College of Pharmacy and Research Institute of Pharmaceutical Sciences,Seoul National University,1 Kwanakro,Seoul 08826,Republic of Korea

Original Research Paper

Surface-modifed liposomes for syndecan 2–targeted delivery of edelfosine

Gayong Shim,Yong Hee Yu,Soondong Lee,Jinyoung Kim, Yu-Kyoung Oh*

College of Pharmacy and Research Institute of Pharmaceutical Sciences,Seoul National University,1 Kwanakro,Seoul 08826,Republic of Korea

A R T I C L EI N F O

Article history:

Received 17 April 2016

Accepted 11 May 2016

Available online 7 June 2016

Ligand density

AG73 peptide

Syndecan 2

Liposome

Anticancer drug

Edelfosine

Here,we report that the modifcation of liposome surfaces with AG73 peptides enhances delivery of the lipophilic anticancer drug,edelfosine,to tumor cells overexpressing the cellsurface receptor,syndecan 2.To test the effect of liposomal surface density of AG73 peptides on cellular uptake,we synthesized AG73 peptide-conjugated polyethylene glycol(MW 2000) lipid and incorporated it into fuorescence dye-labeled anionic liposomes with different ligand densities(1,2,or 5 mol%of total lipids).Cellular uptake of AG73-peptide–modifed liposomes gradually increased in proportion to the surface ligand density.The percentages of cells positive forAG73-modifed,fuorescent-dye–labeled liposomes were 19.8±2.0%,23.1±5.0%, and 99.2±1.0%,for ligand mole percentages of 1,2,and 5,respectively.The cell-targeting ability of AG73-modifed liposomes was not signifcantly altered by the serum content of culture media.In keeping with the observed enhanced cellular uptake,AG73-peptide–modifed liposomes entrapping edelfosine exhibited greater cancer cell-killing effects compared with unmodifed liposomes.Following intravenous administration into tumor-bearing mice, AG73-peptide–modifed liposomes showed 2.1-fold greater accumulation in tumors than unmodifed liposomes.These results support the feasibility of using syndecan 2–directed liposomes for delivery of edelfosine.

?2016 Production and hosting by Elsevier B.V.on behalf of Shenyang Pharmaceutical University.This is an open access article under the CC BY-NC-ND license(http:// creativecommons.org/licenses/by-nc-nd/4.0/).

1.Introduction

Edelfosine,an alkylphospholipid analog anticancer agent,is known to induce apoptosis of cells in solid tumor through an endoplasmic reticulum stress response and interaction with mitochondria[1,2].Although edelfosine has shown promising anticancer effects,the dose-dependent hemolysis of edelfosine and its toxicity toward normal tissues such as bone marrow cells[3]have limited its further use in clinical applications.Thus,tumor-targeted delivery would be helpful for reducing the side effects of edelfosine to normal tissues.Although previous studies have described nanoparticle formulations of edelfosine that reduce toxicity[4,5],there has been little effort to formulate edelfosine in surface-modifed liposomes with appropriate tumor-cell–targeting ligands.

Syndecansaretransmembraneheparansulfate proteoglycans that act as cell surface receptors for a variety of proteins,including growth factors[6],enzymes[7]and extracellular matrix proteins[8,9].Recent studies have demonstrated that syndecans are overexpressed in malignant cells [7,10],where they promote cell adhesion,proliferation[7],and invasion[11].The receptor,syndecan 2,is considered a prognostic marker of various malignant cells,including those of pancreatic adenocarcinomas[12],oral squamous cell carcinomas[13],and colon carcinomas[14].It is known to be upregulated on the surface of cancer cells[10,15]and has been shown to play an important role in the progression of breast carcinoma[11,16].

Here,we tested the syndecan 2–targeting ability of peptideligand–modifed liposomes for tumor-cell–targeted delivery of edelfosine.We demonstrated that syndecan 2–targeting liposomes with 5 mol%density of ligand enhanced the cellular delivery,tumor accumulation,and therapeutic effects of edelfosine.

2.Materials and methods

2.1.Synthesis of lipids tagged with a syndecan 2 ligand

The peptide sequence of the syndecan 2 ligand,AG73,is CGGRKRLQVQSIRT(Peptron Inc.,Daejeon,South Korea).In this sequence,the two glycines and a cysteine act as a spacer and an attachment site for maleimide-functionalized lipids,respectively.AG73 peptide(11 μmol)was dissolved in 10 ml of dimethyl sulfoxide(DMSO),to which dithiothreitol(Sigma-Aldrich,St.Louis,MO,USA)was subsequently added at a fnal concentration of 1 mM.After adding 10 μmol of 1,2-distearoylsn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)2000](ammonium salt)(Avanti Lipids,Birmingham,AL,USA)dissolved in 10 ml of DMSO,the reaction mixture was stirred at 4°C for 24 h,then dialyzed (MWCO 2000 Da;Spectrum Laboratories,Inc.,Rancho Dominguez,CA,USA)against 25%ethanol for 12 h and against deionized water for 24 h.The resulting product was confrmed by1H NMR analysis.

2.2.Preparation of liposomes

Liposomes were prepared as described previously[17]with slight modifcation.For preparation of liposomes,2 μmol each of egg L-α-phosphatidylcholine(Avanti Lipids),eggL-α-phosphatidyl-DL-glycerol(Avanti Lipids),cholesterol(Sigma-Aldrich),and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N[methoxy (polyethylene glycol)2000](PEG2K-DSPE;Avanti Lipids)or AG73-PEG2K-DSPE were mixed at a molar ratio of 2:2:2:0.3 in organic solvents.In some experiments,fuorescent liposomes were prepared by adding 0.2 μmol of 18:1–12:0 Square-685 phosphatidylcholine(Avanti Polar Lipids)orN-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine(triethylammonium salt)(NBD-PE; Molecular Probes,Inc.,Eugene,OR,USA)to the previously described lipid mixtures.Liposomes loaded with edelfosine(Tocris Bioscience,Bristol,UK)were prepared by adding 2 μmol of edelfosine to the previously described lipid mixtures.For comparison with edelfosine-loaded liposomes,1-stearoyl-2-hydroxysn-glycero-3-phosphocholine(Lyso PC;Avanti Polar Lipids)-loaded liposomes were prepared by substituting 2 μmol of Lyso PC for edelfosine.Organic solvents were removed from lipid mixtures using a rotary evaporator.The resulting thin flms were hydrated with 1 ml of HEPES-buffered saline(pH 7.4)and vortexed.The resulting multilamellar vesicles were extruded three times through 0.2-μm polycarbonate membrane flters (Millipore Corp.,Billerica,MA,USA)and then incubated at 4°C before use.

2.3.Syndecan 2 expression on cancer cell surfaces

Flow cytometry was used to evaluate the expression of syndecan 2 on the cell surface.After harvesting,BT20 cells were incubated for 1 h at 4°C in cold phosphate-buffered saline(PBS) containing 3%bovine serum albumin.Next,cells were washed and incubated for 1 h at 4°C with an allophycocyaninconjugated rat anti-human syndecan 2 monoclonal antibody (R&D Systems,Inc.,Minneapolis,MN,USA),diluted 1:50 in cold PBS containing 2%fetal bovine serum(FBS).Antibody-stained cells were then analyzed by fow cytometry using a BD FACSCalibur system equipped with Cell Quest Pro software(BD Bioscience,San Jose,CA,USA).

2.4.Cellular uptake

Cellular uptake of liposomes containing NBD-PE was assessed using fuorescence microscopy and fow cytometry.For fuorescence microscopy,BT20 cells were seeded onto 24-well plates at a density of 5×104cells/well.In some experiments,the medium was replaced with medium containing different concentrations of FBS(10%,20%,or 50%)or with 100% FBS.After 24 h,cells were treated with 20 μl of fuorescent liposomes for 30 min,then washed twice with PBS and observed under a fuorescence microscope(Leica DM IL;Leica,Wetzlar, Germany).Fluorescent-liposome–treated cells were harvested,washed three times with cold PBS containing 2%FBS, and analyzed by fow cytometry.

2.5.Animals

Five-week-old female Balb/c athymic nude mice,obtained from Orient Bio.Inc.(Seungnam,Kyonggi-do,South Korea;approved animal experimental protocol number SNU-150609-2),were used forin vivoexperiments.Animals were raised under standard pathogen-free conditions at the Animal Center for Pharmaceutical Research,Seoul National University.All animal experiments were conducted in accordance with the Guidelines for the Care and Use of LaboratoryAnimals of the Institute of Laboratory Animal Resources,Seoul National University.

2.6.Anticancer activity

The anticancer activity of liposomes containing edelfosine or Lyso PC was tested using a Cell Counting Kit(CCK)assay.BT20cells were seeded onto a 48-well plate at a density of 2×104cells per well,and treated with liposomes containing 5 nM edelfosine or Lyso PC.After 24 h,cell viability was quantifed using a CCK according to the protocol provided by the manufacturer(Dojindo Molecular Technologies,Inc.,Rockville,MD, USA).Cell viability was expressed as a percentage of that measured in control groups.

2.7.In vivomolecular imaging

Molecular imaging was used to trace the distribution of PEG-and AG73-PEG–modifed liposomes(PEG2KLipo and AG73-PEG2KLipo,respectively).Mice were subcutaneously inoculated in the dorsal right side with 4×106BT20 cells.After tumors had grown to approximately 100–150 mm3,200 μl of Square-685 lipid-loaded fuorescent liposomes were intravenously injected.At 2 h post-dose,Square-685 fuorescence was imaged using an eXplore Optix system(Advanced ResearchTechnologies Inc.,Montreal,Canada).A 670-nm pulsed-laser diode was used to excite Square-685 molecules.

2.8.Statistics

Analysis of variance(ANOVA)withpost hocStudent–Newman–Keuls test was used for statistical evaluation of experimental data.All statistical analyses were done using SigmaStat software(version 3.5;Systat Software,Richmond,CA,USA).AP-value＜0.05 was considered statistically signifcant.

3.Results and discussion

3.1.Construction of AG73-PEG2KLipo for syndecan 2 targeting

The premise of these studies is that syndecan 2 overexpressing cancer cells can be targeted byAG73-PEG2KLipo through receptormediated cellular delivery,as shown in Fig.1.To this end,we grafted the AG73 peptide,utilized as a ligand for the receptor, syndecan 2,onto liposomes as described in section 2;liposomes prepared withoutAG73(PEG2KLipo)were used as controls. There was no signifcant difference in particle sizes(Fig.2A) or zeta potentials(Fig.2B)between liposomes.AG73 is a biologically active peptide originating from the cell-adhesion protein laminin α1[18,19]that is known to promote cell attachment[20],angiogenesis[21],tumor growth,and metastasis [22]in various malignant cells through binding to syndecans. Yamada and colleagues[23]have utilized AG73 as a celladhesion ligand to modify hyaluronic acid hydrogels for tissue engineering,and Negishi and colleagues[24]have used ultrasound imaging of AG73-modifed bubble liposomes for tumor diagnosis.

3.2.Cellular delivery of AG73-PEG2KLipo with various densities of ligand

AG73 peptide tagging affected the effciency of cellular uptake of liposomes in syndecan 2–expressing cancer cells.Surface expression of syndecan 2 on BT20 cancer cells was confrmed by fow cytometry(Fig.3A).BT20 cells treated withAG73-PEG2KLipo,labeled with the fuorescent dye,NBD-PE,to allow fuorescence monitoring,showed a gradual increase in cellular uptake effciency as a function of AG73 content on the liposome surface(Fig.3E,F,G),whereas PEG2KLipo was not taken up by cells regardless of PEG2Kcontent(Fig.3B,C,D).AG73-PEG2KLipo modifed with 5 mol%of added ligand achieved the greatest cellular delivery(Figs.3 and 4),with 99.2±1.0%of cells treated with this preparation exhibiting fuorescence(Fig.4C,D) compared with 19.8±2.0%and 23.1±5.0%of cells prepared with 1 mol%(Fig.4A,D)and 2 mol%(Fig.4B,D)of ligand.Ligand modifcation of PEGylated nanoparticles has been suggested as an effective solution to the so-called“PEG dilemma”[25],which refects inhibition of cellular uptake of nanoparticles by the PEG stabilizer.As an example of the latter effect,PEGylated nanoparticles with 5 mol%PEG achieved a signifcantly higher concentration in blood,but lower cellular delivery of cargo,than PEG-unmodifed nanoparticles after systemic injection[26].The AG73 ligand modifcation was able to reverse the effects of PEG on cellular delivery.This ability of AG73 modifcation to effectively overcome the PEG dilemma is important because PEGylation serves valuable functions,providing a hydrophilic,fexible spacer between ligand molecules and liposomes to improve functionality of the ligand and protecting liposomes from interaction with biological fuids[27].

Fig.1–AG73-modifed liposomes for syndecan 2 targeting of edelfosine.AG73-PEG2KLipo for targeting edelfosine to syndecan 2 on cancer cells is depicted.

Fig.2–Characterization of AG73-PEG2KLipo.(A)Particle sizes of PEG2KLipo and AG73-PEG2KLipo were measured by dynamic light scattering.(B)Zeta potentials were determined using an electro-Doppler method.

3.3.Cellular delivery of AG73-PEG2KLipo in biological fuids

The cellular-uptake effciency of AG73-PEG2KLipo was not infuenced by serum-containing conditions that mimic biological fuids(Fig.5).Likewise,the cancer cell-targeting ability of AG73-PEG2KLipo was similarly unaffected by increased serum conditions.Only the 100%serum condition slightly decreased cellular uptake ofAG73-PEG2KLipo(to 82.5%±5.0%).On the basis of these results,AG73 modifcation would not be expected to alter the pharmacokinetic profle of PEG2KLipo.Indeed,several groups have reported that modifcation of liposomes with ligands targeting epithelial cell adhesion molecule[28],epidermal growth factor receptor[29],or Her2/neu[30]did not increase clearance of liposomes,but did increase tumor accumulation compared with unmodifed liposomes.

3.4.Antitumor effect of edelfosine delivered by AG73-PEG2KLipo

Edelfosine-encapsulated AG73-PEG2KLipo showed enhanced cancer cell-killing effects(Fig.6).CCK assays revealed that treatment with edelfosine-loaded liposomes with a PEG2Kor AG73-PEG2Kcontent of 1 or 2 mol%had no signifcant effect on BT20 cell viability.In contrast,treatment with edelfosine-loaded liposomes containing 5 mol%PEG2Kor AG73-PEG2Kcontent exerted tumor cell-killing effects,reducing cell viability to 64.2%±2.5%and 46.1%±9.2%,respectively.Edelfosine acts as an anticancer agent by inducing apoptosis in malignant cells, including breast cell carcinoma[3,31].However,severe side effects of edelfosine have been observed after systemic administration[32].Recent reports have reported that edelfosine can be formulated into lipid bilayers of liposomes owing to its lipid-like structure[5,33,34].Liposomal edelfosine formulations have been shown to prevent against the toxicity of free edelfosine,demonstrating much higher safety than the free drug [5]and an improved therapeutic index as a result of their enhanced toxicity toward resting cancer cells[34].

Fig.3–Syndecan 2–mediated cellular uptake of AG73-PEG2KLipo.(A)The surface expression of syndecan 2 on BT20 cell surfaces was analyzed by fow cytometry.BT20 cells were treated with NBD-PE–loaded PEG2KLipo(B,C,D)or AG73-PEG2KLipo(E,F,G)constructed to contain 1 mol%(B,E), 2 mol%(C,F),or 5 mol%(D,G)PEG2Kor AG73-PEG2K-DSPE. After 30 min,cells were observed under a fuorescence microscope.Scale bar,100 μm.

3.5.Tumor accumulation of AG73-PEG2KLipo

Systemically injected AG73-PEG2KLipo nanoparticles with a 5 mol%AG73-PEG2Kcontent were mainly distributed to tumor tissues of mice xenografted with syndecan 2–overexpressing BT20 cells(Fig.7A).The fuorescence intensity in tumor regionsof AG73-PEG2KLipo–treated mice was 2.1-fold higher than that of PEG2KLipo–treated mice(Fig.7B).PEG2KLipo,lacking the AG73 ligand,also showed some distribution to tumor sites.Such distribution to tumors in the absence of a targeting ligand frequently occurs with nanoparticulate delivery systems owing to the enhanced retention and permeability effect[35].However, tumor-targeting ligands are still needed for improved intracellular delivery to tumor cells[36].Indeed,whereas PEG2KLipotreated mice showed higher distributions to organs other than tumor tissue,AG73-PEG2KLipo nanoparticles were mainly distributed in tumor tissue(Fig.7).

Fig.4–Dependence of cancer cell-targeting effciency on the AG73-PEG2Kcontent of liposomes.BT20 cells were treated with NBD-PE–loaded PEG2KLipo or AG73-PEG2KLipo constructed to contain 1 mol%(A),2 mol%(B),or 5 mol%(C) of PEG2Kor AG73-PEG2K-DSPE.After 30 min,fuorescencepositive populations of cells were analyzed(A–C)and quantifed(D)by fow cytometry.*P＜0.05 vs.other groups (ANOVA and Student–Newman–Keuls test).

Fig.5–Effect of serum content on cellular-uptake effciency of AG73-PEG2KLipo.BT20 cells cultured with different

percentages of serum were treated with NBD-PE–loaded

AG73-PEG2KLipo.After 30 min,fuorescence-positive

populations of cells were analyzed by fow cytometry.

Fig.6–In vitroanticancer effect of edelfosine delivered by AG73-PEG2KLipo.BT20 cells were treated with edelfosine (50 μM)-encapsulated PEG2KLipo or AG73-PEG2KLipo.After 24 h,BT20 cell viability was measured using CCK assays (n=4).

4.Conclusions

We synthesized AG73-peptide–conjugated PEG-lipid and formulatedAG73-grafted liposomes.AG73-PEG2KLipo nanoparticles exhibited ligand density-dependent,enhanced cellular delivery in syndecan 2–overexpressing cancer cellsin vitro,and showed higher tumor accumulation than ligand-unmodifed liposomes after systemic administrationin vivo.In addition, edelfosine-loaded liposomes signifcantly decreased tumor cell viability.These results provide evidence that AG73-PEG2KLipo could serve as a receptor-specifc nanovehicle for delivery of edelfosine to syndecan 2–overexpressing cancer cells.

Acknowledgments

This work has been fnancially supported by a Grant from the Korean Health Technology R&D Project(No.HI15C2842),Ministry of Health&Welfare,Republic of Korea.

Fig.7–In vivodistribution of AG73-PEG2KLipo.Square-685–labeled PEG2KLipo or AG73-PEG2KLipo was intravenously injected into BT20 tumor-bearing mice.(A)After 2 h,thein vivodistribution of liposomes was visualized using a molecular imaging system.(B)Average photon counts at tumor sites were quantifed.*P＜0.05 vs.the PEG2KLipo group(Student’s t-test).

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*< class="emphasis_italic">Corresponding author.

.College of Pharmacy and Research Institute of Pharmaceutical Sciences,Seoul National University,1 Kwanakro,Seoul 08826,Republic of Korea.Tel.:+82 2 880 2493;fax:+82 2 882 2493.

E-mail address:ohyk@snu.ac.kr(Y.-K.Oh).

http://dx.doi.org/10.1016/j.ajps.2016.05.003

1818-0876/?2016 Production and hosting by Elsevier B.V.on behalf of Shenyang Pharmaceutical University.This is an open access article under the CC BY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).