Peptide-22 and Cyclic RGD Functionalized Liposomes for GliomaTargeting Drug Delivery Overcoming BBB and BBTBCuitian Chen,† Ziqing Duan,† Yan Yuan,† Ruixiang Li,† Liang Pang,† Jianming Liang,† Xinchun Xu,*,‡

and Jianxin Wang*,†

†Department of Pharmaceutics, School of Pharmacy, Fudan University & Key Laboratory of Smart Drug Delivery, Ministry ofEducation, Shanghai 201203, People’s Republic of China‡Shanghai Xuhui Central Hospital, Shanghai 200031, People’s Republic of China

*S Supporting Information

ABSTRACT: Chemotherapy outcomes for the treatment of gliomaremain unsatisfied due to the inefficient drug transport across BBB/BBTB and poor drug accumulation in the tumor site. Nanocarriersfunctionalized with different targeting ligands are considered as one ofthe most promising alternatives. However, few studies were reported tocompare the targeting efficiency of the ligands and develop nanoparticlesto realize BBB/BBTB crossing and brain tumor targeting simultaneously.In this study, six peptide-based ligands (Angiopep-2, T7, Peptide-22,c(RGDfK), D-SP5 and Pep-1), widely used for brain delivery, wereselected to decorate liposomes, respectively, so as to compare theirtargeting ability to BBB or BBTB. Based on the in vitro cellular uptakeresults on BCECs and HUVECs, Peptide-22 and c(RGDfK) were pickedto construct a BBB/BBTB dual-crossing, glioma-targeting liposomal drugdelivery system c(RGDfK)/Pep-22-DOX-LP. In vitro cellular uptakedemonstrated that the synergetic effect of c(RGDfK) and Peptide-22 could significantly increase the internalization of liposomeson U87 cells. In vivo imaging further verified that c(RGDfK)/Pep-22-LP exhibited higher brain tumor distribution than singleligand modified liposomes. The median survival time of glioma-bearing mice treated with c(RGDfK)/Pep-22-DOX-LP (39.5days) was significantly prolonged than those treated with free doxorubicin or other controls. In conclusion, the c(RGDfK) andPeptide-22 dual-modified liposome was constructed based on the targeting ability screening of various ligands. The system couldeffectively overcome BBB/BBTB barriers, target to tumor cells and inhibit the growth of glioma, which proved its potential forimproving the efficacy of chemotherapeutics for glioma therapy.

KEYWORDS: glioma, blood−brain barrier, blood−brain tumor barrier, liposome, c(RGDfK), Peptide-22

1. INTRODUCTION

Glioma is one of the most common and aggressive intracranialmalignancies with high morbidity and mortality. The medianoverall survival of glioma patients is 12−18 months and the 5-year survival rate is less than 10%.1,2 Because of the invasivegrowth and an undefined tumor edge, surgery can hardlyremove the tumor completely and the recurrence rate is veryhigh.3,4 Once relapse, the average survival duration is only 1.5years.1 Systemic chemotherapy has been widely used in thetreatment of glioma.5−7 Unfortunately, clinical outcomesremain unsatisfied due to the unfavorable pharmacokinetics,severe side effects and poor drug accumulation in the brainparenchyma.7,8 Recently, the exploitation of nanocarriers fordrug delivery has emerged as a promising alternative to conquerthese problems.9 However, the effective transport of nano-carriers to the glioma site is still limited.10,11

It is essential to deliver anticancer drug loaded nanocarriersacross the blood−brain barrier (BBB) and blood−brain tumorbarrier (BBTB) simultaneously in order to treat glioma. The

BBB, an endothelial cell monolayer associated with pericytesand astrocytes, prevents approximately 98% small moleculesand nearly 100% large molecules from transporting into thebrain.12 Meanwhile, it behaves as the main obstacle fornanocarriers to enter the brain parenchyma in the early stageof glioma.13 With the gradual progression of glioma, theintegrity of BBB is compromised to some extent while theBBTB emerges. The BBTB exists between the brain tumortissues and capillary vessels, which also impedes the effectivetransport of nanocarriers into tumor.14,15 Many efforts havebeen made to overcome BBB or BBTB separately. However, inmost cases, BBB and BBTB exist simultaneously when glioma isdiagnosed.16 Therefore, it is significant to develop a novel drugdelivery system possessing BBB and BBTB dual-targetingability.

Received: December 9, 2016Accepted: January 27, 2017Published: January 27, 2017

Research Article

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© 2017 American Chemical Society 5864 DOI: 10.1021/acsami.6b15831ACS Appl. Mater. Interfaces 2017, 9, 5864−5873

As is reported previously, receptor-mediated transcytosis(RMT) is one of the most common strategies for nanocarriersto penetrate BBB/BBTB.17 Studies have found that braincapillary endothelial cells and endothelial cells of tumorangiogenic vessels express numerous different receptors.14,18

Nanocarriers decorated with specific ligands can efficiently bindto corresponding receptors overexpressed on BBB/BBTB andthen initiate the RMT process, resulting in the enhancement ofdrug penetration across the barriers and accumulation in theglioma site.19 Various ligands have been demonstrated topossess high binding affinities to specific receptors and exhibitobvious brain tumor targeting properties when conjugated tonanoparticles. There are thousands of reports about themodification with targeting ligands on nanocarriers. Almostall the research results are positive and prove that theapplication of ligands could significantly improve the targetingefficiency of the nanosystems. However, it is difficult tocompare the results and determine which ligand is moreeffective for brain delivery, because the important factorsinfluencing the targeting efficiency, such as particle type,formulation parameters and the ligand density, were quitedifferent in various studies.20−22 Therefore, it is essential toevaluate the BBB/BBTB targeting ability of different ligands inthe same nanocarrier, which will pick out the most efficientligand for the establishment of brain targeting drug deliverysystem.Angiopep-2,23 T724 and Peptide-2225 are widely used as

specific ligands for low-density lipoprotein receptor-relatedprotein-1 (LRP1), transferrin receptor (TfR) and low-densitylipoprotein receptor (LDLR), respectively, which are overex-pressed on BBB as well as glioma cells. c(RGDfK)16 (a ligandof integrin αvβ3/αvβ5), Pep-1

26 (a ligand of IL-13Rα2) and D-SP527 (a novel retro-inverso analogue of L-SP5) have beenproved to display dual-targeting bioactivity for both tumorneovasculature (BBTB) and tumor cells. It was reported thatnanocarriers functionalized with any of the above ligands couldbe served as an effective targeting delivery system to deliverchemotherapeutics across BBB or BBTB and accumulate in thetumor site.26−31 However, few studies were reported tocompare the targeting efficiency of these ligands and combinethe specific ones to decorate nanocarriers so as to overcomemultiple barriers.In this study, we try to develop a glioma-targeted delivery

system modified with ligands facilitating across not only BBBbut also BBTB, and targeting tumor cells and neovasculature aswell. Liposomes were chosen as the delivery vehicle because ofthe nontoxic and nonimmunogenic characteristics, the highdrug loading ability and the possibility to be decorated withdifferent ligands. Six peptide-based ligands with differentdensity were screened and compared the targeting efficiency.We divided these functionalized liposomes into two groups:one for the evaluation of BBB targeting capacity (Angiopep-2-LP, T7-LP, Peptide-22-LP) and the other for the investigationof BBTB targeting ability (c(RGDfK)-LP, Pep-1-LP, D-SP5-LP). On the basis of the comparison results, we selected theoptimal ligand from each group and combined them to developa BBB/BBTB dual-crossing, glioma-targeting liposomal drugdelivery system (c(RGDfK)/Pep-22-DOX-LP). To verifyfurther the brain tumor targeting efficacy, in vitro cellularuptake and in vivo imaging study were performed. Eventually,therapeutic effects of different formulations were evaluated onintracranial glioma-bearing mice model, choosing doxorubicin(DOX) as the model drug.

2. MATERIALS AND METHODS2.1. Materials. T7 with a cysteine on the N-terminal (Cys-T7)

(CHAIYPRH) , D - SP5 -Cy s ( D (PRPSPKMGVSVSC)) ,Pep-1(CGEMGWVRC), Angiopep-2-Cys(TFFYGGSRGKRNNFK-TEEYC), c(RGDfK) (c(Arg-Gly-Asp-D-Phe-Lys)) and Peptide-22(NH2-C6-[cMPRLRGC]c-NH2) were synthesized by Bankpeptidebiological Technology Co. Ltd. (Hefei, China) with the purity above95%. Soyabean phosphatidylcholine (SPC) was purchased from LipoidGmbH (Ludwigshafen, Germany). Cholesterol was from SinopharmChemical Reagent Co. Ltd. (Shanghai, China). mPEG2000-DSPE wasobtained by NOF Co. (Tokyo, Japan). DSPE-PEG3400-NHS wasprovided by Nanocs Co. (NewYork, USA). DSPE-PEG3400-Mal wasobtained from Laysan Bio Co. (Arab, AL). 5-Carboxyfluorescein(FAM), Hoechst 33342 and near-infrared dye DiR were received fromFanboBiochemicals (Beijing, China). The 5-(dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) was purchased from sigma(St. Louis, MO, USA). Doxorubicin hydrochloride (DOX·HCl) wasobtained from Melonapharma (Dalian, China).

Human umbilical vascular endothelial cells (HUVECs), braincapillary endothelial cells (BCECs) and human glioblastoma cells(U87s) were obtained from Cell Bank, Chinese Academy of Sciences(Shanghai, China). The cells were cultured in Dulbecco’s ModifiedEagle Medium (DMEM) supplemented with 10% FBS, 1% L-glutamine, 1% nonessential amino acids, 100 U/mL penicillin and100 μg/mL streptomycin at 37 °C under a humidified atmospherecontaining 5% CO2.

Balb/c nude mice (female, 18−20 g) were purchased from ShanghaiSLAC Laboratory Animal Co. Ltd. (Shanghai, China). All animalexperiments were performed in accordance with the principles of careand use of laboratory animals. The protocol of animal experiments wasapproved by the Animal Experimentation Ethics Committee of FudanUniversity.

2.2. Synthesis and Characterization of FunctionalizedPhospholipid. DSPE-PEG3400-T7, DSPE-PEG3400-D-SP5, DSPE-PEG3400-Pep-1, DSPE-PEG3400-Angiopep-2 were synthesized byconjugating DSPE-PEG3400-Mal to the cysteine residue on Cys-T7,D-SP5-Cys, Pep-1 and Angiopep-2, respectively.27 DSPE-PEG3400-Peptide-22 and DSPE-PEG3400-c(RGDfK) were synthesized by thereaction of amino groups on Peptide-22 and c(RGDfK) with the activegroup NHS contained in DSPE-PEG3400-NHS, respectively.

32 In brief,DSPE-PEG3400-Mal and peptide (molar ratio 1:1.5) were dissolved inwarm PBS (pH 7.0) with sonication. DSPE-PEG3400-NHS and peptide(molar ratio 1:2) were dissolved in anhydrous DMF with DIEA (0.5equiv). Mixtures were gently stirred for 24 h at room temperature.Excessive peptides were removed by dialysis against distilled water.Pure products were freeze-dried and stored at −20 °C until use. Thesefunctionalized phospholipids were confirmed by MALDI-TOF massspectrometry (MALDI TOF/TOF, SCIEX TOF/TOF 5800, ABSCIEX, USA).

2.3. Preparation and Characterization of Liposomes. Lip-osomes were prepared by a thin-film hydration and extrusion method.Eight kinds of liposomes were prepared with different lipidcompositions. Blank liposomes (LP), which were not modified withany ligands, were taken as the control and made of SPC, cholesteroland mPEG2000-DSPE (60:33:7, molar ratio). The liposomes modifiedwith different ligands, including c(RGDfK) modified liposomes(c(RGDfK)-LP), D-SP5 modified liposomes (D-SP5-LP), Pep-1modified liposomes (Pep-1-LP), Angiopep-2 modified liposomes(Angiopep-2-LP), T7 modified liposomes (T7-LP) and Peptide-22modified liposomes (Pep-22-LP), were prepared with the sameformulation as blank liposomes except that part of mPEG2000-DSPEwas substituted by DSPE-PEG3400-c(RGDfK), DSPE-PEG3400-D-SP5,DSPE-PEG3400-Pep-1, DSPE-PEG3400-Angiopep-2, DSPE-PEG3400-T7and DSPE-PEG3400-Peptide-22. To search for the optimal amount ofspecific DSPE-PEG3400-ligand in the liposomes, the ratios of 1%, 3%and 5% were compared. Based on the screening results, a kind of dual-modified liposomes, c(RGDfK) and Peptide-22 (c(RGDfK)/Pep-22-LP), composed of SPC, cholesterol, mPEG2000-DSPE, DSPE-PEG3400-

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c(RGDfK) and DSPE-PEG3400-Peptide-22 (60:33:1:3:3, molar ratio)were developed to validate the synergetic effect of two ligands.All lipid materials were dissolved in CHCl3, and followed by rotary

evaporation to form a lipid film using a ZX-98 rotary evaporator(LOOYE, China) at 35 °C. After maintained in a vacuum overnight,the thin film was hydrated with saline at 37 °C and successivelyextruded through polycarbonate membranes with the pore size of 200,100 and 50 nm, using an Avanti Mini Extruder (Avanti Polar Lipids).DiR and FAM loaded liposomes were prepared by the same methoddescribed above except that DiR was added in the organic solution(CHCl3) to form the thin film, whereas FAM was added in thehydration medium (saline). DOX loaded liposomes were preparedusing an ammonium sulfate gradient loading method according to theprevious report.33

Particle size and ζ-potential of various liposomes were measured bya dynamic light scattering detector (Zetasizer, Nano-ZS, Malvern,UK). The morphology of liposomes was detected by transmissionelectron microscopy (TEM, Tecnai G2 F20 S-Twin, FEI, USA). Theencapsulation efficiency and the loading efficiency of DOX weremeasured using a previously reported method.34

2.4. In Vitro DOX Release. The release of DOX was performed bya dialysis method. Briefly, 0.4 mL of liposome suspension or free DOXsolution (0.5 mg DOX/mL) was placed into a dialysis tube (MWCO =3500) and tightly sealed. Then the dialysis tube was immersed into 40mL of PBS (pH 7.4) in a 37 °C shaker for 48 h at 100 rpm. Atpredetermined time points, 0.5 mL of release medium was sampledand immediately replaced with an equal volume of fresh releasemedium. The concentration of DOX was measured by a microplatereader (synergy TM 2, Bio Tek, USA) at Ex = 480 nm and Em = 525nm.2.5. In Vitro Cellular Uptake. To evaluate the BBB targeting

ability of Angiopep-2, T7 and Peptide-22 modified liposomes in vitro,BCECs were seeded into a 12-well plate at a density of 5 × 105 cells/well. After 24 h of incubation, the culture medium was replaced with 5μM FAM-loaded liposomes of different formulations in DMEMcontaining 10% FBS and allowed for further coincubation for 4 h at 37°C. Following that, cells were rinsed with cold PBS twice, thentrypsinized, centrifuged and finally resuspended in 0.3 mL of PBS. Thefluorescence intensity of cells treated with different types and differentligand densities (1%, 3% and 5%) of liposomes was analyzed by a flowcytometer (BD Biosciences, Bedford, MA, USA). Similarly, the BBTBtargeting capacity of c(RGDfK), D-SP5 and Pep-1 modified liposomeswas evaluated on HUVECs and the effect of ligand density (1%, 3%and 5%) on cellular uptake was also investigated by flow cytometry asdescribed previously.To investigated qualitatively the tumor targeting ability of LP,

c(RGDfK)-LP, Peptide-22-LP and c(RGDfK)/Peptide-22-LP, thecellular uptake of the liposomes on U87 cells was studied. U87 cellswere seeded into a 12-well plate at a density of 5 × 104 cells/well.Twenty-four hours later, 5 μM FAM loaded liposomes in DMEMcontaining 10% FBS were added into each well. After 4 h ofincubation, the cells were stained with Hoechst 33342 and then werewashed with cold PBS for three times. Finally, the samples werevisualized by fluorescent microscopy (Leica, DMI4000D, Germany).For quantitative study, flow cytometry was conducted on U87 cellsand the detailed process was in accordance with the above description.2.6. In Vivo Animal Imaging. The intracranial glioma-bearing

mice model was established as previously described.35 Fifteen daysafter inoculation, glioma-bearing mice were intravenously injected viathe tail with DiR loaded LP, c(RGDfK)-LP, Pep-22-LP andc(RGDfK)/Pep-22-LP, respectively, at a dose of 0.3 mg DiR/kg.The distribution of fluorescence was observed at predetermined timepoints (2, 6, 12 and 24 h) using an in vivo imaging system (IVISSpectrum, Caliper, USA). Twenty-four hours later, the mice weresacrificed after heart perfusion with saline. Brains and periphericorgans were harvested and imaged as well.2.7. In Vitro Cytotoxicity. The cytotoxicity of free DOX, DOX-

LP, c(RGDfK)-DOX-LP, Pep-22-DOX-LP and c(RGDfK)/Pep-22-DOX-LP against U87 cells was assessed by MTT assay. Briefly, U87cells were seeded into 96-well plates at a density of 3× 103 cells/well

and cultured for 24 h at 37 °C. Different formulations of doxorubicinloaded liposomes were diluted to predetermined concentrations withcomplete medium and then were added into each well. Forty-8 h later,the medium was removed and the cells were washed twice with PBS,then 100 μL of MTT solution (0.5 mg/mL) was added. After 4 hcoincubation at 37 °C, MTT solution was replaced with 150 μL ofDMSO, followed by gently shaking to dissolve the formazan crystals.The absorbance was measured via a microplate reader (synergy TM 2,BioTek, USA) at 490 nm. The cells without treatment were served ascontrol.

2.8. In Vivo Antiglioma Efficacy. In vivo antitumor efficacy wasperformed in intracranial glioma-bearing mice. Seven days after tumorinoculation, the mice were randomly divided into six groups (n = 6).The mice in blank control group were intravenously injected withsaline and the other five treatment groups were administered with freeDOX, DOX-LP, c(RGDfK)-DOX-LP, Pep-22-DOX-LP and c-(RGDfK)/Pep-22-DOX-LP via tail vein at day 7, 14 and 21 with 5mg DOX/kg, respectively. Body weights were monitored every otherday. Survival time was recorded and analyzed by GraphPad Prism 5(GraphPad Software Inc., California, USA).

2.9. Cardiotoxicity Evaluation. Thirty-six intracranial glioma-bearing mice were divided into 6 groups randomly and given injectionsof saline, free DOX, DOX-LP, c(RGDfK)-DOX-LP, Pep-22-DOX-LPand c(RGDfK)/Pep-22-DOX-LP, respectively, at a dose of 5 mgDOX/kg at day 7, 14 and 21. Two days after the last administration,the mice were sacrificed. The sections of heart were stained withhematoxylin and eosin to evaluate the cardiotoxicity of DOX.

2.10. Statistical Analysis. All data were analyzed using GraphpadPrism 5 software. Statistical comparisons were performed by one-wayANOVA. Data were expressed as mean ± SD. Statistical significancewas defined as *p < 0.05 and extreme significance was defined as **p< 0.01.

3. RESULTS AND DISCUSSION

3.1. Synthesis and Characterization of FunctionalizedPhospholipid. We conjugated T7, D-SP5, Pep-1 andAngiopep-2 to the distal end of DSPE-PEG3400-Mal by Michaeladdition of cysteine residue and maleimide, respectively.Similarly, c(RGDfK) and Peptide-22 were conjugated to thedistal end of DSPE-PEG3400-NHS, respectively, by the reactionof amino groups with active groups NHS. The results ofMALDI-TOF mass spectrometry (see Supporting InformationFigure S1) confirmed the successful synthesis of all the sixproducts, with the observed molecular weight around 5099 Da(DSPE-PEG3400-T7), 5689 Da (DSPE-PEG3400-D-SP5), 5648Da (DSPE-PEG3400-Pep-1), 6797 Da (DSPE-PEG3400-Angio-pep-2), 5180 Da (DSPE-PEG3400-Peptide-22) and 5096 Da(DSPE-PEG3400-c(RGDfK)), which were close to the theoreti-cal molecular weights.

3.2. Evaluation of BBB/BBTB Targeting Ability ofDifferent Ligands Modified Liposomes. We successfullyprepared different ligands modified FAM loaded liposomeswith 1%, 3% and 5% DSPE-PEG3400-ligand. The mean particlesizes of various formulations ranged from 100 to 125 nm, PDIwere less than 0.3 and ζ-potentials were between −15 and −30mV. The BBB targeting ability of Angiopep-2-LP, T7-LP andPep-22-LP was evaluated on brain capillary endothelial cells(BCECs), which was widely used as a model for mimickingBBB.30,36 As shown in Figure 1A, the cellular uptake ofliposomes was enhanced by ligand modification, which wasrespectively owning to the specific binding to low-densitylipoprotein receptor-related protein-1 (LRP1), transferrinreceptors (TfR) and low-density lipoprotein receptor(LDLR) highly expressed on BCECs and then initiated RMTprocess.24,29,36 However, fluorescence intensity of differentligands decorated liposomes differed from each other. We

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speculated that it was correlated with the expression level ofcorresponding receptor on BCECs, the ligand−receptorbinding affinity and the cellular uptake mechanism. Herein,the cellular uptake of Pep-22-LP was significantly higher thanthose of Angiopep-2-LP and T7-LP (p < 0.001), indicating thestrongest potency of Peptide-22 in transporting liposomesacross BBB. Peptide-22, generated by phage display biopan-ning, is a cyclic peptide with properties of good stability, easysynthesis at a relative low cost and lack of immunogenicity.29

Previous studies have revealed that Peptide-22 exhibits specialaffinity for LDLR but without competition with endogenousLDL and could be served as a LDLR-targeting moiety for CNStargeting and delivery.25,29 Furthermore, Pep-22-LP displayed apeptide density-dependent manner. With the increased amountof DSPE-PEG3400-Peptide-22 in the lipid constitution, thecellular uptake was improved as well. Compared with 1%modification ratio, the cellular uptake of 3% DSPE-PEG3400-Peptide-22 modified liposomes was significantly higher (p <0.001). Nevertheless, there was no obvious difference influorescence intensity between 3% and 5% modification ratio ofPeptide-22, which might be due to the receptor saturation.Considering that large amount of DSPE-PEG3400-ligand wouldinfluence the integrity and the stability of liposomes in serum,3% DSPE-PEG3400-Peptide-22 was opted for the followingstudies.Similarly, the BBTB targeting capacity of c(RGDfK)-LP, D-

SP5-LP and Pep-1-LP was evaluated on human umbilicalvascular endothelial cells (HUVECs), which was the commonly

used model for mimicking BBTB.37−39 As shown in Figure 1B,compared with unmodified liposomes, the cellular uptake ofligand decorated liposomes was enhanced, among whichc(RGDfK)-LP was significantly higher than those of DSP-5-LP and Pep-1-LP (p < 0.001). c(RGDfK) is the specific ligandof integrin αvβ3/αvβ5, which is overexpressed on tumorneovasculature (BBTB). Thus, c(RGDfK) could mediateendocytosis to improve the cellular uptake of neovascularendothelial cells.16 Analogously, Pep-1 could also increasecellular internalization through interleukin 13 receptor α2 (IL-13Rα2) mediated endocytosis.40 However, it has been foundthat the expression level of IL-13Rα2 was lower than that ofαvβ3 on HUVECs.39 D-SP5 is a novel retro-inverso analogue ofL-SP5.27 Although the binding mechanism of D-SP5 is notmuch clear, it has been reported that the targeting efficiency ofD-SP5 might be lower on inactivated HUVECs than VEGFstimulated HUVECs.41 Moreover, from the fluorescenceintensity of different density of DSPE-PEG3400-c(RGDfK)modified liposomes, we could find that 3% modification ratioshowed significant increase than 1% (p < 0.001), but there wasno difference between 3% and 5%. Therefore, 3% DSPE-PEG3400-c(RGDfK) was chosen for the following studies.

3.3. Preparation and Characterization of DualLigands-Modified Liposomes. Conventional chemotherapyof glioma has been hampered due to ineffective drug deliveryacross BBB/BBTB.12,42 In most cases, BBB and BBTB existsimultaneously when glioma is diagnosed. Based on the cellularuptake results on BCECs and HUVECs (Figure 1A,B), 3%c(RGDfK) and 3% Peptide-22 were selected to construct adual-modified liposomal drug delivery system, c(RGDfK)/Pep-22-DOX-LP, in which Peptide-22 and c(RGDfK) couldenhance the targeting and permeation of the liposomes of theBBB and the BBTB, respectively. Moreover, both of the ligandscould bind with the specific receptors on the tumor cells furtherand then target the liposomes to glioma, which was of greatsignificance for the specific tumor distribution of the system inthe brain.Appropriate particle size and uniform distribution of

nanoparticles were required for BBB/BBTB permeation andbrain tumor targeting.13,43 The size distribution graph ofc(RGDfK)/Pep-22-DOX-LP is shown in Figure 2A. The meanparticle sizes, ζ-potentials, DOX encapsulation efficiency andloading efficiency of different formulations are listed in Table 1.For all the liposomes with or without ligand modification, themean particle sizes ranged from 95 to 105 nm, PDIs were lessthan 0.1 and the DOX encapsulation efficiency and loadingefficiency were about 95% and 5.5%, respectively. The resultsindicated that the incorporation of DSPE-PEG3400-c(RGDfK)and DSPE-PEG3400-Peptide-22 did not affect the physicalproperties of liposomes significantly. The morphology ofc(RGDfK)/Pep-22-DOX-LP obtained by TEM revealed thatthe liposomes were homogeneously spheroids with moderatedispersivity (Figure 2B). No significant change of particle sizeand PDI of different DOX loaded liposomes was observed in10% FBS-contained medium over 24 h, suggesting goodstability of liposomes in serum-contained medium (seeSupporting Information Figure S2). The average size ofdifferent formulations maintained approximately 110 nm witha narrow distribution within 14 days, revealing good stabilityunder the storage condition (Data not shown).

3.4. In Vitro DOX Release. The in vitro release of DOXfrom liposomes was performed in PBS (0.01 M, pH 7.4). Asshown in Figure 3, compared with the rapid release of free

Figure 1. Cellular uptake of liposomes modified with different DSPE-PEG3400-ligands on BCECs (A) and HUVECs (B) measured by a flowcytometer. Cells were incubated with 5 μM FAM loaded liposomes at37 °C for 4 h (n = 3, mean ± SD). The horizontal ordinate suggestedthe ratio of DSPE-PEG3400-ligand in total lipid of the liposomes. ***Indicates p < 0.001 and N.S. indicates there is no significant difference.

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DOX, all the DOX loaded liposomes exhibited sustainedrelease behaviors that the cumulative DOX release fromliposomes was less than 40% within 48 h and no burst initialrelease was detected. Similar release patterns were observedamong DOX-LP, c(RGDfK)-DOX-LP, Pep-22-DOX-LP andc(RGDfK)/Pep-22-DOX-LP, implying that the modification ofligands did not affect the release behavior of DOX obviously.3.5. Cellular Uptake on U87 cells. The targeting effect of

FAM loaded dual-modified liposomes was investigated by thecellular uptake on U87 cells through fluorescent microscopy(Figure 4A) and flow cytometry (Figure 4B) in vitro. As shownin Figure 4A, compared with unmodified liposomes (LP),c(RGDfK)-LP and Pep-22-LP exhibited stronger greenfluorescence signals inside U87 cells, which indicated thatboth c(RGDfK) and Peptide-22 modification could effectivelyincrease the uptake of liposomes by U87 cells. This result wasconsistent with previous reports. For instance, Kim et al.demonstrated that the cellular uptake of DOX in cRGD-modified liposomes into U87MG was 8-fold higher than that of

unmodified liposomes.44 Zhang et al. found that thefluorescence intensity of C6 cells treated with coumarin-6-labeled peptide-22-decorated nanoparticles were obviouslyhigher than those treated with unmodified NPs.29 Furthermore,the liposomes modified with c(RGDfK) and Peptide-22simultaneously resulted in the strongest fluorescence signalamong four groups, indicating that the synergetic recognition ofintegrin αvβ3 and LDLR on U87 cells could significantlyimprove the internalization of liposomes.Similar results were obtained from flow cytometry (Figure

4B). Compared with LP, the cellular uptake of liposomesmodified with ligands was significantly increased on U87 cells.Moreover, the fluorescence intensity of c(RGDfK)/Pep-22-LPwas 7.57- and 1.58-fold higher than those of c(RGDfK)-LP andPep-22-LP. These data illustrated that dual-modification couldnot only combine the transcytosis capability of c(RGDfK) andPeptide-22 themselves, but also exerted a synergetic effect ontumor cell uptake. The synergetic effect was realized byexpanding the number of the recognized receptors (bindingsites) and strengthening the binding affinity via conjugations oftwo different ligands to the corresponding receptors on thesame cell surface, which made the process of internalizationmore easily.

3.6. In Vivo Imaging Study. During the occurrence anddevelopment of glioma, BBB is gradually impaired and BBTBemerges as the new obstacle for the transport of nanoparticlesinto tumor.45 Therefore, it is essential to construct a novelsystem to realize BBB/BBTB dual-crossing and brain tumortargeting simultaneously. In our study, we evaluated the in vivoglioma targeting efficiency of different liposomes containingnear-infrared dye DiR in nude mice bearing intracranial glioma.The liposomes were administrated 15 days after the inoculationof tumor cells, at the time the BBB and the BBTB existsimultaneously. A real-time distribution of liposomes was

Figure 2. Particle size distribution of c(RGDfK)/Pep-22-DOX-LP (A)determined by DLS. Morphology of c(RGDfK)/Pep-22-DOX-LP (B)obtained by TEM after negative staining with 2% sodiumphosphotungstate solution. Bar: 100 nm.

Table 1. Characterization of Different Doxorubicin Liposomesa

liposomes particle size (nm) PDI ζ-potential (mV) encapsulation efficiency of DOX (%) loading efficiency of DOX (%)

DOX-LP 97.56 ± 0.71 0.058 ± 0.007 −28.57 ± 0.75 97.54 ± 1.13 5.68 ± 0.21c(RGDfK)-DOX-LP 99.59 ± 0.87 0.094 ± 0.005 −17.45 ± 1.25 95.26 ± 3.19 5.54 ± 0.32Pep-22-DOX-LP 101.33 ± 0.40 0.091 ± 0.008 −16.57 ± 0.61 94.04 ± 0.70 5.16 ± 0.15c(RGDfK)/Pep-22-DOX-LP 103.43 ± 0.92 0.098 ± 0.010 −17.40 ± 1.15 96.65 ± 3.13 5.39 ± 0.27

aData represented mean ± SD (n = 3).

Figure 3. In vitro release curves of DOX from solution and liposomes(DOX-LP, c(RGDfK)-DOX-LP, Pep-22-DOX-LP, c(RGDfK)/Pep-22-DOX-LP) in PBS (0.01 M, pH 7.4) at 37 °C (n = 3, mean ± SD).

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observed under IVIS Spectrum system. As shown in Figure 5A,the c(RGDfK)/Pep-22-LP group exhibited the strongestfluorescence signal in the glioma region at all-time pointswhen compared with that of unmodified or single-modifiedgroups, indicating that dual-modification of Peptide-22 andc(RGDfK) could significantly facilitate liposomes to traversethe BBB/BBTB and enhance the accumulation of liposomes inthe tumor site via the synergetic effect of integrin αvβ3 andLDLR mediated endocytosis. The efficient BBB/BBTB trans-porting capability and precise glioma targeting ability ofc(RGDfK)/Pep-22-LP were further verified by ex vivo imagingof the brains 24 h postinjection (Figure 5C). Excised organswere also harvested and imaged (Figure 5B). An Intensefluorescence signal was observed in liver and spleen,corresponding to the main clearance route of liposomes.46

Moreover, no perspicuously difference in fluorescence signal ofperipheric organs was observed among groups, suggesting that

c(RGDfK) and Peptide-22 modification would not change thenonspecific distribution behavior of liposomes.

3.7. In Vitro Cytotoxicity Study. In vitro cytotoxicity offree DOX and different DOX loaded liposomes on U87 cellswas measured by MTT assay (Figure 6 and Table 2). It couldbe concluded from the results that both free DOX and DOXloaded liposomes could inhibit the proliferation of U87 cells ina concentration-dependent manner, proving the anticancereffect of DOX on such kind of brain tumors. As shown in Table2, free DOX (IC50 = 0.325 μg/mL) exhibited strongerinhibitory effect to the proliferation of U87 cells than DOX-LP (IC50 = 0.747 μg/mL), c(RGDfK)-DOX-LP (IC50 = 0.504μg/mL) and Pep-22-DOX-LP (IC50 = 0.389 μg/mL). Thisphenomenon was also observed in other reports,32,47 which wasbecause free DOX could be quickly transported into cellsdirectly via passive diffusion without the drug release process,while liposomes were internalized through endocytosis whichcost time and energy, and then probably underwent a

Figure 4. Qualitative (A) and quantitative (B) cellular uptake of 5 μM FAM loaded liposomes on U87 cells for 4 h. Original magnification = 100×.Data are represented as mean ± SD (n = 3). *** Indicates statistically significant difference (p < 0.001).

Figure 5. In vivo imaging of intracranial glioma-bearing mice at 2, 6, 12 and 24 h after i.v. injection with DiR loaded liposomes (A). Ex vivo imagingof excised organs (B) and brains (C) 24 h postinjection.

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sustained-release process. Moreover, the PEGylation of lip-osomes might retard the interaction between liposomes andcells, resulting in the lower cytotoxicity of unmodified or single-modified liposomes. However, c(RGDfK)/Pep-22-DOX-LPdisplayed the strongest cytostatic activity among the groups,whose IC50 value (IC50 = 0.164 μg/mL) was only half of thefree DOX. The effect was ascribed to the tremendouslyenhanced cellular uptake mediated by the specific bindingbetween c(RGDfK)/αvβ3 and Peptide-22/LDLR. Targetingmore than one receptors in the same cell surface could increasethe number of binding sites, strengthen the binding affinity, andsubsequently deliver more anticancer drugs into tumor cells,thus leading to the enhanced cytotoxicity.48

3.8. In Vivo Antiglioma Effect. To investigate the in vivoantiglioma effect of functionalized liposomes, intracranialglioma-bearing mice were injected with different DOXformulations on day 7, 14 and 21. Figure 7A shows the bodyweight changes of mice during the experiment. Compared withother groups, the mice treated with free DOX exhibited arapidest loss in body weight, which was caused by the severesystemic toxicity of free DOX.49 The gentler loss of bodyweight was observed in the groups administrated with DOX

loaded liposomes, indicating the reduction of systemic sideeffect by liposome encapsulation. As shown in Figure 7B, themodification of c(RGDfK) or Peptide-22 could improve theantiglioma effect of DOX-loaded liposomes, the median survivaltime of mice were notably prolonged. Among all the groups,the mice treated with c(RGDfK)/Pep-22-DOX-LP, i.e., 39.5days, was significantly longer than that of mice treated withsaline (27 days, p < 0.001), free DOX (30.5 days, p < 0.001),DOX-LP (31.5 days, p < 0.001), c(RGDfK)-DOX-LP (34.5days, p < 0.01) and Pep-22-DOX-LP (36 days, p < 0.05),respectively. The results demonstrated that c(RGDfK) andPeptide-22 dual-modified liposomes exhibited a significantimprovement in antiglioma effect than those decorated withany of the single ligand, indicating the synergetic effect of twoligands in treating brain tumors. Nanocarries functionalizedwith targeting ligands were frequently reported for overcomingBBB and targeting the tumor cells in the treatment ofglioma.17,47,50 But there were only some reports aboutmodifying ligands to the surface of nanoparticles to realizesimultaneous BBB/BBTB crossing and brain tumor targeting.This study was the first one to construct c(RGDfK) andPeptide-22 comodified liposomes loaded with antitumor drugs,which could cross the BBB/BBTB simultaneously and deliverdrugs into the tumor.

3.9. Cardiotoxicity Evaluation. DOX, one of the mosteffective chemotherapeutic agents, is used as a first-line drug innumerous types of cancer. However, it exhibits serious adverseeffects, especially lethal cardiotoxicity.51 To evaluate further thesafety of different formulations, sections of heart were stainedwith hematoxylin and eosin. As shown in Figure 8, myocardialhypertrophy was obviously observed in the heart section of freeDOX treated group. However, there was no injury found in thesamples of all the liposome treated groups, which indicated thatthe liposomes could decrease the cardiac toxicity of DOX andhad good in vivo safety.

4. CONCLUSIONSIn this study, we investigated the BBB targeting capacity ofAngiopep-2-LP, T7-LP and Peptide-22-LP and the BBTBtargeting ability of c(RGDfK)-LP, D-SP5-LP and Pep-1-LP,respectively. Based on the cellular uptake results on BCECs andHUVECs, Peptide-22 and c(RGDfK) were picked to develop aBBB/BBTB dual-crossing, glioma targeting liposomal drugdelivery system, c(RGDfK)/Pep-22-DOX-LP. Compared withthe modification of a single peptide, the co-operative effect ofintegrin αvβ3 and LDLR mediated recognition significantlyimproved the internalization and cytotoxicity of DOX loadedliposomes on U87 cells. In vivo biodistribution and antiglioma

Figure 6. Cytotoxicity of free DOX and different DOX liposomesagainst U87 cells 48 h postincubation at 37 °C (n = 4, mean ± SD).

Table 2. IC50 Values of Free DOX and Different DOXLiposomes against U87 Cells

formulations IC50 (μg/mL)free DOX 0.325DOX-LP 0.747c(RGDfK)-DOX-LP 0.504Pep-22-DOX-LP 0.389c(RGDfK)/Pep-22-DOX-LP 0.164

Figure 7. Antiglioma effect of different DOX formulations after intravenous administration to intracranial glioma-bearing mice. (A) Body weightchange and (B) Kaplan−Meier survival curves of model mice (n = 6).

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effects further verified that c(RGDfK)/Pep-22-DOX-LP couldefficiently transport across BBB/BBTB and specificallyaccumulate in the glioma site, and then prolong the survivaltime of intracranial glioma-bearing mice. Taking these together,we claimed c(RGDfK) and Peptide-22 dual-modified liposomescould serve as a promising platform for glioma chemotherapy indifferent progression stages.

■ ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acsami.6b15831.

MALDI-TOF mass spectrum of DSPE-PEG3400-T7,DSPE-PEG3400-D-SP5, DSPE-PEG3400-Pep-1, DSPE-PEG3400-Angiopep-2, DSPE-PEG3400-Peptide-22 andDSPE-PEG3400-c(RGDfK); serum stability of differentDOX-loaded liposomes over 24 h (PDF)

■ AUTHOR INFORMATIONCorresponding Authors*X. Xu. Tel: +86-21-3127-0810. E-mail: xuxinchun@live.cn.*J. Wang. Tel: +86-21-5198-0088. E-mail: jxwang@fudan.edu.cn.ORCIDZiqing Duan: 0000-0002-2323-7137Jianxin Wang: 0000-0003-0879-5883NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis work was supported by the grant from the National BasicResearch Program of China (No. 2013CB 932500) andNational Natural Science Foundation of China (No.81361140344).

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Figure 8. Histochemistry inspection of heart sections stained with hematoxylin and eosin of intracranial glioma-bearing mice after i.v. administrationof different DOX formulations. Red arrows indicate the myocardial hypertrophy. Original magnification = 200×.

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