European Journal of Pharmaceutics and Biopharmaceutics 84 (2013) 517525Contents lists available at SciVerse ScienceDirect

European Journal of Pharmaceutics and Biopharmaceutics

journal homepage: www.elsevier .com/locate /e jpbResearch paper

Surface functionalization of doxorubicin-loaded liposomes with octa-argininefor enhanced anticancer activity

Swati Biswas, Namita S. Dodwadkar, Pranali P. Deshpande, Shruti Parab, Vladimir P. Torchilin Center for Pharmaceutical Biotechnology and Nanomedicine, Northeastern University, Boston, USA

a r t i c l e i n f oArticle history:Received 12 November 2012Accepted in revised form 27 December 2012Available online 17 January 2013

Keywords:DoxorubicinLiposomesOcta-arginineDrug deliveryApoptosis0939-6411/$ - see front matter 2013 Elsevier B.V. Ahttp://dx.doi.org/10.1016/j.ejpb.2012.12.021

Corresponding author. Center for Pharmaceutical Bicine, Northeastern University, 140 The Fenway, 36002115, USA. Tel.: +1 617 373 3206; fax: +1 617 373 7

E-mail address: v.torchilin@neu.edu (V.P. Torchilina b s t r a c t

Doxorubicin-loaded PEGylated liposomes (commercially available as DOXIL or Lipodox) were surfacefunctionalized with a cell-penetrating peptide, octa-arginine (R8). For this purpose, R8-peptide wasconjugated to the polyethylene glycoldioleoyl phosphatidylethanolamine (PEGDOPE) amphiphilicco-polymer. The resultant R8PEGPE conjugate was introduced into the lipid bilayer of liposomes at2 mol% of total lipid amount via spontaneous micelle-transfer technique. The liposomal modificationdid not alter the particle size distribution, as measured by Particle Size Analyzer and transmission elec-tron microscopy (TEM). However, surface-associated cationic peptide increased zeta potential of themodified liposomes. R8-functionalized liposomes (R8-Dox-L) markedly increased the intracellular andintratumoral delivery of doxorubicin as measured by flow cytometry and visualizing by confocal laserscanning microscopy (CLSM) compared to unmodified Doxorubicin-loaded PEGylated liposomes(Dox-L). R8-Dox-L delivered loaded Doxorubicin to the nucleus, being released from the endosomes athigher efficiency compared to unmodified liposomes, which had marked entrapment in the endosomesat tested time point of 1 h. The significantly higher accumulation of loaded drug to its site of action forR8-Dox-L resulted in improved cytotoxic activity in vitro (cell viability of 58.5 7% for R8-Dox-L com-pared to 90.6 2% for Dox-L at Dox dose of 50 lg/mL for 4 h followed by 24 h incubation) and enhancedsuppression of tumor growth (348 53 mm3 for R8-Dox-L, compared to 504 54 mm3 for Dox-L treat-ment) in vivo compared to Dox-L. R8-modification has the potential for broadening the therapeuticwindow of pegylated liposomal doxorubicin treatment, which could lead to lower non-specific toxicity.

2013 Elsevier B.V. All rights reserved.1. Introduction

Liposomes, nanosized phospholipid vesicles, have found wideapplication in drug delivery as pharmaceutical carriers [1].The advantages of using liposomes as a drug delivery vehicleinclude the biocompatibility, biodegradability, monodispersity,non-immunogenicity, and high capacity for entrapment of bothwater-soluble and insoluble drugs. Liposomes and other nanopar-ticles can preferentially deliver chemotherapeutic drugs to a tumorsite due to their small size and long systemic circulation. Nanopar-ticles take advantage of a tumors leaky blood vessels associatedwith enlarged gaps between endothelial cells that range from100 to 400 nm that allow increased extravasation of nanosizedpharmaceutical drug carriers into a tumor site [24]. In spite ofthe various advantages of liposomal nanocarriers including a size(100 nm) which allows preferential accumulation of liposomesin a tumor and reduced toxic effects of chemotherapy on normalll rights reserved.

iotechnology and Nanomed-Huntington Ave., Boston, MA509.).tissues, the drawbacks including rapid renal clearance and recogni-tion by the reticulo-endothelial system have prompted liposomalsystem modification to obtain higher therapeutic efficacy. To addressthe rapid renal clearance issue, the liposomes can be coated withpolyethylene glycol (PEG) to make them long-circulating, whichallows them to accumulate in the areas with a compromised, leakyvasculature [58]. Pegylated liposomal doxorubicin (Dox), commer-cially available as Doxil/Caelyx or Lipodox, is an example of thislong-circulating liposome formulation. Dox-loaded pegylated lipo-somes have been indispensible for application in metastatic, breast,and ovarian cancer [9]. This formulation decreases the toxic effectsassociated with free Dox administration. However, PEG-modifiedliposomes have poorer cellular internalization [10]. The therapeuticefficacy of pegylated liposomal Dox was not dramatically increaseddue mainly to poor cell penetration and slower drug release fromthe liposomes [9,11]. Therefore, it is obvious that improved intracel-lular delivery would lead to the increased cytosolic drug concentra-tion required for enhanced drug action.

The emerging concept for improvement of drug action involvesefficient intracellular or specific organelle-targeted drug delivery[1215]. Due to the inability of the nanocarriers to passively crossthe cell membrane barrier as do small molecule drugs (which

http://dx.doi.org/10.1016/j.ejpb.2012.12.021mailto:v.torchilin@neu.eduhttp://dx.doi.org/10.1016/j.ejpb.2012.12.021http://www.sciencedirect.com/science/journal/09396411http://www.elsevier.com/locate/ejpb

518 S. Biswas et al. / European Journal of Pharmaceutics and Biopharmaceutics 84 (2013) 517525require energy-dependant endocytosis for internalization), cellpenetration enhancers have been utilized that accelerate cytoplas-mic delivery [16,17]. In this regard, many peptide sequences havebeen identified that promote translocation of a variety of cargosacross the cell membrane [18]. The most commonly used cell-penetrating peptide (CPP) is TATp, which is derived from the86-mer trans-activating transcriptional activator (TAT protein),encoded by the human immunodeficiency virus type-1 (HIV-1)[16,1921]. The poly-arginine CPP also has translocation activityvery similar to TATp [22]. In particular, synthetic oligoargininepeptides mimic the HIV-1 TAT protein (Tat-(48-60)). The optimumchain length of poly-arginine for promotion of efficient transloca-tion is 8 arginine units (R8) [22].

R8 has been used as a penetration enhancer in some of the pre-viously reported studies. R8-conjugated Smac peptide suppressedthe inhibitor of apoptosis (IAP) proteins [23]. Octaarginine andpH-sensitive fusogenic peptide-modified nanoparticles were uti-lized for liver gene delivery [24]. The Harashima group studiedthe intracellular fate of R8-modified liposomes [25,26]. Khalilet al. demonstrated that liposomes modified with a high densityof R8 were taken up mainly by macropinocytosis [27]. A possibleinternalization pathway for the R8-modified liposomes with a fus-ogenic composition was investigated in our recent study [28].

To enhance the therapeutic efficacy by addressing the drawbackof poor penetration of cell membranes, we modified a pegylatedliposomal doxorubicin, Lipodox. In this study, for the first time,the possibility of enhanced anticancer activity of a relatively shortCPP, R8-modified pegylated liposomal Dox, was investigated.2. Materials and methods

2.1. Materials

2.1.1. ChemicalsPegylated Liposomal Doxorubicin (Lipodox, 2 mg/mL of Dox)

was purchased from Sun Pharmaceutical Ind. Ltd. (Gujarat,India). 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000](ammonium salt) (PEG2KDOPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)was from Avanti Polar Lipids (AL, USA). Octa-arginine peptide(RRRRRRRR, M.W. 1267.46 Da) was synthesized by the Tufts Uni-versity Core Facility (Boston, MA). NPCPEG2KNPC was obtainedfrom Laysan Bio (AL, USA). Thiazoyl blue tetrazolium bromide(MTT) was purchased from Sigma-Aldrich (St. Louis, MO). AnnexinV-Alexa Fluor 488 conjugate, Annexin-binding buffer 5 concen-trate, Hoechst 33342, and Transferrin-Alexa Fluor 488 were pur-chased from Molecular Probes (Eugene, OR). Para-formaldehydewas from Electron Microscopy Sciences (Hatfield, PA). The Trypanblue solution was purchased from Hyclone (Logan, UT). Fluoro-mount-G was from Southern Biotech (Birmingham, AL).

2.1.2. Cell linesThe murine mammary carcinoma cell line, 4T1, and normal

mouse fibroblast cells, NIH-3T3, were purchased from the Ameri-can Type Culture Collection (Mansas, VA). Dulbeccos modified Ea-gles media (DMEM) and heat-inactivated fetal bovine serum (FBS)was obtained from Gibco (Carlsbad, CA). Concentrated penicillin/streptomycin stock solution was from CellGro (Herndon, VA).All other chemical and solvents were of analytical grade, purchasedfrom Sigma-Aldrich, and used without further purifications.

2.1.3. AnimalsFemale BALB/c mice (68 weeks old) were purchased from

Charles River Laboratories, MA, USA. All animal procedures wereperformed according to an animal care protocol approved byNortheastern University Institutional Animal Care and Use Com-mittee. Mice were housed in groups of 5 at 1923 C with a 12 hlight-dark cycle and allowed free access to food and water.2.2. Methods

2.2.1. Synthesis of R8PEG2KDOPEpNPPEG2KDOPE, the starting material to synthesize R8

PEG2KDOPE, was synthesized and purified according to anestablished procedure with modification [15,29]. Briefly, into thesolution of NPCPEG2KNPC (1 g, 0.5 mmol) in chloroform, a DOPE(37.2 mg, 0.05 mmol) solution in chloroform and 20 lL of triethyl-amine were added drop wise. The reaction mixture was stirredovernight at room temperature. On the following day, the reactionmixture was evaporated at reduced pressure using a rotary evapo-rator and freeze-dried. The dry, crude reaction mixture was dis-solved in 0.01 M HCl solution and purified by gel filtrationchromatography using a Cl4B column with the 0.01 M HCl as elu-ent. The purified fractions were freeze-dried, weighed, and dis-solved in chloroform to obtain a 10 mg/mL solution for storage at80 C. For the synthesis of R8PEG2KDOPE, R8 (7.4 mg,5.86 lmol) and triethylamine (10 lL) dissolved in DMF (200 lL)were added to a mixture of pNPPEG2KDOPE (10 mg, 3.9 lmol)in 1 mL chloroform. The reaction mixture was stirred at room tem-perature overnight. On the following day, the chloroform wasevaporated on a rotary evaporator at reduced pressure andfreeze-dried. This mixture was dissolved in PBS, pH 8.4, stirred atroom temperature for 4 h, and dialyzed against water overnightusing a cellulose ester membrane (MWCO. 2000 Da). The dialysatewas freeze-dried to obtain a solid white fluffy product which wasdissolved in methanol at 5 mg/mL and stored at 80 C.2.2.2. Modification of liposomes with PEG2KDSPE or R8PEG2KDOPEPegylated Dox-loaded liposomes (Lipodox) were modified

with R8PEG2KDOPE by the post-insertion method [3032]. Lipo-dox was provided as a sterile, translucent, liposomal dispersion of10 mL vial. The Doxorubicin HCl was at concentration 2 mg/mL at apH of 6.5. The liposomes were composed of N-(carbonyl-methoxy-polyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phospho-ethanolamine sodium salt (mPEGDSPE), 3.19 mg/mL; fullyhydrogenated soy phosphatidylcholine (HSPC), 9.58 mg/mL; andcholesterol, 3.19 mg/mL. The inserted co-polymer was 2 mol% ofthe total lipids in the liposomes. Briefly, Lipodox) solution(1000 lL) was added to the dry lipid film of PEG2KDSPE(0.90 mg, 0.45 lmol) or R8PEG2KDOPE (1.64 mg, 0.45 lmol).The modified liposomal solutions, Dox-L (modified with PEG2KDSPE) and R8-Dox-L (modified with R8PEG2KDOPE), werevortexed for 5 min and stirred overnight at 4 C for completehydration of the lipid film.

Empty liposomes were prepared with the following the lipidcomposition: fully hydrogenated soy phosphatidylcholine(9.58 mg/mL), cholesterol (3.19 mg/mL), and mPEGDSPE(3.19 mg/mL). The liposome suspension was stirred overnight at4 C with R8PEG2KDOPE (2 mol% of total lipids).2.2.3. Liposomal characterizationThe liposomal size and size distribution were measured by dy-

namic light scattering (DLS) using a Coulter N4-Plus SubmicronParticle Sizer (Coulter Corporation, Miami, FL). Size distributionwas confirmed by using transmission electron microscopy (TEM)(Jeol, JEM-1010, Tokyo, Japan). Liposome surface charge was mea-sured with a Zeta Phase Analysis Light Scattering (PALS) Ultrasen-sitive Zeta Potential Analyzer (Brookhaven Instruments, Holtsville,NY).

S. Biswas et al. / European Journal of Pharmaceutics and Biopharmaceutics 84 (2013) 517525 5192.2.4. Cell cultureCancer cells (4T1) and normal mouse fibroblasts (NIH-3T3)

were grown in DMEM with 2 mM L-glutamine, supplemented with10% (v/v) heat-inactivated FBS, 100 units/mL penicillin G, and100 lg/mL streptomycin. Cultures were maintained in a humidi-fied atmosphere at 37 C with 5% CO2.

2.2.5. Cell association with liposomes by FACS analysisThe cell association with liposomes was assessed by FACS anal-

ysis. After the initial passage in 75 cm2 tissue culture flasks (Corn-ing Inc., NY), 4T1 cells (4 105/well) were seeded in 6-well tissueculture plates. The following day, the cells were incubated withDox-L and R8-Dox-L at a Dox concentration of 6 lg/mL in 2 mLof serum-free media for 1 h and 4 h incubation periods. The mediawere removed; the cells were washed several times, trypsinized,suspended in 1 mL PBS, and then centrifuged at 1000 RPM for5 min. The cell pellet was suspended in PBS, pH 7.4, before analysisfor Dox fluorescence using a BD FACS Caliber flow cytometer. Thecells were gated using forward (FSC-H)-versus side-scatter (SSC-H) to exclude debris and dead cells before analysis of 10,000 cellcounts.

2.2.6. Cellular internalization by confocal microscopyCellular uptake of liposomes was visualized with confocal

microscopy. After the initial passage in tissue culture flasks, 4T1cells (4 104 cells) were grown in complete media on circular cov-er glasses placed in 12-well tissue culture plates in complete med-ia. The following day, cells were incubated with Dox-L or R8-Dox-Lat a Dox concentration of 6 lg/mL for 1.5 h in serum-free media.After the incubation period, the cells on the cover-slips werewashed with PBS (four times) and Transferrin-Alexa Fluor 488was added at 10 lg/mL for 20 min, followed by Hoechst 33342 at5 lg/mL for 5 min, washed thoroughly with PBS, and fixed with4% para-formaldehyde for 10 min at room temperature. The cov-er-slips were washed thoroughly with PBS and mounted cell-sidedown on superfrost microscope slides with fluorescence-free glyc-erol-based mounting medium (Fluoromount-G; Southern Biotech-nology Associates) and viewed with a Zeiss confocal laser scanningmicroscope (Zeiss LSM 700) equipped with UV (Ex/Em. 385/470 nm), FITC (Ex/Em. 548/595 nm), and a rhodamine filter (Ex/Em. 548/719 nm) for imaging. The LSM picture files were analyzedusing Image J software.

2.2.7. Early apoptotic marker determination, Annexin V assayThe procedure for Annexin V labeling was carried out according

to the manufacturers protocol. Briefly, 4T1 cells were seeded in12-well plates at 8 104/well. After incubation for 4 h with Dox-L and R8-Dox-L at Dox concentration of 15 lg/mL, the cells wereincubated for an additional 18 h, trypsinized, washed with coldbinding buffer, and re-suspended in binding buffer (200 lL) withor without Annexin V-Alexa Fluor 488 conjugate (15 lL) for15 min in dark. The Dox-L and R8-Dox-L treated cells withoutthe Annexin V-Alexa Fluor 488 conjugate treatment were usedfor compensating the interference of Dox fluorescence in FACSstudy. The cells were diluted with binding buffer to a total volumeof 400lL and analyzed immediately by flow cytometry.

2.2.8. MTT assayThe 4T1 cells were seeded in 96 well microplates in phenol red-

free DMEM media at a density of 5 103 and 3 103 cells/well for24 and 48 h, respectively. On the following day, the cells wereincubated with Dox-L or R8-Dox-L at Dox concentrations up to100 lg/mL for 4 h, the media were removed, and the cells wereincubated for an additional 24 and 48 h in fresh complete media.After incubation, the old media were removed and the cells weretreated with MTT solution (5 mg/mL) in serum/phenol red-freeDMEM for 4 h. At the end of the incubation, cell viability was esti-mated by the ability of the cells to reduce the yellow dye, MTT, to apurple formazan product. The media were removed and replacedwith 100 lL of SDS solution (20%) in 0.01 N HCl for 4 h to dissolvethe formazan crystals. The absorbance was read at 570 nm using amicroplate reader (Synergy HT multimode microplate reader, Bio-tek Instrument, Winooski, VT). Blank readings obtained from thetreatment well with no cells were subtracted from each reading.For the evaluation of empty liposome cytotoxicity, 4T1 and NIH-3T3 cells were seeded in 96 well plates at 5 103 cells/well. Onthe following day, the cells were treated with empty or R8-modi-fied liposomes at a lipid concentration range of 0125 lg/mL for24 h. The cell viability was determined by MTT assay followingthe above outlined protocol.

2.2.9. In vivo tumor xenograftA subcutaneous tumor was established in the left flank of the

BALB/c mice by inoculating 2 106 4T1 cells (suspended in100 lL PBS). The time for detectable appearance of the tumorwas usually 1520 days. The length and width of the tumor weremeasured with calipers at 3 days intervals, and the tumor volumewas calculated using the formula (width2 length)/2.

2.2.10. Doxorubicin localization in tumorThe 4T1 tumor-bearing mice with an average tumor volume of

200 mm3 were administered Dox-L or R8-Dox-L i.v at a Dox dose of10 mg/kg or PBS. The animals were sacrificed with CO2 at 4, 24 h or72 h after injection and the tumors isolated. The isolated tumorswere washed quickly with PBS, pH 7.4, and frozen immediatelyby immersion in tissue freezing media and stored at 80 C. Tumorslices (8 lm) of the frozen tumors were cryo-sectioned using aCryotome Cryostat, mounted on superfrost plus slides, treated withHoechst 33342 at 5 lg/mL for 5 min, washed and fixed in 4% para-formaldehyde for 10 min at RT, and visualized using fluorescencemicroscopy.

2.2.11. Tumor volume reduction studyIntravenous treatment with PBS, Dox-L, or R8-Dox-L was

started after the tumor volume reached 50100 mm3 at Dox dosesof 0.5 mg/kg. Mouse groups were as follows: (i) PBS (controls); (ii)Dox-L; and (iii) R8-Dox-L (n = 6 per group). Injections via tail veinwere once every 3 days. The tumor volume and body weight wererecorded at 3 days interval for all tumor-bearing mice for 16 daysuntil the tumor size of control group animals reached 1000 mm3.

2.2.12. TUNEL assayA TUNEL assay was performed on the frozen tumor sections to

measure the pro-apoptotic effect at 24 and 72 h following Dox-Lor R8-Dox-L treatment at a Dox dose of 100 lg/mL. Tumor slices(8 lm) were cryo-sectioned, mounted on superfrost plus slides,fixed in 4% para-formaldehyde for 10 min at RT and permeabilizedwith proteinase K (20 lg/mL) for 15 min at RT. A TUNEL assay wasperformed on tissue sections using the FragEl DNA fragmenta-tion detection Kit following manufacturers instructions for frozensections. The TUNEL positive cells were examined with fluores-cence microscope equipped with a FITC-filter. Four random imagesobtained from two different tumors for each treatment group wereanalyzed using Spot Advanced software.

2.2.13. Statistical analysisThe data were assessed for statistical significance using

Students paired t-test and p values calculated with the GraphPad Prism 5 software (GraphPad Software, Inc; San Diego, CA).All numerical data are expressed as mean SD, n = 34, from threedifferent experiments. P values 60.05 were considered statistically

520 S. Biswas et al. / European Journal of Pharmaceutics and Biopharmaceutics 84 (2013) 517525significant. , , in figures indicated p values 60.05, 0.01, and0.001, respectively.3. Results

3.1. Physicochemical characterization of liposomes

Average particle sizes of Dox-L and R8-Dox-L at day +1 aftersynthesis were 90.9 17.4 and 86.7 23.9 nm and at day +15 were84.4 24.2 and 86.7 23.9 nm, respectively (Supplementary Fig. 1A

B

C

(PEG-PE)

Dox-L

O2N O OO

On O

ONH

OO

NPC-P

R8

NH

OO

On O

ONH

OO

R8-PE

R8

Fig. 1. Schematic representation of (A) the synthesis of R8-conjugated PEGPE and (B)micelle-transfer technique. (C) TEM image of control and modified liposomes (left, Dox-L,in this figure legend, the reader is referred to the web version of this article.)(S-1)). The result was confirmed using transmission electronmicroscopy for Dox-L and R8-Dox-L (Fig. 1). The average zeta po-tential values of Dox-L and R8-Dox-L at day +1 were35.68 4.31 and 14.24 1.16 nm and at day +15 were33.0 8.43 and 11.66 4.31 nm, respectively (Fig. S-1).3.2. Cellular association of liposomes

A time-dependent (1 h and 4 h) cell association study wasperformed using flow cytometry and confocal laser scanningDoxorubicin-loaded liposomes in HBS, pH 7.4

(R8-PEG-PE)

R8-Dox-L

PO

OH O

O

OO

EG2K-PE

DMF, Et3N, 12 h

PO

OH O

O

OO

G2k-PE

the preparation of the unmodified and R8-modified Dox-loaded liposomes by theand right R8-Dox-L). Scale bar. 100 nm. (For interpretation of the references to color

S. Biswas et al. / European Journal of Pharmaceutics and Biopharmaceutics 84 (2013) 517525 521microscopy (Fig. 2). The geometric means of Dox fluorescence forDox-L and R8-Dox-L were 8.77 0.12 and 14.11 0.53 for 1 hand 18.77 0.98 and 37.29 0.90 for 4 h, respectively. The cellassociation of Dox fluorescence was 2.14-fold and 2.64-fold higherfor R8-Dox-L compared to Dox-L at 1 and 4 h exposures, respec-tively. Confocal microscopy confirmed the FACS analysis result thatthe cells treated with R8-Dox-L demonstrated higher Dox fluores-cence compared to Dox-L at both the time points.

3.3. Doxorubicin localization in tumor

Dox fluorescence in tumor sections treated with R8-Dox-L washigher at all the time points (4, 24, and 72 h) compared to Dox-LA

B

Control Dox-

Dox-L-4h R8-Dox-L

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orub

icin

DN

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Ho

Fig. 2. Cellular uptake of Dox-L and R8-Dox-L in vitro and in vivo by 4T1 monolayersfluorescence due to Dox-labeling by FACS analysis, upon incubation with Dox-L and R8-Dwith Dox-L and R8-Dox-L (Dox dose, 6 lg/mL), visualized by confocal laser scanning mxenograft, showing the localization of Dox (right) upon the intravenous administration o548/595 nm). Nuclei were stained with Hoechst 33342 and tracked in UV (Ex/Em. 385/47mean was analyzed by Students t-test, Indicates p < 0.001.(Fig. 2). The Dox accumulation in tumor was highest after 24 hfor both the treatment, which eventually reduced by 72 h as indi-cated by eventual decreased fluorescence of the Dox signal.

3.4. Assessment of intracellular co-localization

To assess the intracellular localization of Dox delivered by Dox-L or R8-Dox-L, we performed a co-localization analysis using nucleistaining and an endosomal marker. The yellow and purple spots inthe merged pictures indicate the overlap of green with red andblue with red fluorescence, respectively. The confocal fluorescencemicrographs of Dox-L or R8-Dox-L dosed cells stained for nucleiand endosome visualization are represented in Fig. 3A. R8-Dox-LL-1h R8-Dox-L-1h

-4h

Dox-L R8-Dox-Lechst 33342 Doxorubicin Hoechst 33342 Doxorubicin

Geo

Mea

n Fl

uore

scen

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Contr

ol 1 h 4 h0

10

20

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DoxilR8-Doxil

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and tumor xenografts. (A) Representative dot plot of 4T1 cells and comparison ofox-L, equivalent to 6 lg/mL of Dox for 1 h and 4 h. (B) Images of 4T1 cells, incubatedicroscopy (left). Fluorescence microscopy images of the sections of a 4T1 tumor

f liposomal Dox at 10 mg/kg. The Dox signal was tracked in the red channel (Ex/Em.0 nm). Objective, 40. Scale bar, 25 lm. The significance of difference between the

A

B

Dox

-LR

8-D

ox-L

1 2 52

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6

Dox-L R8-Dox-L

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Nucleus Endosome0.0

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R8-Dox-L******

Fig. 3. Intracellular fate of Dox delivered by Dox-L and R8-Dox-L. (A) Confocal microscopy images of 4T1 cells, treated with Dox-L and R8-Dox-L at Dox dose of 6 lg/mL for1 h: (1) nuclei stained by Hoechst 33342 at 1 lg/mL for 5 min; (2) Dox-signal; (3) endosomes stained by Transferrin-Alexafluor 488 at 10 lg/mL for 20 min; (4) mergedpicture of 1 and 2, representing fluorescence localization of Dox-signal in nuclei; (5) merged picture of 2 and 3, representing co-localization of Dox in endosomes; (6) mergedpicture of all the fluorescence. (B) Representative view of the orthogonal sections of the merged image of the cells in the XY plane, obtained by Zeiss LSM image browsersoftware (left). Yellow and purple signals in the merged images indicate the co-localization of the red and green, red, and blue fluorescence, respectively. Analysis offluorescence intensity-co-localization (Pearsons coefficient), obtained from the merged pictures (n = 6) from Dox-L and R8-Dox-L-treated cells, by Image J software (Right).

522 S. Biswas et al. / European Journal of Pharmaceutics and Biopharmaceutics 84 (2013) 517525treatment led to strong accumulation of Dox in the nuclei as indi-cated by strong purple spots in the merged pictures of column 1and 2 resulting from the co-localization of red and blue signals,whereas treatment with Dox-L had relatively poor Dox internaliza-tion. Merged pictures of column 2 and 3 indicate co-localization ofDox with the endosomes, represented in column 5. Dox-L-treatedcells demonstrated higher intensity of yellow signal indicative ofendosomal co-localization in the merged picture compared toR8-Dox-L treatment (column 5). A orthogonal section of themerged picture of Dox-L or R8-Dox-L treated cells in a XY planewas used to detail localization behavior (Fig. 3B). Pearsons coeffi-cient obtained after analyzing the merged pictures by Image J soft-ware were plotted to compare co-localization. Pearsonscoefficients for the co-localization of Dox signal with nuclei andendosomal stain were 0.28 0.03/0.96 0.002 and 0.81 0.01/0.50 0.08 for Dox-L/R8-Dox-L treatment, respectively.3.5. Assessment of early apoptosis marker

Binding of fluorescently labeled Annexin V to the cell surfacebound early apoptotic marker, phosphatidyl serine, was used todetect early apoptosis. The representative histogram and quadrantplot obtained by FACS analysis are shown in Fig. 4A. The histogramshowed higher shift in the fluorescence intensity of R8-Dox-L com-pared to Dox-L. Quadrant statistics demonstrated that 79.8% of thetotal cell population treated with R8-Dox-L shifted to the lowerright quadrant compared to 8.2% with Dox-L treatment due tothe higher Annexin V labeling.3.6. Assessment of cytotoxicity

The cytotoxic effect of Dox delivered to the cells by Dox-L or R8-Dox-L was evaluated by MTT assay. R8-Dox-L demonstrated highercytotoxicity compared to Dox-L. No cytotoxicity was observed 12 hafter the treatment. However, R8-Dox-L demonstrated 63.6 8.3%and 46.5 5.3% cell viability compared to 85.2 3.4% and70.4 1.1% for Dox-L at the highest tested Dox dose of 100 lg/mLat 24 and 48 h, respectively. To prove that the cytotoxic effect ofR8-modified liposomes is because of the toxicity of Dox, we testedthe Dox-free liposomes for their effect on cell viability (Fig. 5).The unmodified empty liposomes and R8-modified liposomes dem-onstrated no apparent cytotoxicity up to 125 lg/mL for 24 h.

3.7. Assessment of in vivo therapeutic efficacy

3.7.1. Tumor volume reduction studyThe result from the tumor volumes recorded over the course of

tumor growth reduction study demonstrated that the R8-Dox-Ltreatment suppressed the tumor growth significantly more thanthe Dox-L treatment (Fig. 6). Over the 16-day period, the tumorvolume increased from 64.6 4.3 to 348.0 53.4 mm3 for R8-Dox-L, whereas for Dox-L, the volume increased from 66.6 9.7to 504.2 54.9 mm3. The body weight was uniform throughoutthe study indicating no apparent general toxicity (data not shown).

3.7.2. TUNEL assayNuclear DNA fragmentation, a marker of apoptosis, was

followed by TUNEL assay of the cryostat sections of the frozen

A

B

Untreated cellsAnnexin V positive controlPlain Dox-LR8-Dox-L

Histogram Plot

Control AnV control Dox-L R8-Dox-L

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Doxorubicin (g/mL)

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ell v

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***

Doxorubicin (g/mL) Doxorubicin (g/mL)

Fig. 4. Effect of Dox-loaded liposomes on cell death and initiation of apoptosis in vitro and in vivo. (A) Representative FACS histogram plot showing the effect of Dox-formulations on initiation of apoptotic activity in vitro (left). The apoptotic marker phosphatidyl serine, expressed on the cell surface due to initiation of apoptosis, waslabeled with its ligand, FITC-labeled Annexin V, and the fluorescence of the ligand was tracked to detect the apoptotic cell population by flow cytometry. (B) Assessment ofcell viability of 4T1 cells treated with Dox-loaded liposomes at Dox concentration of 0100 lg/mL for 12, 24, and 48 h. The significance of difference between the mean wasanalyzed by Students t-test, , indicates p < 0.001 and 0.01, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the webversion of this article.)

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.25 62.5 12

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Fig. 5. Effect of empty (Dox-free) plain and R8-modified liposomes on cell viability. Cells were incubated with the formulations for 24 h at a Lipid dose range 0125 lg/mL.The percentage cell viability was determined by considering untreated cells as 100% viable.

S. Biswas et al. / European Journal of Pharmaceutics and Biopharmaceutics 84 (2013) 517525 523tumors isolated from the mice of PBS, Dox-L, or R8-Dox-L treat-ment groups at 24 and 72 h time points. The fluorescencemicroscopy image demonstrated significantly higher apoptoticcell death in tumors treated with R8-Dox-L compared to Dox-Lat 24 and 72 h as indicated by green dots (TUNEL positive cells)attributed to FITC-labeled TdT. No green dots indicative ofTUNEL positive cells were observed in the tumor sections fromthe Dox-L treatment group.

A

B Dox-L R8-Dox-L

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Fig. 6. (A) Comparison of the efficiency of the formulations in inhibiting tumorgrowth. The 4T1-tumor-bearing mice were injected i.v with the formulations at aDox dose of 0.5 mg/kg at alternate days starting from day 1 after tumor volumereached 50100 mm3 (total eight injections). The tumor volume was measuredperiodically and plotted as a function of time. Data are expressed as mean SEM,n = 6, and p < 0.05, Students t-test. (B). Detection of apoptotic cells in frozen tumorsections treated with Dox-L and R8-Dox-L, determined by TUNEL assay andvisualized by fluorescence microscopy. The left panel shows the sections stainedwith Hoechst 33342 and the right panel shows the TUNEL staining. Magnificationx20 objective.

524 S. Biswas et al. / European Journal of Pharmaceutics and Biopharmaceutics 84 (2013) 5175254. Discussion

Dox is considered one of the most effective first line antican-cer chemotherapeutic drugs, since it has been effective againstmany types of cancer including leukemia, lymphomas, breast, uter-ine, ovarian, and lung cancers [33,34]. Dox prevents the replicationand transcription in rapidly growing cancer cells by intercalatingbetween base pairs of the DNA strands. A positively charged man-nose amine in Dox allows efficient binding to the negativelycharged nucleic acid phosphate diester groups and the anthraqui-none planar ring structure enables Dox to intercalate into the dou-ble stranded DNA. In addition, Dox inhibits the enzymetopoisomerase II and prevents the relaxation of super-coiledDNA, another way of blocking transcription and replication. Apartfrom this, a major biological effect of Dox is that it forms iron-med-iated oxygen free radicals that cause oxidative damage to DNA,proteins, and cell membrane lipids. Dox has a high affinity for neg-atively charged cardiolipin, a mitochondrial membrane lipid.Therefore, this free radical production and the affinity for mito-chondrial membrane protein associated with Dox treatment canlead to mitochondria-rich myocardial tissue damage and severecardiotoxicity.

The unique advantages of using liposomal Dox are the de-creased presence of free Dox in the circulation and its preferentialaccumulation in the tumor area which reduce cardiotoxicity signif-icantly. However, conventional liposomes are attacked by plasmaopsonins leading to membrane destabilization. Modified liposomesprepared by steric stabilization (pegylation) reduce non-specificinteractions with cellular proteins. However, pegylated liposomalsystem reduces liposomal uptake by the target cells. Suppressionof intracellular free Dox concentration is considered a barrier toachievement of the optimum therapeutic efficacy of this otherwisehighly potent anticancer drug. To address this issue, various cellpenetration enhancers such as certain peptides have been usedto increase the cytosolic translocation of the drug loaded nanocar-riers. The synthetic, relatively short peptide, octa-arginine (R8)which resembles the peptide sequences of HIV-1TAT peptide, hasfound application as efficient cell penetrating peptide [23,26,27].Previous report indicated that the cellular uptake mechanism ofhigh density arginine-rich peptide-modified liposomes was macr-opinocytosis, whereas low-density R8-modification led to clath-rin-mediated endocytosis [27]. The uptake mechanism influencedtheir intracellular trafficking, resulting in marked difference inthe efficacy of loaded cargo. The highly charged basic octa-argininemoiety has demonstrated well established cell transport proper-ties. While investigating intracellular trafficking of D, andL-enantiomer of octa-arginine, it was reported that D-octa-arginine(D-R8) was additionally bound to the nucleolus, whereas both ofthem translocated in the cytoplasm and labeled nucleus [35].However, the role of octa-arginine as a promoter for nuclear deliv-ery has to be investigated in detail.

In our study, to improve the cell penetration and therapeuticefficacy of doxorubicin-loaded pegylated liposomes (Doxil, Lipo-dox) (Dox-L), we anchored R8 to the surface by inserting a R8-conjugated PEGDOPE co-polymer. R8-Dox-L had a significantlyhigher cellular association compared to Dox-L at both the testedtime points of 1 and 4 h. To assess the intracellular localizationof Dox delivered by Dox-L and R8-Dox-L, a co-localization analysisusing a nuclear stain and an endosomal marker was performed.The treatment with R8-Dox-L generated a marked accumulationof Dox in nuclei as indicated by the bright purple color of the nu-cleus resulting from the co-localization of blue and red signals.Dox-L failed to deliver Dox to the nuclei at +1 h. However, withDox-L, a strong green fluorescence was observed in the mergedpicture of Dox and endosomes indicating that the higher Dox accu-mulation was confined to the endosomes. Endosomal entrapmentof Dox was also observed in R8-Dox-L treatment. However, the ex-tent of the endosomal localization was much lower than observedwith Dox-L treatment. With Dox-L treatment, the top, right handpicture of slices through different planes in the orthogonal sectionsof the merged images indicated that the yellow signal is from in-side the cells, whereas cells treated with R8-Dox-L lacked yellowsignal. Pearsons coefficients obtained by analysis of the mergedimages of the cells indicated that R8-Dox-L treated cells had veryhigh co-localization with nuclei and less endosomal entrapmentcompared to Dox-L. Such a dramatic improvement in the efficacyof the modified nanocarrier represents a significant step for modi-fication of pegylated liposomal Dox.

The clear improvement in the intracellular delivery efficiencywas expected to lead to a more efficient anti-tumor drug action.The ability of the formulations to induce apoptosis by detectionof cell surface-associated phosphatidyl serine was evaluated. Themechanism underlying the initiation of apoptosis involves thetranslocation of phosphatidylserine receptor to the outer cell sur-face, which binds to its ligand, annexin V. Apoptosis is detectedand quantified by the degree of labeling of the cells with the An-nexin V, Alexa Fluor 488 conjugate. The treatment with R8-Dox-L induced significantly higher apoptosis in vitro and in vivo, underspecified conditions than with Dox-L treatment. The cytotoxic abil-ity of Dox-L due to the effect of R8-modification was also investi-gated. The data suggest that the treatment with R8-Dox-Ldramatically increases the Dox concentration in the cell, whichgives rise to higher cytotoxicity than Dox-L. The cytotoxic effectof R8-modified liposomes was not because of the toxicity of theliposomal carriers. The formulations were non-toxic at a lipid doserange of 0-125 lg/mL for 24 h. In vitro tumor growth inhibitionstudy indicated higher retardation of tumor growth rate by R8-Dox-L treatment than by Dox-L suggesting that the modification

S. Biswas et al. / European Journal of Pharmaceutics and Biopharmaceutics 84 (2013) 517525 525on the nanocarrier surface allowed R8-Dox-L to deliver more Doxin the tumor cells resulting in higher therapeutic efficacy com-pared to Dox-L. The TUNEL assay proved that the R8-Dox-L treat-ment induced higher apoptosis than Dox-L treatment at specifiedtime points.

5. Conclusion

We surface-modified PEGylated Dox-loaded liposomes, Lipo-dox, with a relatively short synthetic cell-penetrating peptide,octa-arginine (R8), using R8PEGDOPE. The modification resultedin significant increase in Dox delivery to the cells compared tounmodified Dox liposomes. The R8-modified liposomes deliveredmore Dox to the cytoplasm which eventually gets accumulatedin the nuclei, the site of Dox action, with less Dox entrapped inthe endosomes. The higher intracellular Dox delivery led to morepronounced in vitro cancer cell apoptosis, greater cytotoxicity,and the suppression of tumor growth in mice. Further modifica-tions could be performed to shield cell penetrating peptides inthe systemic circulation to achieve a higher in vivo anti-tumor effi-cacy [32]. However, this simple surface modification of liposomalDox addresses the issue of poor penetration of PEGylated lipo-somes and provides a method that can significantly improve itstherapeutic efficacy. Such a modified liposomal Doxorubicin for-mulation could have a significant impact in cancer chemotherapy.

Acknowledgements

This work was supported by the NIH Grants RO1 CA121838 andRO1 CA 128486 to Vladimir P. Torchilin. We also thank Dr. WilliamHartner for his helpful advice in editing the manuscript.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.ejpb.2012.12.021.

References

[1] V.P. Torchilin, Recent advances with liposomes as pharmaceutical carriers, Nat.Rev. Drug Discov. 4 (2005) 145160.

[2] A. Gabizon, H. Shmeeda, Y. Barenholz, Pharmacokinetics of pegylatedliposomal Doxorubicin: review of animal and human studies, Clin.Pharmacokinet. 42 (2003) 419436.

[3] A.A. Gabizon, Selective tumor localization and improved therapeutic index ofanthracyclines encapsulated in long-circulating liposomes, Cancer Res. 52(1992) 891896.

[4] T.M. Allen, C. Hansen, F. Martin, C. Redemann, A. Yau-Young, Liposomescontaining synthetic lipid derivatives of poly(ethylene glycol) show prolongedcirculation half-lives in vivo, Biochim. Biophys. Acta 1066 (1991) 2936.

[5] V.P. Torchilin, A.L. Klibanov, L. Huang, S. ODonnell, N.D. Nossiff, B.A. Khaw,Targeted accumulation of polyethylene glycol-coated immunoliposomes ininfarcted rabbit myocardium, FASEB J. 6 (1992) 27162719.

[6] V.P. Torchilin, J. Narula, E. Halpern, B.A. Khaw, Poly(ethylene glycol)-coated anti-cardiac myosin immunoliposomes: factors influencing targeted accumulation inthe infarcted myocardium, Biochim. Biophys. Acta 1279 (1996) 7583.

[7] V.P. Torchilin, V.G. Omelyanenko, M.I. Papisov, A.A. Bogdanov Jr., V.S.Trubetskoy, J.N. Herron, C.A. Gentry, Poly(ethylene glycol) on the liposomesurface: on the mechanism of polymer-coated liposome longevity, Biochim.Biophys. Acta 1195 (1994) 1120.

[8] V.P. Torchilin, Passive and active drug targeting: drug delivery to tumors as anexample, Handb. Exp. Pharmacol. (2010) 353.

[9] I. Judson, J.A. Radford, M. Harris, J.Y. Blay, Q. van Hoesel, A. le Cesne, A.T. vanOosterom, M.J. Clemons, C. Kamby, C. Hermans, J. Whittaker, E. Donato diPaola, J. Verweij, S. Nielsen, Randomised phase II trial of pegylated liposomaldoxorubicin (DOXIL/CAELYX) versus doxorubicin in the treatment of advancedor metastatic soft tissue sarcoma: a study by the EORTC Soft Tissue and BoneSarcoma Group, Eur. J. Cancer 37 (2001) 870877.

[10] S.T. Duggan, G.M. Keating, Pegylated liposomal doxorubicin: a review of its usein metastatic breast cancer, ovarian cancer, multiple myeloma and AIDS-related Kaposis sarcoma, Drugs 71 (2011) 25312558.[11] M.E. OBrien, N. Wigler, M. Inbar, R. Rosso, E. Grischke, A. Santoro, R. Catane,D.G. Kieback, P. Tomczak, S.P. Ackland, F. Orlandi, L. Mellars, L. Alland, C.Tendler, Reduced cardiotoxicity and comparable efficacy in a phase III trial ofpegylated liposomal doxorubicin HCl (CAELYX/Doxil) versus conventionaldoxorubicin for first-line treatment of metastatic breast cancer, Ann. Oncol. 15(2004) 440449.

[12] V.P. Torchilin, Recent approaches to intracellular delivery of drugs and DNAand organelle targeting, Annu. Rev. Biomed. Eng. 8 (2006) 343375.

[13] S. Biswas, N.S. Dodwadkar, P.P. Deshpande, V.P. Torchilin, Liposomes loadedwith paclitaxel and modified with novel triphenylphosphoniumPEGPEconjugate possess low toxicity, target mitochondria and demonstrateenhanced antitumor effects in vitro and in vivo, J. Control. Release 159(2012) 393402.

[14] S. Biswas, N.S. Dodwadkar, A. Piroyan, V.P. Torchilin, Surface conjugation oftriphenylphosphonium to target poly(amidoamine) dendrimers tomitochondria, Biomaterials 33 (2012) 47734782.

[15] S. Biswas, N.S. Dodwadkar, R.R. Sawant, A. Koshkaryev, V.P. Torchilin, Surfacemodification of liposomes with rhodamine-123-conjugated polymer results inenhanced mitochondrial targeting, J. Drug Target. 19 (2011) 552561.

[16] S.R. Schwarze, S.F. Dowdy, In vivo protein transduction: intracellular deliveryof biologically active proteins, compounds and DNA, Trends Pharmacol. Sci. 21(2000) 4548.

[17] B. Gupta, T.S. Levchenko, V.P. Torchilin, Intracellular delivery of largemolecules and small particles by cell-penetrating proteins and peptides,Adv. Drug Deliv. Rev. 57 (2005) 637651.

[18] H. Nagahara, A.M. Vocero-Akbani, E.L. Snyder, A. Ho, D.G. Latham, N.A. Lissy, M.Becker-Hapak, S.A. Ezhevsky, S.F. Dowdy, Transduction of full-length TATfusion proteins into mammalian cells: TAT-p27Kip1 induces cell migration,Nat. Med. 4 (1998) 14491452.

[19] E. Vives, P. Brodin, B. Lebleu, A truncated HIV-1 Tat protein basic domainrapidly translocates through the plasma membrane and accumulates in thecell nucleus, J. Biol. Chem. 272 (1997) 1601016017.

[20] A.D. Frankel, C.O. Pabo, Cellular uptake of the tat protein from humanimmunodeficiency virus, Cell 55 (1988) 11891193.

[21] V.P. Torchilin, T.S. Levchenko, R. Rammohan, N. Volodina, B. Papahadjopoulos-Sternberg, G.G. DSouza, Cell transfection in vitro and in vivo with nontoxicTAT peptideliposomeDNA complexes, Proc. Natl. Acad. Sci. USA 100 (2003)19721977.

[22] S. Futaki, T. Suzuki, W. Ohashi, T. Yagami, S. Tanaka, K. Ueda, Y. Sugiura,Arginine-rich peptides. An abundant source of membrane-permeable peptideshaving potential as carriers for intracellular protein delivery, J. Biol. Chem. 276(2001) 58365840.

[23] L. Yang, T. Mashima, S. Sato, M. Mochizuki, H. Sakamoto, T. Yamori, T. Oh-Hara,T. Tsuruo, Predominant suppression of apoptosome by inhibitor of apoptosisprotein in non-small cell lung cancer H460 cells: therapeutic effect of a novelpolyarginine-conjugated Smac peptide, Cancer Res. 63 (2003) 831837.

[24] I.A. Khalil, Y. Hayashi, R. Mizuno, H. Harashima, Octaarginine- and pH sensitivefusogenic peptide-modified nanoparticles for liver gene delivery, J. Control.Release 156 (2011) 374380.

[25] A. El-Sayed, I.A. Khalil, K. Kogure, S. Futaki, H. Harashima, Octaarginine- andoctalysine-modified nanoparticles have different modes of endosomal escape,J. Biol. Chem. 283 (2008) 2345023461.

[26] I.A. Khalil, K. Kogure, S. Futaki, H. Harashima, Octaarginine-modifiedliposomes: enhanced cellular uptake and controlled intracellular trafficking,Int. J. Pharm. 354 (2008) 3948.

[27] I.A. Khalil, K. Kogure, S. Futaki, H. Harashima, High density of octaargininestimulates macropinocytosis leading to efficient intracellular trafficking forgene expression, J. Biol. Chem. 281 (2006) 35443551.

[28] A. Koshkaryev, A. Piroyan, V.P. Torchilin, Bleomycin in octaarginine-modifiedfusogenic liposomes results in improved tumor growth inhibition, Cancer Lett.

[29] V.P. Torchilin, T.S. Levchenko, A.N. Lukyanov, B.A. Khaw, A.L. Klibanov, R.Rammohan, G.P. Samokhin, K.R. Whiteman, P-NitrophenylcarbonylPEGPE-liposomes: fast and simple attachment of specific ligands, includingmonoclonal antibodies, to distal ends of PEG chains via p-nitrophenylcarbonyl groups, Biochim. Biophys. Acta 1511 (2001) 397411.

[30] T.M. Allen, P. Sapra, E. Moase, Use of the post-insertion method for theformation of ligand-coupled liposomes, Cell. Mol. Biol. Lett. 7 (2002) 889894.

[31] T. Ishida, D.L. Iden, T.M. Allen, A combinatorial approach to producingsterically stabilized (Stealth) immunoliposomal drugs, FEBS Lett. 460 (1999)129133.

[32] E. Koren, A. Apte, A. Jani, V.P. Torchilin, Multifunctional PEGylated 2C5-immunoliposomes containing pH-sensitive bonds and TAT peptide forenhanced tumor cell internalization and cytotoxicity, J. Control. Release 160(2012) 264273.

[33] R.B. Weiss, The anthracyclines: will we ever find a better doxorubicin?, SeminOncol. 19 (1992) 670686.

[34] G. Minotti, P. Menna, E. Salvatorelli, G. Cairo, L. Gianni, Anthracyclines:molecular advances and pharmacologic developments in antitumor activityand cardiotoxicity, Pharmacol. Rev. 56 (2004) 185229.

[35] M.M. Fretz, N.A. Penning, S. Al-Taei, S. Futaki, T. Takeuchi, I. Nakase, G. Storm,A.T. Jones, Temperature-, concentration- and cholesterol-dependenttranslocation of L- and D-octa-arginine across the plasma and nuclearmembrane of CD34+ leukaemia cells, Biochem. J. 403 (2007) 335342.

http://dx.doi.org/10.1016/j.ejpb.2012.12.021

Surface functionalization of doxorubicin-loaded liposomes with octa-arginine for enhanced anticancer activity1 Introduction2 Materials and methods2.1 Materials2.1.1 Chemicals2.1.2 Cell lines2.1.3 Animals

2.2 Methods2.2.1 Synthesis of R8PEG2KDOPE2.2.2 Modification of liposomes with PEG2KDSPE or R8PEG2KDOPE2.2.3 Liposomal characterization2.2.4 Cell culture2.2.5 Cell association with liposomes by FACS analysis2.2.6 Cellular internalization by confocal microscopy2.2.7 Early apoptotic marker determination, Annexin V assay2.2.8 MTT assay2.2.9 In vivo tumor xenograft2.2.10 Doxorubicin localization in tumor2.2.11 Tumor volume reduction study2.2.12 TUNEL assay2.2.13 Statistical analysis

3 Results3.1 Physicochemical characterization of liposomes3.2 Cellular association of liposomes3.3 Doxorubicin localization in tumor3.4 Assessment of intracellular co-localization3.5 Assessment of early apoptosis marker3.6 Assessment of cytotoxicity3.7 Assessment of in vivo therapeutic efficacy3.7.1 Tumor volume reduction study3.7.2 TUNEL assay

4 Discussion5 ConclusionAcknowledgementsAppendix A Supplementary materialReferences