6 nmbyDLS (Figure 2,

inset). It should be noted that the smaller apparentmicellar

size from TEM than that determined by DLS should be

attributed to the shrinkage of micelle during the prepara-

tion of TEM specimen.

Figure 2. Typical TEM micrograph and diameter determined byTEM or DLS (inset) of mPEG-b-O(D,L-Leu) micelle.

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Scheme 2. Schematic illustrations for fabrication ofM-DOX, and its circulation, intratumoral accumulation, endocytosis, and controlled DOXrelease after i.v. administration.


www.mbs-journal.de

As shown in Figure S3, Supporting Information, the

cytocompatibility ofmicelle against bothMG63 and Saos-2

cells after incubation for 72hwereassessedbyaMTTassay.

The high cell viabilities of above two osteosarcoma cells

(>90%) indicated the little cytotoxicity of micelle with

concentration up to 40.0mg L�1. It enhanced its promising

application as a potential nanovehicle for controlled drug

delivery.[16]

Figure 3. Release profile of DOX from M-DOX in PBS at pH 7.4,37 8C. Data were presented as mean� SD (n¼ 3).

3.2. In Vitro DOX Encapsulation, Release, and

Cellular Proliferation Inhibition

As shown in Scheme 2, DOX, a widely used anthracycline

antineoplastic drug, was representatively encapsulated

into micelle through a nanoprecipitation approach to

improve the chemotherapy efficacy and reduce the severe

side effects.[9,16] The DLC and DLE ofM-DOXwere 12.0� 0.5

and 54.6� 3.6wt%, respectively. The relative high DLE

might be ascribed to the noncovalent interactions, e.g.,

hydrogen bond and hydrophobic interactions, between

DOX and oligopeptide backbone (Scheme 2).[31,33] As

depicted in Figure 3, the in vitro DOX release profile of

M-DOX in 72hwas revealed in PBS at pH 7.4. The sustained

release behavior of DOX without significant burst release

was observed. It was because that the noncovalent

interactions endowed M-DOX with exceptional capability

for smoothly releasing the payload.[34] In addition, as

depicted in Figure S4, Supporting Information, M-DOX also

exhibitedasphericalmorphologywithanaveragediameter

of around 50nm,while theDh fromDLS determinationwas



86� 3.5 nm. Similarly as the blank micelle, the smaller

apparent micellar scale evaluated by TEM than that from

DLS should be ascribed to the shrinkage of micelle during

the preparation of TEM specimen. Furthermore, the

appropriate size of micelle would endow it with excellent

ability to accumulate in tumor region through the EPR

effect.[25]

Furthermore, the cellular uptake and intracellular DOX

release of M-DOX toward MG63 and Saos-2 cells were

subsequently assessed by both CLSM and FCM. For CLSM

and FCM detections, MG63 and Saos-2 cells were cultured

with F-DOX orM-DOXwith 10.0mg L�1 DOX equivalent for

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Figure 4. Typical CLSMmicroimages of MG63 and Saos-2 cells incubated with F-DOX or M-DOX for 2 h. From left to right, the microimagesrepresented DAPI-dyed cellular nuclei (blue), Alexa 488-stained F-actin (green), intracellular DOX fluorescence (red), and the overlays ofabove three microimages.

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C. Li et al.

334

2h. As shown in Figure 4, stronger DOX fluorescence

intensity in nucleiwas detected inM-DOXgroup compared

with that of F-DOX group toward both two types of

osteosarcoma cells. The results should be attributed to the

efficient endocytosis and intracellular DOX release of M-

DOX, and rapid cellular efflux rate of F-DOX.[50,51] Similarly

as the result of CLSM, the DOX fluorescence intensities in

both MG63 and Saos-2 cells cultured with M-DOX were

higher compared with that incubated with F-DOX in

FCM assays (Figure 5). The above data demonstrated that

M-DOX exhibited efficiently selective intracellular DOX

release, and rightfully generate higher intracellular drug

concentration than F-DOX.

More interestingly, M-DOX exhibited more efficient

inhibition capability against the proliferation of both

MG63 and Saos-2 cells compared to F-DOX, which were



assessed by MTT assays (Figure 6). Similarly to the reason

for the different intracellular DOX concentrations, the

lower cytotoxicity of F-DOX should be assigned to its

quicker intracellular metabolic rate and cellular efflux.

Moreover, the half maximal inhibitory concentrations

(IC50s) of both F-DOX and M-DOX toward two kinds of

osteosarcoma cells were estimated from Figure 6. M-DOX

exhibited half lower IC50s (i.e., 81.7 and 15.9 mg L�1,

respectively) toward bothMG63 and Saos-2 cells compared

with that of F-DOX (i.e., 165.5 and 34.7mg L�1, respectively)

in the same test condition. It quantificationally confirmed

that M-DOX possessed stronger inhibition capability

against the proliferation of osteosarcoma cells relative to

F-DOX. The obtained data revealed that the encapsulation

of DOX with mPEG-b-O(D,L-Leu) micelle might reduce the

drug leakage in circulation system, enhance the selective

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Figure 5. FCM profiles of MG63 (A) and Saos-2 cells (B) culturedwith PBS (a; control), F-DOX (b) or M-DOX (c) for 2 h. Figure 6. In vitro suppression efficacies of F-DOX and M-DOX

toward MG63 (A) or Saos-2 cells (B) after incubation for 72 h.Data were presented as mean� SD (n¼ 3).


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accumulationthroughtheEPReffect in lesionsites, improve

the therapeutic efficacy and downregulate the side effects

of drug in vivo.

3.3. In Vivo DOX Distribution and Tumor

Suppression

The ex vivo fluorescence imaging of isolatedmajor internal

organs (that is, heart, liver, spleen, lung, and kidney) and

tumors were performed to detect the biodistribution of

DOX toward bothMG63 and Saos-2-xenografted nudemice

at 6 and 12h post-injection (Figure 7). After the tail vein

injection of F-DOX or M-DOX for 6 h, strong DOX fluores-

cence was observed in both liver and kidney (Figure 7A),

indicating that both the DOX formulations were mainly

metabolized by the two organs.[52,53] Interestingly, com-

pared with that of F-DOX, weaker DOX fluorescence in

kidney after intravenous injection ofM-DOXwas observed.

The phenomenon should be ascribed to the significantly

improved half-life of M-DOX in the blood circulation.[53]

With the evolution of time from 6 to 12h, almost all the



tested regions, especially for tumors, presented increased

DOX fluorescence intensities after the injection of M-DOX,

while decreased intensities were observed in F-DOX group.

It indicated that mPEG-b-O(D,L-Leu) micelle was able to

delay pharmacokinetic and optimize the biodistribution of

payload, which provided obvious benefit to enhance the

tumor accumulation of DOX and reduce its systemic

toxicity.

As shown in Figure 7B, all the fluorescence intensity of

DOXwas semi-quantitatively analyzed. The average signal

intensity revealed that mPEG-b-O(D,L-Leu) micelle dropped

the maximum concentration of DOX in heart by 33.8 and

35.3%, and inkidney for19.3 and11.4%toMG63andSaos-2-

borne nude mice in the test duration, respectively. It

indicated that M-DOX exhibited reduced dose-dependent

side effects in heart and kidney including cardiomyopathy,

congestive heart failure, kidney intoxication, etc. compared

toF-DOX.[26,54]Notably,M-DOXdeliveredDOXtoMG63and

Saos-2 tumors with about 1.5 and 1.4 times higher amount

than those of F-DOX throughout the test interval,

15, 15, 328–341


Figure 7. Representative DOX fluorescence images (A) and average intensity (B and C) of ex vivo main internal organs and tumors fromBALB/c nude mice bearing MG63 (B) or Saos-2 tumors (C) at 6 or 12 h post-injection of F-DOX or M-DOX. All statistical data were presentedas mean� SD (n¼6; #p<0.01, �p<0.001).

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C. Li et al.

336

respectively. With enhanced DOX accumulation, M-DOX

might show elevated chemotherapy efficacy, whereas

F-DOX might present mediocre ability to inhibit tumor

growth attributed to the decreased fluorescence intensity

as the passage of time.[42,55]

Benefited from the improved pharmacokinetic and

biodistribution, M-DOX might demonstrate superior che-

motherapy efficacy without undesired side effects.[56] To

verify this hypothesis, the in vivo antitumor efficacies of F-

DOX and M-DOX were meticulously investigated toward

both MG63 and Saos-2-xenografted nude mouse models.

PBS was applied as control. As soon as the tumor volume

grew to�80mm3, 0.2mL of PBS, or PBS solution of F-DOXor

M-DOX (4.0mgDOXperkgbodyweight)was intravenously



injected four times every 3 days, respectively. The tumor

volumes were monitored in real-time in the 12 days of

treatment period. As depicted in Figure 8A, the superiority

of M-DOX in MG63 tumor suppression began to show up

relative to those of PBS and F-DOX after around 7 days

of first treatment (p< 0.001). In contrast, as shown in

Figure 8B, Saos-2 tumor was more sensitive to DOX, and

M-DOXshowedobvious advantageof efficacyalmost at the

beginning of medication (on Day 2; p< 0.01). On Day 12

after first injection, i.e., the last moment of therapy, both

MG63 and Saos-2 tumor volumes of control groups rapidly

grew to more than 1 100 mm3, while the average tumor

volumes of F-DOX andM-DOXwere only 62.0 and 28.8% (to

MG63tumor), or59.1and30.2% (toSaos-2 tumor)of those in

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Figure 8. In vivo antitumor efficacies of F-DOX and M-DOXtoward MG63 (A) and Saos-2-borne BALB/c nude mice (B) withPBS as control. Data were presented as mean� SD (n¼6;#p<0.01, �p<0.001).


www.mbs-journal.de

control groups (p< 0.001; Figure 8). More interestingly,

moreefficient inhibition toward twokindsofallogeneically

planted osteosarcomaswas observed inM-DOX group, and

the average tumor volumeswere only 0.5-fold of those in F-

DOX group. The improved antitumor capability of M-DOX

might benefit from the stable structure in blood circulation

and sustained intracellular release property after the

accumulation in tumor tissue via the EPR effect and

endocytosis. In contrast, the worst antitumor efficacy of F-

DOX in vivo should be attributed to the quick excretion by

glomerular filtration.[55]

In addition, the antitumor efficacies of different for-

mulations were further assessed by the histopathological

and in situ apoptosis detections of isolated tumors at

the end of all treatment (that is, on Day 12). For H&E

staining, the nuclei are labelled bluish violet by alkaline

hematoxylin, while cytoplasm and extracellular matrix

are stainedpink by acidic eosin.Moreover, thenecrotic cells

did not have clear cellular morphologies, the chromatin

became darker, aggregated to gobbet or diffused separately

outside the cell, and the nuclei became pyknotic and even



absence. As shown in Figure 9, for both MG63 and Saos-2

tumors, clear cell morphology, more chromatin and

binucleolates, and almost no necrotic area were observed

in tumor cells of control group, which indicated a vigorous

tumor growth.[53] Giving hope is that the average numbers

of tumor cells decreased apparently. Concurrently, the

tissue necrosis, chromatin scatter, and nuclear condensa-

tion, fragmentation, and disappearance were presented in

F-DOX and M-DOX-treated groups with progressively

worsening trend. These all confirmed that M-DOX had

great potential in the chemotherapy of malignancy.

Furthermore, as depicted in Figure 10, the relative necrosis

areas ofM-DOXwere�1.6 times larger than those of F-DOX

for both MG63 and Saos-2 tumors (p< 0.001), which

exhibited the same rule with the inhibition profiles of

tumor growth in vivo after the administration with F-DOX

and M-DOX.

Moreover, various degrees of in situ cell apoptosis of

both MG63 and Saos-2 tumors after the treatments with

various therapeutic agents were analyzed by a TUNEL

assay.Apoptosis activatesDNAenzymeandgeneratesDNA

fragmentation in the nuclei of tumor cells, a marker of late

apoptosis,whichcanbedyedgreenfluorescencebyaTUNEL

kit. As shown in Figure 9, less apoptotic cells were observed

in tumor tissue treatedwith PBS, while different degrees of

cellular apoptosiswere detected in both F-DOX andM-DOX

groups. In consistent with the results of H&E assays,

M-DOX inducedmore cell apoptosis than F-DOX. As above-

mentioned, the fascinating efficacy ofM-DOX should result

from the improved stability in circulation systemandmore

durable intracellular DOX release.[42]

3.4. In Vivo Security Detection

In vivo security of antitumor drugs is another critical

evaluation index for clinical chemotherapy, which is

directly linked to the survival of malignancy patients.[57,58]

In this study, the safety ofM-DOXwasdetected through the

detection of body weight change and histopathological

analyses of internal organs (i.e., heart, liver, spleen, lung,

and kidney).

Body weight loss of animal is an important indicator of

the performance of the organism toxicity of chemotherapy

drugs.[59] As shown in Figure 11, the body weights of nude

mice bearing both MG63 and Saos-2 tumors after the four

times administration of F-DOX at a dosage of 4.0mgkg�1

bodyweight showed a decrease of over 20%with respect to

the beginning body weight. In addition, the nude mice

showed very weak. It should be attributed to the sharply

rising DOX concentration in plasma that damage heart,

liver, or kidney in a certain extent.[60] Fortunately, for the

above-mentioned two kinds of osteosarcoma models, the

ameliorated body weight loss was observed in M-DOX

group with regard to that in F-DOX group at the end of all

15, 15, 328–341


Figure 9. Ex vivo histopathological and in situ cell apoptosis analyses of MG63 and Saos-2 tumor sections after treatments with F-DOX orM-DOX with PBS as control. Magnification: 200�.

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C. Li et al.

Macromol. Biosci. 2015, 15, 328–341

� 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim338 www.MaterialsViews.com

Figure 11. Body weight changes of MG63 (A) and Saos-2-xenografted female BALB/c nude mice (B) treated with F-DOXorM-DOXwith PBS as control. Datawere presented asmean� SD(n¼6; �p<0.01).

Figure 10. Relative necrotic area of MG63 and Saos-2 tumorsections from H&E staining after treatments with F-DOX or M-DOX with PBS as control. Data were presented as mean� SD(n¼6; �p<0.001).


www.mbs-journal.de



treatments (i.e., on Day 12; p< 0.01). The obtained results

showed that the encapsulation and designated release of

antineoplastic drugs with oligopeptide micelle decreased

the side effects and improved its security in vivo.

Organ injury after the long-term administration of

antitumor drugs is a major factor for the decrease of body

weight and survival rate.[53] In this work, several suscepti-

ble organs, such as heart, liver, spleen, lung, and kidney,

were harvested, sectioned, and stained by H&E on Day 12

after all treatments. As shown in Figure 12, no significant

morphological variations were detected in control group

relative to those in F-DOXandM-DOXgroups.However, it is

not optimistic that the administrations of both F-DOX and

M-DOX induced various degrees of organ damage, especial-

ly to heart, liver, and kidney. The specific histopathological

response mainly including the following aspects: i)

myocardial cells degenerated with obvious pitch; ii) liver

cells increasedandcompressed, and thecytoplasmrevealed

blatant pale staining; iii) kidney exhibited renal interstitial

hemorrhage and abnormal shape of glomerulus. Thankful-

ly, M-DOX exhibitedmoremoderate degree of organ injury

than that of F-DOX indicating that M-DOX had better

security than F-DOX, and exhibited favorable clinical

application prospect of chemotherapy.

4. Conclusion

In summary, the block copolymer containing hydrophilic

mPEG and hydrophobic O(D,L-Leu) was synthesized, which

spontaneously self-assemble into micelle in aqueous

solution with narrow Dh at �120nm. The moderate size

endowed themicelle with excellent feasibility for selective

drug delivery because of the enhanced accumulation in

tumor tissue through the EPR effect. Benefited from the

hydroxyl, carboxyl, and amino groups in molecular

skeleton, DOX, a model antineoplastic agent, was loaded

into micelle assisted by the noncovalent interactions, e.g.,

hydrogen bond and hydrophobic interactions, between

DOX and oligopeptide backbone with a relative high DLE

(i.e., 54.6� 3.6wt%) and a sustained release profile. The

encapsulation with micelle gave DOX enhanced cellular

internalization and proliferation inhibition. More impor-

tantly, M-DOX exhibited about 1.5-fold accumulation in

tumor tissue, around two times suppression ability for

tumor growth, and reduced toxicities to almost all

susceptible organs toward both MG63 and Saos-2-xeno-

grafted nude mouse models compared with F-DOX. In

addition, the obtained drug delivery system could be

extended to a abroad range of antitumor drugs. With

convenient preparation, favorable biocompatibility, effi-

cient drug loading and directional release, appropriate

biodistribution, and excellent antitumor efficacy and

security, the fascinating polypeptide drug delivery system

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Figure 12. Histopathological analyses of main visceral organ sections from MG63 and Saos-2-xenografted female BALB/c nude mice aftertherapy with F-DOX or M-DOX using PBS as control. Magnification: 200�.

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C. Li et al.

340

presented great potential for clinical chemotherapy of

malignancy.

Acknowledgements: This work was financially supported by theNational Natural Science Foundation of China (Projects 51303174,51473165, 51273196, 51203153, 51321062, 51233004, and51390484) and the Scientific Development Program of JilinProvince (20140520050JH).

Received: August 2, 2014; Revised: October 12, 2014; Accepted:October 30, 2014; Published online: November 7, 2014; DOI:10.1002/mabi.201400356

Keywords: chemotherapy; diblock copolymers; drug deliverysystems; nanotechnology; noncovalent interaction

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Micellization of antineoplastic agent to significantly upregulate efficacy and security

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