Cancer Letters 335 (2013) 191200Contents lists available at SciVerse ScienceDirect

Cancer Letters

journal homepage: www.elsevier .com/locate /canletOcta-arginine-modified pegylated liposomal doxorubicin: An effectivetreatment strategy for non-small cell lung cancer0304-3835/$ - see front matter 2013 Elsevier Ireland Ltd. All rights reserved.http://dx.doi.org/10.1016/j.canlet.2013.02.020

Corresponding author. Address: Center for Pharmaceutical Biotechnology andNanomedicine, Northeastern University, 140 The Fenway (Rm. 225), 360 Hunting-ton Ave., Boston, MA 02115, United States. Tel.: +1 617 373 3206; fax: +1 617 3737509.

E-mail address: v.torchilin@neu.edu (V.P. Torchilin).Swati Biswas, Pranali P. Deshpande, Federico Perche, Namita S. Dodwadkar, Shailendra D. Sane,Vladimir P. Torchilin Center for Pharmaceutical Biotechnology and Nanomedicine, 360 Huntington Avenue, 140 The Fenway, Northeastern University, Boston, MA 02115, United States

a r t i c l e i n f o a b s t r a c tArticle history:Received 22 November 2012Received in revised form 9 January 2013Accepted 8 February 2013

Keywords:LiposomesLung cancerDoxorubicinOcta-arginineSpheroidsTumorThe present study aims to evaluate the efficacy of octa-arginine (R8)-modified pegylated liposomal doxo-rubicin (R8-PLD) for the treatment of non-small cell lung cancer, for which the primary treatment modal-ity currently consists of surgery and radiotherapy. Cell-penetrating peptide R8 modification ofDoxorubicin-(Dox)-loaded liposomes was performed by post-insertion of an R8-conjugated amphiphilicPEGPE copolymer (R8-PEGDOPE) into the liposomal lipid bilayer. In vitro analysis with the non-smallcell lung cancer cell line, A549 confirmed the efficient cellular accumulation of Dox, delivered by R8-PLDcompared to PLD. It led to the early initiation of apoptosis and a 9-fold higher level of the apoptotic reg-ulator, caspase 3/7 (9.24 0.34) compared to PLD (1.07 0.19) at Dox concentration of 100 lg/mL. Thetreatment of A549 monolayers with R8-PLD increased the level of cell death marker lactate dehydroge-nase (LDH) secretion (1.2 0.1 for PLD and 2.3 0.1 for R8-PLD at Dox concentration of 100 lg/mL) con-firming higher cytotoxicity of R8-PLD than PLD, which was ineffective under the same treatment regimen(cell viability 90 6% in PLD vs. 45 2% in R8-PLD after 24 h). R8-PLD had significantly higher penetrationinto the hypoxic A549 tumor spheroids compared to PLD. R8-PLD induced greater level of apoptosis toA549 tumor xenograft and dramatic inhibition of tumor volume and tumor weight reduction. The R8-PLD treated tumor lysate had a elevated caspase 3/7 expression than with R8-PLD treatment. This sug-gested system improved the delivery efficiency of Dox in selected model of cancer which supports thepotential usefulness of R8-PLD in cancer treatment, lung cancer in particular.

2013 Elsevier Ireland Ltd. All rights reserved.1. Introduction

Lung carcinoma is the most frequent cancer in the world, withan incidence of 1.5 million new cases per year, accounting forapproximately one-third of all cancer-related deaths [1,2]. Non-small cell lung cancer (NSCLC) is prevalent (85%) among all lungcancer types. 6580% of all lung cancer patients are diagnosed atan advanced stage of local carcinoma or metastasis [3,4]. Surgeryremains the most successful treatment option for patients withearly detection of the disease [1]. However, the rate of recurrenceis 90% in the first 5 years after surgery [2,5,6]. There is only a 5%absolute benefit of adding chemotherapy to surgery, a minimalgain in overall patient survival rates [711]. Another chemother-apy-related problem is that the relapsed tumors acquire resistanceto the administered chemotherapy. Therefore, there is an unmetneed for developing more effective treatment regimens for NSCLC.

The platinum containing drugs, cisplatin and carboplatin areconsidered the first choice of chemotherapy for NSCLC [12]. Paclit-axel or docetaxel in combination with either of the two platinumdrugs is used as a second-line treatment [13,14]. Doxorubicin,alone, or in combination, are currently in clinical trials for ad-vanced stages of NSCLC [1517]. However, treatment with Dox isassociated with numerous side-effects including severe cardiotox-icity [18,19].

The use of liposomes has advantages in cancer treatment due totheir enhanced permeability and retention related to their smallsize (100 nm) that allows passage through leaky tumor bloodvessels and accumulation preferentially in the tumor [20,21]. Inthis regard, liposomes need to be modified to impart the propertyof long systemic circulation that leads to eventual accumulation inthe tumor. Nonetheless, the nanocarriers drug load has to be re-leased at the tumor site, has to be efficiently cellular-internalizedand followed by release from endosomes to demonstrate drug effi-cacy [22]. For long circulation, liposomes are coated with hydro-philic polymers, mainly polyethylene glycol (PEG). However, thePEG-modified liposomes lead to reduced cellular internalization.

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192 S. Biswas et al. / Cancer Letters 335 (2013) 191200Long-circulating pegylated liposomal doxorubicin (commer-cially available as Doxil or Lipodox) is an example of a stealthliposome, an indispensible treatment means for metastatic, breastand ovarian cancers [23]. Doxil represents an improved formula-tion of free drug with better pharmacokinetic profile and reducedcardiotoxicity. However, its therapeutic efficacy is not dramaticallyincreased and adverse side-effects remain, indicating a furtherimprovement of this formulation. Addressing the issue of poor cel-lular internalization is one of the approaches used to improve thetherapeutic window of the drug.

Intracellular or organelle-specific targeting is an emerging con-cept for improvement of drug action of the nanocarriers [22,2426]. Since the nanocarriers utilize energy-dependent endocytosisas a major pathway for cellular internalization rather than randomdiffusion typical for small drug molecules, a cell penetration en-hancer would dramatically improve their cytoplasmic delivery[2730]. In this regard, many peptide sequences have been identi-fied that cause translocation of a variety of cargos across the cellmembrane [28]. Poly-arginine, a relatively short cell-penetratingpeptide, with an optimum chain length of 8-arginine units hasbeen successfully utilized for intracellular delivery of therapeutics[3134]. A possible internalization pathway of the R8-modified lip-osomes has also been investigated [35].

In this study, we have utilized R8 modified liposomes toenhance the penetration of lung cancer cells. R8-conjugatedamphiphilic poly(ethylene glycol)dioleoyl phosphatidylethanol-amine (PEGDOPE) conjugate, R8-PEGDOPE was incorporated inthe lipid bilayer of PLD. PEGDOPE copolymer is much used forliposomes modification due to its biocompatibility and non-immunogenicity [20]. The hydrophobic lipid moiety in theconjugate could be easily embedded in the liposomal bilayer toallow surface modifications. The PEG-spacer imparts easy R8-accessibility for the cell surface interaction. The therapeuticefficacy of R8-modified pegylated liposomal Doxorubicin (PLD)was assessed in human alveolar adenocarcinoma cell line toevaluate the potential benefit of R8-PLD in the debilitating NSCLCtreatment.2. Materials and methods

2.1. Materials

Octa-arginine peptide (R8, MW. 1267.46 Da) was synthesized by the TuftsUniversity Core Facility (Boston, MA). Pegylated liposomal doxorubicin (Lipodox,2 mg/mL of Dox) was purchased from Sun Pharmaceutical India Ltd (Gujarat, India).1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene gly-col)-2000](ammonium salt) (PEG2KDOPE), 1,2-dioleoyl-sn-glycero-3-phosphoeth-anolamine (DOPE) was purchased from Avanti Polar Lipids (AL, USA).NPCPEG2KNPC was purchased from Laysan Bio (AL, USA). thiazoyl blue tetrazo-lium bromide (MTT) was purchased from SigmaAldrich (St. Louis, MO). MicroBCA protein assay kit, Apo-ONE Homogeneous Caspase-3/7 Assay and CytoTox96 Non-Radioactive Cytotoxicity Assay kits were purchased from Promega(Madison, WI). Annexin V Alexa Fluor 488 conjugate, Annexin-binding buffer5 concentrate and Hoechst 33342 were purchased from Molecular Probes Inc.(Eugene, OR). Para-formaldehyde was from Electron Microscopy Sciences (Hatfield,PA). Fluoromount-G was from Southern Biotech (Birmingham, AL). The Trypan bluesolution was obtained from Hyclone (Logan, UT).2.2. Cell lines

The human alveolar adenocarcinoma cell line, A549 was purchased from theAmerican Type Culture Collection (Manasas, VA). Dulbeccos modified Eagles media(DMEM) and heat-inactivated fetal bovine serum (FBS) were obtained from Gibco(Carlsbad, CA). Concentrated penicillin/streptomycin stock solution was from Cell-Gro (Herndon, VA). All other chemical and solvents were of analytical grade, pur-chased from SigmaAldrich and used without further purifications. A549 cells weregrown in DMEM with 2 mM L-glutamine, supplemented with 10% (v/v) heat-inacti-vated fetal bovine serum, 100 units/mL penicillin G and 100 lg/mL streptomycin.Cultures were maintained in a humidified atmosphere at 37 C and 5% CO2.2.3. Synthesis of R8-PEG2KDOPE

For this purpose, pNP-PEG2KDOPE was synthesized and purified according toan established procedure with modification [24,36]. Briefly, into the solution ofNPCPEG2KNPC (1 g, 0.5 mmol) in chloroform, a DOPE (37.2 mg, 0.05 mmol) solu-tion in chloroform mixed with 20 lL of triethylamine was added drop wise. Thereaction mixture was stirred overnight at room temperature. On the followingday, the reaction mixture was evaporated using a rotary evaporator and freeze-dried to remove traces of solvent. The dry, crude reaction mixture was dissolvedin HCl solution (0.01 M) and purified by gel filtration chromatography using aCl4B column. A HCl solution was used as an eluent. The pure fractions werefreeze-dried, weighed and dissolved in chloroform to obtain a 10 mg/mL solutionfor storage at 80 C. For the synthesis of R8-PEG2KDOPE, into a solution ofpNP-PEG2KDOPE (10 mg, 3.9 lmol) in chloroform (1.0 mL), R8 (7.4 mg, 5.86 lmol)and triethylamine (10 lL) dissolved in DMF (200 lL) was added. The reaction mix-ture was stirred overnight at room temperature. The chloroform was evaporated byrotary evaporation and freeze-dried. The dry reaction mixture was dissolved in PBS,pH 8.4 and stirred at room temperature for 4 h and dialyzed against water using acellulose ester membrane (MWCO. 2000 Da) overnight. The dialysate was freeze-dried to obtain a solid white fluffy product which was dissolved in methanol at5 mg/mL and stored at 80 C.

2.4. Modification of liposomes

Using the post-insertion method, [3739] we decorated Lipodox with R8-PEG2KDOPE conjugate. The inserted conjugate was 2 mol% of the total lipids.Briefly, Lipodox (1.0 mL) was added into the dry lipid film of PEG2KDSPE(0.90 mg, 0.45 lmol) or R8-PEG2KDOPE (1.64 mg, 0.45 lmol). The liposomal sus-pension, PLD and R8-PLD were vortexed for complete hydration of the lipid filmand stirred overnight at 4 C.

2.5. Characterization of liposomes

Surface-modified liposomes (PLD and R8-PLD) were analyzed for size and zeta-potential. The liposomal size and size distribution was measured by dynamic lightscattering (DLS) using a Coulter N4-Plus Submicron Particle Sizer (Coulter Corpo-ration, Miami, FL). Liposome surface charge was measured using a Zeta Phase Anal-ysis Light Scattering (PALS) Ultrasensitive Zeta Potential Analyzer instrument(Brookhaven Instruments, Holtsville, NY).

2.6. Cell association of liposomes

Cell association of liposomes was assessed by FACS analysis. After the initialpassage in T-75 cm2 tissue culture flasks (Corning Inc., NY), A549 cells (0.4 106/well) were seeded in 6-well tissue culture plates. The following day, the cells wereincubated with PLD or R8-PLD at a Dox concentration of 6 lg/mL in 2 mL of serum-free media for 1 h and 4 h incubation periods. The media were removed, the cellswashed several times, trypsinized, suspended in 1 mL PBS and then centrifugedat 1000 RPM for 5 min. The cell pellet was suspended in PBS, pH 7.4 before analysisof the cells labeled with Dox fluorescence using a BD FACS Caliber flow cytometer.The cells were gated using forward (FSC-H)-vs. side-scatter (SSC-H) to exclude deb-ris and dead cells before analysis of 10,000 cell counts.

2.7. Cellular internalization of liposomes

Cellular uptake of liposomes was analyzed by visualization with confocalmicroscopy. After the initial passage in tissue culture flasks, A549 cells (4 104)were grown on circular cover glasses placed in 12-well tissue culture plates in com-plete media. The following day, cells were incubated with PLD or R8-PLD at a Doxconcentration of 6 lg/mL for 1.5 h in serum-free media. After the incubation period,the cells on the cover-slips were washed 4 times with PBS, treated with Hoechst33342 at 5 lg/mL for 5 min, washed thoroughly and fixed with 4% para-formalde-hyde for 10 min at room temperature. The cover-slips were again washed with PBSand mounted cell-side down on superfrost microscope slides with fluorescence-freeglycerol-based mounting medium (Fluoromount-G) and viewed with a Zeiss LSM700 Confocal Laser Scanning Microscope equipped with UV (ex/em. 385/470 nm)and rhodamine filter (ex/em. 548/719 nm) for imaging. The z-stacked images (z115, slice thickness, 0.75 lm) were obtained by capturing serial images of thexy planes by varying the focal length of the same to image along consecutive z-axes. The LSM picture files were analyzed using Image J software. Nuclear localiza-tion of the Dox signal delivered by PLD or R8-PLD was assessed by determiningPearsons and Manders coefficients of colocalization using Image J software.

2.8. Formation of spheroids

Spheroids of 800900 lm diameter were formed from 10,000 A549 cells in 96-well plates according to Yang et al. with modifications as follows [40,41]. A549 cellswere maintained as monolayers before detachment with trypsin to generate a sin-gle-cell suspension. Then, 10,000 cells in 100 lL of media were added to each well

S. Biswas et al. / Cancer Letters 335 (2013) 191200 193of a 96 well plate previously coated with 50 lL of DMEM 1.5% agarose. Finally, theplates were centrifuged 15 min at 1500 rcf to form spherical cell mass which wasincubated for approximately 4 days with no medium change. Spheroid formationwas monitored using a Nikon Eclipse E400 microscope at 10 magnification andwith a Spot Insight 3.2.0 camera with Spot Advanced software (Spot Imaging).Spheroids of 800900 lm size were used for experiment.2.9. Liposomal penetration of spheroids

Spheroids were incubated with PLD and R8-PLD at a Dox concentration of100 lM for 1 h or 4 h, washed with PBS and viewed with a Zeiss LSM 700 ConfocalLaser Scanning Microscope equipped with rhodamine filter (ex/em. 548/719 nm)using a 10 objective. The Z-stacked images of spheroids (z 116) were obtainedby capturing serial images of the xy planes by varying the focal length to imagealong consecutive z-axis. The LSM picture files of spheroids were analyzed for quan-tifying the mean intensity using Image J software.2.10. Annexin V assay

The procedure for Annexin V labeling was carried out according to the manufac-turers protocol. A549 cells were seeded in 12-well plates at 8 104/well. Afterincubation of A549 cells for 4 h with PLD or R8-PLD at a Dox concentration of15 lg/mL, the cells were incubated for an additional 18 h, trypsinized, washed withcold binding buffer, and re-suspended in Annexin V-Alexa Fluor 488 conjugate(15 lL)-added binding buffer (200 lL) for 15 min in darkness. The cells were dilutedwith binding buffer at total volume 400 lL and analyzed immediately by flowcytometry.2.11. Cytotoxicity studies

2.11.1. MTT assayFor cytotoxicity assays including MTT, LDH release and caspase assay, A549

cells were seeded into 96 well microplates at a density of 5 103 and 3 103cells/well for 24 and 48 h respectively in phenol red-free DMEM media. On the fol-lowing day, the cells were incubated with PLD or R8-PLD at Dox concentrations of0100 lg/mL for 4 h. Media was removed and the cells were incubated for an addi-tional 24 and 48 h in fresh complete media. After the incubation period, the mediawas removed and the cells were treated with 3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyl-2H-tetrazolium bromide (MTT) solution (5 mg/mL) in serum-free DMEMfor 4 h. At the end of the incubation period, cell viability was estimated by the abil-ity of the cells to reduce the yellow dye, MTT to a purple formazan product. Themedia was replaced with 100 lL of SDS solution (20%) in 0.01 N HCl for 4 h to dis-solve the formazan crystals. The absorbance was read at 570 nm using a microplatereader (Synergy HT multimode microplate reader, Biotek Instrument, Winooski,VT). Blank readings obtained from the treatment well with no cells were subtractedfrom each reading.2.11.2. LDH releaseThe A549 cells were treated with PLD or R8-PLD at a Dox concentration range of

0100 lg/mL for 4 h followed by removal of the media and further incubation for24 and 48 h. Released lactate dehydrogenase (LDH) in the media was measuredwith a Cytotox 96 Non-Radioactive Cytotoxicity kit (Promega) following manufac-turers instruction. LDH release was normalized to total LDH content following celllysis with a medium containing 0.9% Triton X-100. The plate was read with a Multi-mode microplate reader (Synergy HT; Biotek; absorbance. 340 nm).2.11.3. Caspase assayAfter the treatment of A549 cells with PLD or R8-PLD at Dox concentration

range of 0200 lg/mL for 4 h and incubating the cells for additional 24 and 48 hin phenol red-free complete medium, the samples were analyzed using theApo-ONE Homogeneous Caspase 3/7 Assay (Promega) following manufacturersinstructions. Briefly, following the incubation period, caspase substrate was dilutedin Apo-ONE Homogeneous Caspase-3/7 Buffer and 100 lL added to cells in 100 lLmedia and kept at room temperature with additional gentle shaking. The plateswere read after 4 h using the microplate reader at ex/em. 499/521 nm. The valueswere directly proportional to the amount of apoptotic induction. A blank (allreagents without cells) reading was subtracted from each reading.

For the determination of caspase 3/7 level in the A549 tumor xenograft, tumorswere lysed using a hand-held tissue homogenizer (Qiagen, CA, USA) in PBS, pH 7.4at 0 C and protein content was measured using micro BCA protein assay usingmanufacturers protocol. 25 lg of protein in 100 lL (PBS, pH 7.4) was added to wellsof a 96-well plate. Samples were analyzed immediately as above.2.12. Animals

Immunodeficient NU/NU nude mice (46 weeks old) were purchased fromCharles River Laboratories, MA, USA. All animal procedures were performed accord-ing to an animal care protocol approved by Northeastern University InstitutionalAnimal Care and Use Committee. Mice were housed in groups of 5 at 1923 C witha 12 h lightdark cycle and allowed free access to food and water.

2.13. In vivo tumor xenograft

A subcutaneous tumor was initiated by inoculating A549 human alveolar ade-nocarcinoma cells (2 106, suspended in 100 lL PBS) over the left flank. The timefor the appearance of the tumor was usually 7 days. However, after an initial phaseof slow growth, tumors increased grow and reached a volume of 400 mm3 after60 days from implantation. The length and width of the tumor was measured bycalipers at 3 days interval and the tumor volume was calculated using the formula:(width2 length)/2. Treatment was started when the tumors reached a size of400 mm3.

2.14. Assessment of tumor volume reduction and TUNEL assay

To measure the pro-apoptotic effect following PLD or R8-PLD treatment, theTUNEL assay was performed on the frozen tumor sections. Mice (n = 3 in eachgroup) bearing tumor of 400 mm3 volume were injected twice with PLD or R8-PLD at a Dox dose of 2 and 10 mg/kg at days 1 and 3 respectively. The tumor volumewas measured on days 1, 3, and 6. The animals were euthanized with CO2, on day 6and the tumors were isolated. The tumors were washed quickly with PBS, pH 7.4,weighed and frozen immediately after the extraction by immersing in tissue freez-ing media and stored at 80 C. Tumor slices (8 lm) were cryo-sectioned usingCryotome Cryostat, mounted on superfrost plus slides, fixed in 4% paraformalde-hyde for 10 min at RT, permeabilized with proteinase K (20 lg/mL) for 15 min atRT. A TUNEL assay was performed on the sections using the FragElTM DNA fragmen-tation detection kit following manufacturers instructions for frozen sections. TheTUNEL positive cells were detected by fluorescence microscopy equipped with aFITC-filter. Four random images obtained from two different tumors for each treat-ment group were analyzed using Spot advanced software.

2.15. Statistical analysis

The data were tested for statistical significance using Students t-tests. p values,calculated with the Graph Pad prism 5 software (GraphPad Software, Inc, San Diego,CA). All numerical data are expressed as mean SD, n = 3 or 4, from 3 differentexperiments. Any p values less than 0.05 was considered statistically significant., , in figures indicated p values

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Fig. 1. Comparison of cellular uptake of R8-modified and unmodified pegylated liposomal doxorubicin by flow cytometry. The A549 cells were incubated with PLD and R8-PLD at 6 lg/mL of Dox for 1 and 4 h, after which the FACS analysis was performed. The cell-associated Dox fluorescence was measured. (A) Representative dot plot obtainedfrom FACS analysis showing the cell population labeled with Dox after incubation with PLD or R8-PLD at 1 h and 4 h time points. (B) Comparison of the geometric mean offluorescence of the cells treated with PLD or R8-PLD (left panel) and the representative histogram plot obtained from histogram statistics of the FACS analysis (right panel).The data are mean SD, averaged from three separate experiments. The significance of difference between the mean was analyzed by Students t-test, p < 0.001.

194 S. Biswas et al. / Cancer Letters 335 (2013) 191200internalization of Dox in R8-PLD compared to PLD. Localization ofthe Dox signal to the nucleus was observed in the merged picturesas indicated by purple signal, whereas most of the Dox signal deliv-ered via PLD was in the cytosolic compartment. The averaged Pear-sons and Manders coefficients obtained by the Image J analysis ofthe three center z-stacked images were 0.40 0.10, 0.49 0.03 forPLD and 0.71 0.04, 0.9 0.11, respectively (Fig. 2B).

3.4. Accumulation of liposomes in spheroids

Spheroids are a robust model to study the tumor penetrationbehavior of drugs/drug delivery systems in cancer since spheroidsto a great extent mimic both the architecture of tumors and thepenetration barriers of solid tumors where drugs are mostly con-fined to the outer cell layers. The Z-stacked images of spheroidsshowed higher accumulation of R8-PLD compared to PLD at boththe time points (Fig. 3). The formulations had limited penetrationin 1 h. However, R8-PLD demonstrated very high accumulation inthe center zone of the spheroids at 4 h, as shown by the center slice(100 lm from top). Mean intensity (arbitrary units) of the Dox sig-nal in the center slice of the PLD and R8-PLD treated spheroids,quantified by Image J analysis was 29.7 1.2, 71.7 1.5 for 1 hand 105 5, 178.6 3.2 for 4 h, respectively.

3.5. Assessment of early apoptosis

Binding of the fluorescently labeled Annexin V to the cell sur-face-bound early apoptotic marker, phosphatidyl serine is usedto detect early apoptosis. Initiation of apoptosis causes transloca-tion of phosphatidyl serine from inner cytoplasmic membrane tothe outer surface. The FACS analysis result demonstrating the com-parison of up-regulation of this early apoptotic marker upon treat-ment with PLD and R8-PLD has been illustrated in Fig. 4. Aftertreatment, both PLD and R8-PLD initiated apoptosis, demonstrat-ing a shift in the cell population as shown by the increased labelingof the cells with the Annexin V-Alexa Fluor-488 conjugate. How-ever, R8-PLD treated cells had significantly higher total geometricmean fluorescence (182.67 10.8) compared to PLD treated cells(129.80 5.36). The lower right quadrant was additionally gatedto analyze the phosphatidyl serine expression, 8.7 1.5% of the cellpopulation was there for R8-PLD compared to 3.6 0.5% for PLD.3.6. Assessment of cell viability by MTT assay

To determine whether the initiation of apoptosis leads to de-creased cell viability, an MTT assay was performed (Fig. 5). A statis-tically significant decrease in cell viability was observed with R8-PLD treatment compared to PLD at Dox concentration of 6.25 lg/mL and greater. R8-PLD treatment showed a 55% decrease in cellviability compared to 10% with PLD at the highest used Dox con-centration of 100 lg/mL (% cell viability for R8-PLD treatment was45.35 2.14%, compared to 89.6 5.9% for PLD at 100 lg/mL ofDox concentration). Slow decrease in the cell viability(62.89 1.95% to 45.35 2.14% at 24 h and 53.5 5.71% to46.10 2.56% at 48 h) was observed with R8-PLD treatment atDox concentration between 6.25 and 100 lg/mL.3.7. Assessment of other cell death markers

Release of lactate dehydrogenase (LDH) in the media by apopto-tic/necrotic cells and up-regulation of apoptotic protein, caspase 3/7 in the cells were quantified (Fig. 6). The R8-PLD-treated cells hadsignificantly higher level of LDH release compared to PLD at alltested Dox concentrations. There was a Dox-concentration depen-dent increase in LDH release in R8-PLD-treated cells. No noticeableincrease in LDH release was observed in PLD treatment even athighest Dox concentration of 100 lg/mL (Fig. 6A). There was 1.9-fold increase in the level of LDH release in R8-PLD compared toPLD at Dox concentration of 100 lg/mL. Fig. 6B illustrates caspase3/7 activity after PLD and R8-PLD treatment. At a Dox concentra-tion of 200 lg/mL, the R8-PLD treatment increased caspase 3/7 le-vel 5.5-fold compared to PLD treatment (13.72 0.15 for R8-PLDvs. 2.48 0.54 for PLD). Likewise, a LDH release profile of the Doxconcentration-dependent increase in the caspase 3/7 protein levelwas observed in R8-PLD.

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Fig. 2. Assessment of cellular internalization by confocal microscopy. (A) Confocal laser scanning micrographs of A549 cells obtained from z-experiment. A549 cells wereincubated with PLD or R8-PLD for 90 min, were taken at a fixed XY plane along a consecutive Z-axes (Z 5, 7, 9, 11 from total 16 slices). The nuclei were stained with Hoechst33342. The lower panel represents merged pictures of Dox and Hoechst 33342 fluorescence. Scale bar: 20 lm. (B) Assessment of nuclear localization of Doxorubicin deliveredby PLD and R8-PLD. The merged (nuclei stained and PLD/R8-PLD treated) pictures of the center z-slices were analyzed with Image J software and the colocalizationcoefficients were obtained. The data are mean SD, averaged from three images of the same treatment. p < 0.05, paired Students t-test.

S. Biswas et al. / Cancer Letters 335 (2013) 191200 1953.8. Assessment of in vivo therapeutic efficacy

The tumor volumes and weights of post-mortem tumors weremeasured. The initial tumor volume at day 1 was 397.4 5.9,405.0 3.5, 397.8 4.7 mm3, which was increased at day 3 to496.38 10.5, 475.70 15.2 and 451.25 8.2 mm3 for PBS, PLDand R8-PLD treated mice, respectively. On the day of euthanize(day 6), strong suppression of tumor volume was observed withR8-PLD treatment. On day 6, the tumor volumes reached to674.16 20.5, 600.00 25.0, 368.56 18.5 mm3 for PBS, PLD andR8-PLD treatment, respectively. The tumor weight after isolationwas 0.5802 0.20, 0.5561 0.1 and 0.355 0.05 g for PBS, PLDand R8-PLD treatment, respectively. Detection of apoptotic cellsby TUNEL assay, the measurement of the level of caspase 3/7 inPLD, R8-PLD or PBS-treated solid A549 tumors were detected inthe apoptotic cells in tumor sections by TUNEL staining. The cell-nuclei of tumors treated with PBS or PLD treatment exhibited nogreen fluorescence attributable to FITC-labeled TdT, while the tu-mors treated with R8-PLD had significantly higher amounts ofgreen dots representative of apoptotic nuclei (Fig. 7). The level ofcaspase 3/7 expression in R8-PLD-treated tumor lysate was signif-icantly higher (1.1-fold) compared to PBS and PLD treatment. Thus,R8-modified PLD allowed for a strong pro-apoptotic effect of Dox inan in vivo mouse A549 tumor xenograft model.

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Fig. 3. Accumulation of PLD and R8-PLD in A549 spheroids by confocal laser scanning microscopy. (A) Selected Z-stacked micrographs of A549 spheroids, taken at consecutiveZ-axes were shown. (B) Graphical representation of mean intensity of the Doxorubicin signal of the center slice (100 lm inside from top) of PLD and R8-PLD dosed spheroidsquantified with Image J software. The cells were treated with PLD and R8-PLD at 100 lM of Dox for 1 and 4 h. Images were selected out of 16 Z-slices. Scale bar: 200 lm. Thedata in figure B are mean SD, averaged from nine different regions of interest from the images of two spheroids. p < 0.001, paired Students t-test.

196 S. Biswas et al. / Cancer Letters 335 (2013) 1912004. Discussion

Determining an effective treatment strategy for cancer includ-ing NSCLC is challenging as among all the patients with lung can-cer, 55% are diagnosed at an advanced stage. Surgery, radiotherapyand chemotherapy are being utilized as treatment options for lungcancer with high chances of relapse [42]. Platinum-based chemo-therapy is the first-line chemotherapy for NSCLC patients. Othercytotoxic agents such as docetaxel, pemetrexed, gefetinib are op-tions for second or third-line therapy. However, the high incidenceof relapsed tumor growth necessitates work on development of aneffective chemotherapeutic treatment regimen [4]. Dox, combinedwith platinum-based therapy or radiotherapy is currently in clini-cal trials for locally advanced NSCLC [4345]. Dox is one of themost effective anticancer drugs ever developed and considered afirst-line chemotherapeutic [46]. However, Dox, like other chemo-therapeutic drugs, has many adverse effects, the most debilitatingis cumulative dose-dependant cardiotoxicity [18,19]. Pegylatedliposomal Doxorubicin (PLD) (commercially available as Doxil,Lipodox), is a nano-sized Dox-loaded carrier, which accumulatesspecifically in tumor sites by the enhanced permeability and reten-tion effect, reduces the toxicity of free Dox [2023,47,48]. Coatingof liposomes with biocompatible, non-immunogenic polymer PEGimparts non-recognition by plasma serum opsonins that rendersprolonged plasma circulation, which is advantageous for tumortargeted nanocarrier accumulation via EPR effect. However,

A Annexin V-Negative Cells

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Fig. 4. Comparison of up-regulation of an early apoptotic marker, phosphatidyl serine, upon treatment with PLD and R8-PLD by flow cytometry. The A549 cells were treatedwith PLD or R8-PLD at 30 lg/mL of Dox for 4 h, then incubated for 18 h in complete media. The cells were treated with Annexin V-Alexa Fluor 488 conjugate, the ligand ofphosphatidyl serine. The phosphatidyl serine receptor translocates from cytoplasm to the outer cell surface on the initiation of apoptosis. The labeling of cells with Annexin V-Alexa Fluor 488 conjugate was measured by FACS analysis. (A) Representative dot plot showing the cell population with or without Annexin V labeling. (B) Geometric mean offluorescence of PLD/R8-PLD dosed cells treated with or without Annexin V Alexa Fluor 488 conjugate, and (C) Representative histogram plot of cells treated with PLD or R8-PLD followed by Annexin V-Alexa 488. Data in (B) are mean SD, averaged from three separate experiments. p < 0.01, analyzed by the paired Students t-test.

24 h-A549

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Fig. 5. Assessment of Dox-induced cell death of A549 cells treated with PLD and R8-PLD. Cells were incubated with PLD or R8-PLD at a Dox concentration of 0100 lg/mL for4 h followed by the incubation period of 24 and 48 h before cell viability was measured. The data are mean SD, averaged from three separate experiments. p < 0.001,paired Students t-test.

Doxorubicin (g/mL)

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Fig. 6. The effect of PLD and R8-PLD on Dox-induced LDH release (A) and caspase 3/7 activity (B) in A549 cells. The cells were treated with PLD or R8-PLD at varied Doxconcentration for 4 h, media removed and cells incubated for 48 h in complete media before analyzing the media for LDH release and the cell lysate for caspase 3/7 activity.Each bar represents mean SD of n = 3 from three separate experiments. The significance of differences between the means was analyzed by the paired Students t-test.p < 0.001.

S. Biswas et al. / Cancer Letters 335 (2013) 191200 197PEGylation hinders intracellular delivery in the cancer cells afteraccumulation at the tumor site and the maximum therapeutic ben-efit of Dox is not achieved. This necessitates further modification ofPLD for cytosolic delivery to improve the effectiveness of thishighly potent anticancer drug.In an attempt to promote cellular internalization, we surface-modified PLD with the cell-penetrating octa-arginine (R8) peptide.The relatively short, synthetic R8 peptide resembles the peptide se-quence of HIV-1TAT peptide. This highly charged basic peptide hasdemonstrated efficient cell transport properties [31,32,34,35]. The

A

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Fig. 7. Assessment of the in vivo therapeutic efficacy of R8-modified PLD compared to unmodified PLD i.v. in A549-tumor xenograft. (A) Tumor volume and weight (arrowsindicate treatment). (B) Apoptosis analysis. Apoptotic cells were detected in frozen tumor sections, determined by TUNEL assay and visualized by fluorescence microscopy.The upper panel shows the sections stained with Hoechst 33342 and the right panel shows the TUNEL staining. Magnifications-20 objectives. (C) Measurement of caspase 3/7 level in A549 tumor. The tumors isolated from the treatment groups of PBS, PLD or R8-PLD were homogenized, centrifuged and a tumor lysate equivalent to 25 lg ofproteins was analyzed using the Apo-ONE Homogeneous Caspase 3/7 Assay kit. and , P < 0.01and 0.001, unpaired Students t-test. indicates p < 0.001 compared to PBS.

198 S. Biswas et al. / Cancer Letters 335 (2013) 191200R8-conjugated amphiphilic PEGDOPE co-polymer was success-fully inserted into the liposomes lipid bilayer. This modificationof the surface of PLD with a cationic peptide increased the positivecharge on the surface of R8-PLD compared to PLD. However, thesize remained unchanged. As expected, R8-PLD had a time-depen-dent higher cell association compared to PLD, as estimated bytracking the cell associated Dox fluorescence. The cellular internal-ization, PLD- and R8-PLD-treated cells visualized under confocalmicroscopy using z-stacked imaging indicate that R8-PLD had ahigher cytosolic Dox delivery compared to PLD and indicated theco-localization of Dox at its nuclear site of action. Higher co-local-ization coefficients (Pearsons and Manders) in R8-PLD treatedcells obtained by Image J analysis of the center slices clearlypointed out the higher intracellular Dox-delivery efficiency of R8-PLD compared to PLD. Even though, R8 has been recognized as acell penetration enhancer in previous studies, however, the intra-cellular trafficking of R8 after cellular internalization remain rela-tively unexplored. While investigating intracellular trafficking ofD, and L-enantiomer of R8, Fretz et al. reported that D-R8 was addi-tionally bound to the nucleolus, whereas both of them translocatedin the cytoplasm and labeled nucleus [49]. Therefore, the enhancedDox delivery to the nucleus by R8-PLD compared to PLD could bedue to enhanced intracellular delivery coupled with R8-mediatednuclear targeting. However, the role of R8 as a promoter for nucleardelivery needs detailed investigation.

Over the past several years, spheroids, a 3D multilayer, spheredcell culture system has garnered much attention as an importanttool to study several drug developmental stages including drugtransport, binding, therapy resistance as well as cell invasion incancer [41,5053]. Spheroids supplement monolayer-based assaysas they better mimic the complexities of the tumor tissues. Themulticellular spheroids resemble avascular tumor modules of solidtumors, the cells in spheroids resorting to an in vivo-like differen-tiation pattern due to the resemblance in pathophysiological mili-eu conditions such as extracellular matrix assembly as well as cellmatrix and cellcell interactions [50]. Due to similarities of spher-oids with in vivo tumor tissues, spheroids have been considered asan inevitable tool to utilize in the therapeutic development beforeturning to whole animal studies [50,51,5456]. We studied liposo-mal accumulation in spheroids and demonstrated that R8-PLD hadallowed for much higher Dox penetration compared to PLD as fol-lows from the enhanced Dox signal in the center slices of the z-stacked images (Fig. 3). The higher cellular uptake of R8-PLD ledto higher apoptosis mediated by Dox compared to PLD. The resultdemonstrated that the R8-PLD treated cells have higher expressionof the protein phosphatidyl serine compared to PLD. The resultantinduction of early apoptosis was consistent with higher cytotoxic-ity. The result demonstrated that R8-PLD induced higher Dox-med-iated cytotoxicity compared to PLD. The induction of apoptosis isindicated by the elevated release of cell death marker LDH andthe up-regulation of the pro-apoptotic protein, caspase 3/7. R8-PLD demonstrated higher release of LDH and up-regulation of cas-pase 3/7 in A549 monolayer compared to PLD. The in vivo experi-ment using A549 tumor xenograft demonstrated that R8-PLDsignificantly reduces the tumor growth compared to the PLD treat-ment. Higher induction of apoptosis by R8-PLD treatment wasdemonstrated by TUNEL assay compared to PLD. Tumor lysate ofR8-PLD treated A549 tumor bearing mice had increased caspase3/7 expression compared to PLD treatment due to the higher intra-cellular Dox delivery achieved by R8-PLD over PLD.

S. Biswas et al. / Cancer Letters 335 (2013) 191200 199In conclusion, this study has identified a novel liposomal drugdelivery system, R8-modified PLD, for improved chemotherapy ofNSCLC. R8-PLD addresses the issue of poor cell penetration byPLD to increase its therapeutic efficacy. The study provides a ratio-nale for the continued investigation of R8-PLD as a promising anticancer therapy.Acknowledgements

The work was supported in part by NIH Grants RO1 CA121838and RO1 CA128486 to Vladimir P. Torchilin. We thank Dr. WilliamHartner for his help in editing the manuscript.References

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Octa-arginine-modified pegylated liposomal doxorubicin: An effective treatment strategy for non-small cell lung cancer1 Introduction2 Materials and methods2.1 Materials2.2 Cell lines2.3 Synthesis of R8-PEG2KDOPE2.4 Modification of liposomes2.5 Characterization of liposomes2.6 Cell association of liposomes2.7 Cellular internalization of liposomes2.8 Formation of spheroids2.9 Liposomal penetration of spheroids2.10 Annexin V assay2.11 Cytotoxicity studies2.11.1 MTT assay2.11.2 LDH release2.11.3 Caspase assay

2.12 Animals2.13 In vivo tumor xenograft2.14 Assessment of tumor volume reduction and TUNEL assay2.15 Statistical analysis

3 Results3.1 Physico-chemical characterization of liposomes3.2 Cell association of liposomes3.3 Cellular internalization of liposomes3.4 Accumulation of liposomes in spheroids3.5 Assessment of early apoptosis3.6 Assessment of cell viability by MTT assay3.7 Assessment of other cell death markers3.8 Assessment of in vivo therapeutic efficacy

4 DiscussionAcknowledgementsReferences