Simultaneous active intracellular delivery of doxorubicin and C6-ceramide shifts the...

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Journal of Controlled Release xxx (2014) xxx–xxx

COREL-07389; No of Pages 10

Contents lists available at ScienceDirect

Journal of Controlled Release

j ourna l homepage: www.e lsev ie r .com/ locate / jconre l

Simultaneous active intracellular delivery of doxorubicin andC6-ceramide shifts the additive/antagonistic drug interaction ofnon-encapsulated combination

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Nuno A. Fonseca a,b, Lígia C. Gomes-da-Silva a, Vera Moura a,c, Sérgio Simões a,b, João Nuno Moreira a,b,⁎a CNC—Centre for Neurosciences and Cell Biology, University of Coimbra, Largo Marquês de Pombal, 3004-517 Coimbra, Portugalb FFUC—Faculty of Pharmacy, University of Coimbra, Pólo das Ciências da Saúde, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugalc TREAT U, SA, Parque Industrial de Taveiro, Lote 44, 3045-508 Coimbra, Portugal

⁎ Corresponding author at: Center for NeurosciencesCoimbra, Largo Marquês de Pombal, 3004-517 Coimbra, P

E-mail addresses: [email protected] (N.A. F(L.C. Gomes-da-Silva), [email protected] (V. Moura),[email protected] (J.N. Moreira).

http://dx.doi.org/10.1016/j.jconrel.2014.09.0240168-3659/© 2014 Elsevier B.V. All rights reserved.

Please cite this article as: N.A. Fonseca, et al.,onistic drug interaction of non..., J. Control. R

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Article history:Received 26 May 2014Accepted 25 September 2014Available online xxxx

Keywords:Combinatorial ratiometric drug designC6-ceramideNanoparticlesNucleolinF3-targeted delivery

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Drug resistance remains the Achilles tendon undermining the success of chemotherapy. It has been recognizedthat success requires the identification of compounds that, when combined, lead to synergistic tumor inhibitionwhile simultaneously minimizing systemic toxicity. However, in vivo application of such protocols is dependenton the ability to deliver the appropriate drug ratio at the tumor level. In this respect, nanotechnology-based de-livery platforms, like liposomes, offer an elegant solution for the in vivo translation of such strategy.In this work, we propose the active intracellular delivery of combinations of doxorubicin and the pro-apoptoticsphingolipid, C6-ceramide, using our previously described cytosolic triggered release-enabling liposomes,targeting nucleolin with the F3 peptide.Combination of doxorubicin (DXR):C6-ceramide (C6-Cer) at 1:2 molar ratio interacted synergistically againstdrug resistant/triple negativeMDA-MB-231 breast cancer cells, as well as drug sensitiveMDA-MB-435Smelano-ma cells. Cell viability studies indicated that F3-targeted liposomes encapsulating DXR:C6-Cer 1:2 molar ratio (p[F3]DC12) performed similarly as targeted liposomal DXR (p[F3]SL), encapsulating twice the amount of DXR, atthe IC50, for an incubation time of 24 h. Importantly, F3-targeted liposomes encapsulating DXR:C6-Cer 1:2 molarratio (p[F3]DC12) enabled a cell death above 90% at 24 h of treatment against both DXR-resistant and sensitivecells, unattainable by the F3-targeted liposomal doxorubicin. Furthermore, a F3-targeted formulation encapsulat-ing amildly additive/antagonistic DXR:C6-Cer 1:1molar ratio (p[F3]DC11) enabled an effect above 90% for an in-cubation period as short as 4 h, suggesting that the delivery route at the cell level may shift the nature of druginteraction. Such activity, including the one for p[F3]DC12, induced amarked cell and nucleus swelling at similarextent, consistent with necrotic cell death.Overall, these results demonstrated that F3-targeted intracellular delivery of different DXR/C6-Cer ratios, withdiversed drug interactions, enabled a highly relevant increased efficacy against chemotherapy resistant cells.

© 2014 Elsevier B.V. All rights reserved.

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UNC1. Introduction

As global cancer burden continues to increase, 1.6 million newcancer cases were expected in 2013, from which approximately0.58 million deaths were estimated only in US [1,2]. Chemotherapystill represents the cornerstone treatment of disseminated malignan-cies. Nonetheless, drug resistance remains as the Achilles tendonundermining the success of chemotherapy, mainly owing to molecularand cellular heterogeneity within the tumor microenvironment [3,4].

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and Cell Biology, University ofortugal.onseca), [email protected]@ci.uc.pt (S. Simões),

Simultaneous active intracelluelease (2014), http://dx.doi.o

One of the attempts to solve this problem is the combination of drugsnear to their maximum tolerated dose (MTD), often exposing patientsto unacceptable toxicity. Alternatively, it has been recognized that suc-cess requires the identification of compounds that, when combined,lead to synergistic tumor inhibition while simultaneously minimizingsystemic toxicity [5,6].

One of the most important pathways deregulated in cancer is thePI3K/Akt signaling cascade [7]. In severalmurinemodels of cancer, inhi-bition of PI3K/Akt signaling retards tumorigenesis by restoring the apo-ptotic sensitivity of cancer cells to chemotherapeutic agents [8]. In thisrespect, ceramides are one of the most promising drugs, which havebeen described to inhibit the PI3K/Akt pathway both in HL-60 leukemiacells [9] and in PC-3 prostate cancer cells [10]. However, the intracellu-lar activity of ceramides is challenged by glucosylceramide synthase(GCS), an enzyme that converts pro-apoptotic ceramides into inactive

lar delivery of doxorubicin and C6-ceramide shifts the additive/antag-rg/10.1016/j.jconrel.2014.09.024

http://dx.doi.org/10.1016/j.jconrel.2014.09.024

mailto:[email protected]






http://www.sciencedirect.com/science/journal/01683659

www.elsevier.com/locate/jconrel


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glucosylceramides [11–13]. In addition, GCS has been related withdoxorubicin (DXR) resistance, by modulation and depletion ofdoxorubicin-induced ceramide levels [12,14]. This intrinsic relationsupports the addition of exogenous ceramides concomitantly withdoxorubicin, to counteract GCS-mediated pro-apoptotic sphingolipiddepletion, which along with ceramide-mediated PI3K/Akt modulationcould enable increased efficacy at lower chemotherapeutic doses [15].Additionally, it has been demonstrated that ceramides impair angiogen-esis, the formation of new blood vessels from pre-existing ones, an im-portant component in the tumor microenvironment dynamics whichsupports tumor growth [4,16,17]. Therefore, impairing cellular and bio-logical functions of tumors using synergistic combinations of drugs likeceramides and doxorubicin, could translate into an increased gain in ef-ficacy upon acting at different levels of the tumor microenvironment.

However, in vivo application of such protocols is highly dependenton the ability to synchronize the pharmacokinetics of each individualdrug present in the combination, thus enabling the tumor accumulationof a synergistic drug cocktail [18], namely at the intracellular level oftargeted tumor cells. Nanotechnology-based delivery platforms, suchas liposomes, upon the extracellular release of a encapsulated drugcombination, have shown increased efficacy inmurinemodels of cancer[18,19] aswell as in afirst-in-man study against acutemyeloid leukemia[20,21].

Considering the state-of-the-art, the present work aims at selectinga synergistic drug combination between C6-ceramide and doxorubicinand assessing the impact of its intracellular delivery using the F3peptide-targeted pH-sensitive lipid-based nanoparticle, developed byour group [22]. The F3 peptide enables specific recognition of nucleolin,a protein highly expressed in cancer cells and endothelial cells of tumorangiogenic blood vessels [23–26]. Synergy will be established using themedian-effect analysis proposed by Chou and Talalay [27–29], againstcancer cell models, including a drug resistant breast cancer triple nega-tive model and a drug sensitive melanoma model.

2. Materials and methods

2.1. Materials

Doxorubicin hydrochloride (DXR) was from IdisPharma (UK).Calcein, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES),2-(N-morpholino)ethanesulfonic acid (MES), disodium ethylenedi-aminetetraacetate dehydrate (EDTA), Trizma®Base, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), sodiumchloride, 3β-hydroxy-5-cholestene-3-hemisuccinate (CHEMS) andcholesterol (CHOL) were purchased from Sigma-Aldrich (USA). Thelipids 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2k), 1,2-distearoyl-sn-glycero-3-phosphoethanol-amine-N-[maleimide(polyethylene glycol)-2000] (DSPE-PEG2k-maleimide), and N-hexanoyl-D-erythro-sphingosine (C6-Ceramide)were obtained from Avanti Polar Lipids (USA). F3 (KDEPQRRSARLSAKPAPPKPEPKPKKAPAKK) and the non-specific (NS) peptideswere cus-tom synthetized by Genecust (Luxemburg).

2.2. Cells

MDA-MB-231 triple negative breast cancer cell line and MDA-MB-435Smelanoma cell line [30] (acquired from ATCC, USA) were culturedin RPMI 1640 (Sigma-Aldrich, USA) supplemented with 10% (v/v) ofheat-inactivated fetal bovine serum (FBS) (Invitrogen, USA), 100 U/mlpenicillin, 100 μg/ml streptomycin (Lonza, Switzerland) and main-tained at 37 °C in a 5% CO2 atmosphere. As the cells we have workedwith grow adherent to the plastic of the cell culture flask, all the exper-iments have been performed 24 h after cell seeding, to enable cell at-tachment before the corresponding incubationwith the tested samples.

Please cite this article as: N.A. Fonseca, et al., Simultaneous active intracelluonistic drug interaction of non..., J. Control. Release (2014), http://dx.doi.o

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2.3. In vitro screening for synergy between DXR and C6-ceramide

Eight thousandMDA-MB-231 breast cancer orMDA-MB-435Smela-noma cells/well were incubated with serial dilutions of doxorubicin orC6-Ceramide, alone or in combination, at fixed molar ratios, for 24 h at37 °C in an atmosphere of 5% CO2. Following incubation, cell cultureme-dium was exchanged for fresh one and the experiment was furtherprolonged up to 72 h. Cell viability was then evaluated using the MTTassay as previously described [31].

2.4. Preparation of liposomes

pH-sensitive liposomes, with or without ceramide, were composedof DOPE:CHEMS:DSPC:CHOL:DSPE-PEG2k:C6-ceramide at 4:2:1:1:0.8:2(18 mol.% of C6-ceramide) or 4:2:2:2:0.8:0 molar ratio, respectively.

Dried lipid filmswere hydrated at 60 °Cwith ammonium sulfate (pH8.5) and the resulting liposomes were extruded through 80 nm poresize polycarbonate membranes using a LiposoFast Basic mini extruder(Avestin, Canada). The buffer was exchanged in a Sephadex G-50 gelcolumn (Sigma-Aldrich, USA) equilibrated with Trizma®Base sucrose(10%, w/v, buffered at pH 9.0). Remote encapsulation of DXR (9 or18 mol.% of total lipid, for the DXR:C6-ceramide 1:2 or 1:1 molar ratio,respectively) was carried out through ammonium sulfate gradientmethod, upon incubation with liposomes for 1.5 h at 60 °C [32]. Non-encapsulated DXR was removed using a Sephadex G-50 gel columnequilibrated with 25 mM HEPES, 140 mM NaCl buffer (HBS, pH 7.4).

To further prepare targeted liposomes, DSPE–PEG2k–F3 conjugatewas produced. Briefly, thiolated derivative of F3 peptide was generatedby reaction at room temperature with 2-iminothiolane (Sigma-Aldrich,USA) in 25mMHEPES, 140mMNaCl, 1 mMEDTAbuffer (pH8.0) for 1 hin an inert N2 atmosphere. Thiolated derivatives were then incubatedovernight at room temperature with DSPE–PEG2k–maleimide micellesin 25 mM HEPES, 25 mM MES, 140 mM NaCl, and 1 mM EDTA (pH7.0). The resulting micelles of DSPE–PEG2k–peptide conjugates werepost-inserted onto the liposomal membrane at 2 mol.% relative tototal lipid (TL), upon incubation with pre-formed liposomes, for 1 h at50 °C.

To prepare calcein-loaded liposomes, both lipid films were insteadhydrated with a 40 mM isosmotic calcein solution in 25 mM HEPES,140 mMNaCl buffer (pH 7.4), and extruded as described above. Follow-ing removal of calcein excess, through a Sephadex-G50 column equili-brated with 25 mM HEPES, 140 mM NaCl buffer (pH 7.4), liposomeswere immediately submitted to the post-insertion procedure as previ-ously described.

2.5. Liposome characterization

Liposome size and polydispersion index (PDI) were measured bylight scattering with a N5 particle size analyzer (Beckman Coulter,USA). Final total lipid concentrations were determined upon quantifica-tion of cholesterol using Infinity® Cholesterol kit (ThermoScientific,USA). Encapsulated doxorubicin was assayed at 492 nm from a standardcurve, after liposomal solubilization with 90% absolute ethanol, and theloading efficiency (%) was calculated from the equation [(DXR/TL)final /(DXR/TL)initial] × 100.

To assess drug retention, an aliquot of F3-targeted and non-targetedliposomes, encapsulating either DXR or a combination of DXR andC6-Cer, was incubated in HEPES buffer saline pH 7.4 (HBS), 90%RPMI 1640 culturemedium supplementedwith 10% FBS (Invitrogen)or 90% of non-inactivated bovine serum, at 37 °C. At different time-points (0, 4 and 24 h), DXR fluorescence dequenching was measuredin a Spectramax fluorimeter (λex = 485 nm; λem = 590 nm) (Molec-ular Devices, USA). Drug retention of DXR (% of control) was calculat-ed using the following formula: 100 − [(TestRFUn − MeanRFU0) /(MeanRFUctr − MeanRFU0)] × 100, where TestRFUn and MeanRFU0

stand for the fluorescence of tested sample at different time points



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and time 0 h, respectively and MeanRFUctr is the fluorescence corre-sponding to 100% of release, following incubation with 0.25% (v/v) ofTriton X-100.

2.6. C6-ceramide effect on liposomal pH sensitivity

Two hundred thousand MDA-MB-231 breast cancer and MDA-MB-435S melanoma cells were incubated with 50 μM (total lipid)of liposomal calcein for 1 h at 37 °C and immediately analyzed througha FACScalibur flow cytometer (BD Biosciences, USA). Free calcein(10 μM)was included as control. A total of 20,000 eventswere analyzedwith Cell Quest Pro software (BD Biosciences, USA).

2.7. Evaluation of cytotoxicity of liposomal drug combinations

Different concentrations of F3 peptide-targeted or non-targetedliposomes, containing either single DXR (p[F3]SL or pSL, respective-ly) or combined with C6-ceramide at 1:1 (p[F3]DC11 and pDC11, re-spectively) or 1:2 molar ratio (p[F3]DC12 or pDC12, respectively),were incubated with 8000 MDA-MB-231 breast cancer or MDA-MB-435S melanoma cells/well, for 1, 4 or 24 h, at 37 °C in an atmo-sphere of 5% of CO2. F3 peptide-targeted and non-targeted liposomalC6-ceramide were included as controls (p[F3]SL(C6) and pSL(C6),respectively). Afterwards, cell culture medium was exchanged forfresh one and the experiment was prolonged for a total of 96 h. Cellviability was then evaluated using the MTT assay as previouslydescribed [31].

In order to assess the mechanism of cell death induced by the intra-cellular delivery of DXR:C6-Cer combination, cell death was evaluatedby flow cytometry as previously described, upon incubation of 35,000adherent cells/well with each formulation at 2 μM DXR for 4 h at37 °C [33]. Assessment of cell morphology following DAPI (Applichem,Germany) staining was also performed. Images were captured using a5× objective mounted in an Axiovert 200M microscope equipped withan AxioCamHR (Carl Zeiss, Germany). Image analysis was carried outusing FIJI software (National Institutes of Health, USA).

2.8. Median effect analysis of doxorubicin and C6-ceramide combinations

The nature of the interaction between doxorubicin and C6-ceramide(synergism, additivity or antagonism)was evaluated using themedian-effect model developed by Chou and Talalay [27–29]. The model relieson themedian-effect Eq. (1) to describe any dose–response relationship

f af u

¼ DD50

� �m

ð1Þ

where fa and fu represent the fraction affected and unaffected, respec-tively (i.e. the response), D the dose responsible for a given fa, D50 themedian-effect dose and m the sigmoidicity of a dose–response curve.The acquired data from dose-response studies, using single drugs ortheir combination, was fitted to the median-effect plot Eq. (2)

log1

1− f að Þ� �

¼ m log Dð Þ−m log D50ð Þ ð2Þ

enabling one to estimate m, the slope of the plot, and D50, the dose re-sponsible for 50% of the effect.

These parameters establish a dose–response model for each drug orcombination tested, enabling the determination of the CombinationIndex (CI) using the Eq. (3)

CI ¼ D1 þ D2

Dxð Þ1 þ Dxð Þ2ð3Þ

where (Dx)1 and (Dx)2 are the doses of each single drug responsible fora certain effect fa, and D1 and D2 are the doses of each drug in a given


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mixture enabling the same effect fa [28]. As a measure of the nature ofinteraction, a CI value b1,≈1 or N1 is indicative of synergism, additivityor antagonism, respectively [27,28]. Further calculations enable one toestimate the Dose Reduction Index () by the Eq. (4)

DRIn ¼ Dxð ÞnDn

ð4Þ

for the drug n, understood as the drug fold decrease in a combination ascompared to the same drug given alone, for a determined effect level[27,28].

3. Results

3.1. Cytotoxicity of individual drugs against cancer cell lines of diverse his-tological origin

Assessment of single cytotoxicity of the drugs in study is a require-ment for the establishment of their nature of interaction using themedian-effect analysis [28]. Fulfilling that requisite, triple negativebreast cancer MDA-MB-231 and melanoma MDA-MB-435S [30] celllines were incubated with serial dilutions of either DXR or C6-ceramide for 24 h at 37 °C, in an atmosphere of 5% CO2. The formerwas clearlymore resistant to DXR than the latter (IC50= 1.66 μM versusIC50=0.40 μM; Fig. 1, A versus C), in contrast to the higher sensitivity toC6-ceramide (IC50 = 6.15 μM versus IC50 = 11.5 μM; Fig. 1, B versus D,respectively). Expectedly, the described inactive analogue, C6-dihydroceramide, presented a significantly lower activity than C6-ceramide (corresponding to a 10-fold higher IC50), rendering it uncon-sidered for subsequent studies (data not shown).

3.2. Establishment of synergistic combinations of doxorubicin and C6-ceramide

In order to assess the nature of interaction between the two drugs,different doxorubicin (DXR):C6-ceramide (C6-Cer) ratios, from 1:40 to5:1, were tested, using the MDA-MB-231 breast cancer and MDA-MB-435S melanoma cell lines. Data generated from the in vitro screening,were analyzed using the median-effect method described by Chou andTalalay [27,28]. With this method, the combination index (CI), a mea-surement of the nature of interaction between the two drugs, hasbeen determined. A combination index of b1, ≈1 or N1, correspondsto a synergistic, additive or antagonistic interaction, respectively.

The results indicated that the combination of doxorubicin and C6-ceramide exhibited synergistic activity (Fig. 2). Interestingly, the na-ture of the interaction varied according the fraction affected (fa)(percentage of cell death), i.e. the same drug ratio may present si-multaneously an antagonistic/additive or synergistic interaction de-pending on the levels of fa, i.e. cell death (Fig. 2). Surprisingly, theDXR:C6-ceramide molar ratio 1:2 revealed to interact synergisticallyat all fa levels, an effect that was independent of the cell line tested(Fig. 2A and B). Such result translated into a dose-reduction index(DRI) between 4- and 6-fold for doxorubicin. Additionally, other testeddrug ratios also presented relevant DRI values (Fig. 2C and D). However,the nature of the interaction of those did not hold among all fa range.Based on these results, the synergistic DXR:C6-ceramide molar ratio1:2 was selected for further co-encapsulation into liposomes, alongwith the 1:1 molar ratio (mildly additive/antagonistic), as a controlfor the synergy effect.

3.3. Effect of bilayer-incorporated ceramide on intracellular triggereddelivery of pH-sensitive liposomes

The work by Moura et al. (2012), using F3-targeted pH-sensitiveliposomes contaning DXR, has demonstrated in a murine model ofMDA-MB-435 tumors, the therapeutic advantage of endowing a



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Q6 Fig. 1. Cytotoxicity of free doxorubicin (DXR) and C6-ceramide (C6-Cer) against breast andmelanoma cancer cell lines.DXR or C6-ceramide (in serially diluted concentrations)were incubatedfor 24 h at 37 °C with either MDA-MB-231 (A and B, respectively) orMDA-MB-435 (C and D, respectively) breast cancer ormelanoma cell lines respectively, and the experiment was fur-ther prolonged for 72 h after which cell death was assessed by the MTT assay. Dose–response curves represent the mean± SEM of 3–4 independent experiments for each concentrationtested. Doted-line: 50% of cell death.


nanoparticle with the capacity to enable intracellular triggered re-lease of the encapsulated payload. Therefore, before proceedingwith the loading of the established drug ratio into the pH-sensitive

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Fig. 2. In vitro screening for synergy of different doxorubicin (DXR):C6-ceramide (C6-Cer) ratios. Ffrom 1:40 to 5:1, the nature of the interaction between the two drugs was assessed against MDwell as the Dose Reduction Index (DRI) of doxorubicin, at IC50 (fa 0.5) or IC70 (fa 0.7), for bothbetween drugs was used, where CI values b1, N1 or≈1 indicate synergy, antagonism and adddependent experiments performed in triplicate. Dose reduction index (DRI) as a measure of a dnation, both producing the similar effect “fa”. Mean DRI ± SEM was calculated from data of 3–


D Pliposomes, the influence of the presence of ceramide in pH-sensitivity

was assessed. Liposomes were thus loaded with calcein at a self-quenching concentration as previously described [22].

E

ollowing incubation with different doxorubicin (DXR):C6-ceramide (C6-Cer) molar ratios,A-MB-231 breast cancer or MDA-MB-435S melanoma cell lines (A and B, respectively) ascell lines (C and D respectively). Combination Index (CI) as a measure of the interactionitivity, respectively. Mean Combination Index ± SEM was calculated from data of 3–6 in-ose fold-reduction is obtained by the ratio of each drug alone versus the drug in a combi-6 experiments, each performed in triplicate.



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As such, it was possible to assess the impact on the efficiency of thepayload release following the probe dequenching. F3 peptide-targeted li-posomes containing calcein, with or without C6-ceramide (p[F3]SL(C6)andp[F3]SL, respectively), presented a similar extent of intracellular pay-load release (Fig. 3), with greater efficiency than the corresponding non-targeted counterparts (pSL(C6) and pSL). Not less significant, cell incu-bated with an excess of free calcein presented 4-fold higher signal thanthe non-targeted formulation, but significantly lower than the targetedones (Fig. 3C and D). The first indicated that calcein was clearly retainedinside the liposomes,while the second points to thewidespread signal oftargeted formulations as result of extensive intracellular delivery.

Overall, these results demonstrated that bilayer-incorporated C6-ceramidedid not affect the ability of pH-sensitive F3-targeted liposomesto promote intracellular triggered delivery of the payload located in itsaqueous core.

3.4. Characterization of pH-sensitive F3-peptide targeted liposomesco-encapsulating doxorubicin and C6-ceramide

As shown previously, the DXR:C6-cer 1:2 molar ratio presented asynergistic interaction between the two drugs, contrasting to the 1:1molar ratio, which was mildly additive or antagonist depending on the

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Fig. 3. Effect of membrane-incorporated ceramide on liposomal pH-sensitivity. Two hundred thouwith excess of free calcein (10 μM) or with pH-sensitive liposomes containing calcein either atargeted, pSL(C6), or F3-targeted, p[F3]SL(C6)), at 50 μM of lipid for 1 h at 37 °C and immediplots of event distribution for each tested condition. Calcein geometric mean fluorescence, nor231 and MDA-MB-435S cell lines (C and D, respectively). Data represent the mean ± SEM of 3


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cell line (Fig. 2). Therefore, pH-sensitive F3-peptide targeted and non-targeted liposomes containing the indicated DXR:C6-Cer ratio, wereprepared by incorporating C6-ceramide in the liposomal bilayer at thementioned fixed molar ratios and further characterized from a physicalstandpoint.

The presence of C6-ceramide in the bilayer of liposomes hadmin-imal impact in parameters like loading efficiency, mean size andpolydispersion index, when compared to the targeted (p[F3]SL)and non-targeted (pSL) counterparts without ceramide (Fig. 4A, B andC). Interestingly, non-targeted formulations were more heterogeneousthat the F3-targeted liposomes in terms of mean size (Fig. 4C). Furtherevidence of improved stability arouse from drug release studies per-formed at 37 °C. None of the nanosystems tested, either F3-targetedor non-targeted, presented significant drug release in 4 h, regardlessthe incubation medium. At 24 h of incubation, either in cell culture me-dium (Fig. 4E) or serum (Fig. 4F), all formulations, either targeted ornon-targeted, presented a similar extent of drug retention. The experi-ment performed in serum, evidenced a decrease on the ability to retainthe encapsulated DXR, with values that varied between 55 and 84%, forboth targeted and non-targeted liposomes. Thiswas not statistically dif-ferent from the counterparts without C6-ceramide (Fig. 4F). Strikingly,the stability study performed in HBS (Fig. 4D) clearly demonstrated

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sand of MDA-MB-231 breast cancer and MDA-MB-435S melanoma cells were incubatedlone (non-targeted, pSL, or F3-targeted, p[F3]SL) or co-encapsulated with ceramide (non-ately analyzed through a FACScalibur flow cytometer. (A) and (B) are representative dotmalized against the respective signal of the untreated control, is presented for MDA-MB-experiments (one-way ANOVA p b 0.001; ns p N 0.05 Tukey's multiple comparison test).



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Fig. 4. Characterization of liposomes encapsulating doxorubicin (DXR):C6-ceramide (C6-Cer) ratios.Different liposomal formulations incorporating doxorubicin and C6-ceramide at 1:1 or 1:2molar ratios, either targeted (p[F3]DC11 and p[F3]DC12, respectively) or non-targeted (pDC11 and pDC12, respectively) have been characterized, in comparison with the correspondingcontrols containing only DXR, either targeted (p[F3]SL) or non-targeted (pSL), in terms of loading efficiency (A),mean size (B), and polydispersion index (C). (D), (E) and (F) represent thedrug retention capacity of each liposomal formulation when incubated at 37 °C in HBS pH 7.4, RPMI 1640 supplemented with 10% FBS or in 90% non-inactivated serum, respectively. Datarepresent mean ± SEM from 3 to 8 independent experiments (one-way ANOVA ***p b 0.0001, **p b 0.01, *p b 0.05; ns p N 0.05 by Tukey's multiple comparison test).


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1:1 molar ratio) and p[F3]DC12 (targeted liposomes encapsulating theDXR:C6-cer at 1:2molar ratio) presented a similar extent of drug reten-tion, relative the corresponding controls without ceramide (p[F3]SL),and were more stable than the non-targeted counterparts (pDC11 andpDC12, respectively), in accordance with the PDI results (Fig. 4C).

3.5. In vitro cytotoxic of liposomal targeted combinations of doxorubicinand C6-ceramide

In order to evaluate the cytotoxic potential of the targeted drugcombinations, the impact of each formulation on the in vitro viabilityof MDA-MB-231 and MDA-MB-435 cells was assessed. Incubation withp[F3]DC11 or p[F3]DC12 enabled an unique result for an incubationtime as short as 4 h, leading to a 90% decrease on the viability of bothcell lines. This level of decreased viability was not reached following in-cubationwith the counterpart without ceramide (p[F3]SL) in any of thecell lines tested, not even for a 24 h incubation (Fig. 5, and Table 1). In-terestingly, the presence of C6-cer in non-targeted formulations eliciteda decrease in cell viability higher than 90% both in MDA-MB-231 andMBA-MB-435 cells, for 24 h incubations. In any case, these non-targeted formulations presented IC50 and IC90 values 2-fold higherthan the targeted counterparts (Fig. 5 and Table 1).

For short incubation time points (1 h), the previously described ad-vantage arising from the presence of C6-cer was not evident.Within the


selected concentrations of DXR, the value of 50% of cell deathwas barelysurpassed. Nevertheless, the IC50 values determined for targeted lipo-somes were 2-fold lower relative to the non-targeted liposomes, an ef-fect that was independent of the incorporation of C6-Cer (Fig. 5 andTable 1).

Overall, these results demonstrated that the co-encapsulation of theDXR:C6-Cer combination within F3-targeted liposomes increased theircytotoxic activity, even with half of the amount of DXR loaded per lipo-some (corresponding to the doxorubicin:C6-ceramide molar ratio of1:2), which ultimately may result in increased targeted cell deathwhile minimizing collateral toxic effects of doxorubicin.

3.6. Effect of the developed synergistic targeted drug combinations on cellmorphology

In order to gain insight into the mechanism of action of the devel-oped nanoparticles, cell morphology was assessed using fluorescencemicroscopy. The collected data indicated that F3-targeted formulationsaltered the distribution of nuclear sizes, inducing nuclear and cell swell-ing to a greater extent than the non-targeted formulations (Fig. 6 andFig. S5, Supplemental Data). The presence of C6-ceramide, in targetedor non-targeted liposomes, did affect neither the nuclear distributionnor the mean nuclear size (Fig. 6B, C, D, E and F). Not less important isthe fact that the liposomes encapsulating the DXR:C6-Cer 1:2 molarratio (p[F3]DC12) presented the same pattern of performance as the



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Fig. 5. Cytotoxicity of combinations of doxorubicin and C6-ceramide encapsulated in different liposomal formulations. Cells were incubated for 1, 4 and 24 h with F3-targeted lipo-somal DXR (p[F3]SL) or DXR:C6-Cer combination at a molar ratio of 1:1 or 1:2 (p[F3]DC11 and p[F3]DC12, respectively), at DXR serially diluted concentrations, and the experimentwas further prolonged for total of 96 h, after which cell death was assessed. Results were compared to the respective non-targeted counterparts (pSL, pDC11 and pDC12).Figures represent the dose–response curves for the indicated incubation time points for MDA-MB-231 triple negative breast cancer cell line, and MDA-MB-435S melanoma cell line.The data points represent the mean ± SEM of 3 independent experiments for each concentration tested. Doted-line and full line represent 50 and 90% of cell death, respectively.

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DC11) (Fig. 6F). A similar profile was obtained using the drug sensitivemelanomamodel, MDA-MB-435S (not shown). Overall, these data sug-gested that the F3-targeted formulations were more efficient in induc-ing nuclear and cell swelling, consistent with necrosis-induced celldeath [34].

4. Discussion

Despite all efforts, cancer drug resistance remains a distressing prob-lem in oncology. In order to overcome this barrier, chemotherapeuticsare used in combination, normally at their maximum tolerated dose(MTD), which ultimately is not beneficial to the patient as it representsan increased risk of severe toxicity. Nonetheless, it has been recognized

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Table 1Cytotoxicity of different liposomal DXR:C6-ceramide combinations against MDA-MB-231 and

Drug Cell line

MDA-MB-231

1 h 4 h 24 h

IC50 (μM) IQ90 (μM) IC50 (μM) IC90 (μM) IC50 (

Liposomal doxorubicin p[F3]SL 29.53 – 3.73 – 0.89pSL N50 – 7.16 – 3.65

Liposomal combination p[F3]DC11

25.87 – 6.35 40.79 0.85

pDC11 N50 – 16.21 – 1.47p[F3]DC12

23.60 – 8.85 – 0.90

pDC12 40.12 – 18.13 – 3.28

Data represent the IC50 and the IC90 of the mean dose–response curves calculated through linrespectively.


that success demands the identification of drugs that, when combined,lead to synergistic tumor inhibition while minimizing systemic toxicity[5,6]. It has been acknowledge that the nature of the interaction be-tween those drugs varies according to themeasured effect on cell viabil-ity (fraction affected, fa), as well as with the pre-established drugmolarratio, in line with similar studies using drug combinations likeirinotecan/floxuridine or cytarabin/daunorubicin [18,19]. Nonetheless,such scenario is far from ideal as synergistic effects would be limitedto a percentage of tumor cell death above a certain threshold, whichcould be problematic in an in vivo setting. Therefore, ideally a drug com-bination should present a synergistic interaction at all levels of fa. In thisrespect, we have identified in the present work the DXR:C6-Cer 1:2molar ratio as synergistic, regardless the cell line or the level of fa, en-abling at least a 4–6 fold dose reduction of DXR.

MDA-MB-435 cell lines.

MDA-MB-435S

1 h 4 h 24 h

μM) IQ90 (μM) IC50 (μM) IC90 (μM) IC50 (μM) IC90(μM) IC50 (μM) IC90 (μM)

– 9.51 – 8.14 – 0.65 –

– 47.18 – N50 – 6.68 –

10.51 15.98 – 4.88 20.56 1.31 8.71

14.22 35.27 – 15.93 – 2.77 11.8513.60 11.58 – 10.92 – 0.89 17.47

21.19 32.94 – N50 – 3.70 31.74

ear interpolation of the dose values immediately below or above the 50% and 90% effect,



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Fig. 6. Effect of F3-targeted combinations of doxorubicin and C6-ceramide on cell morphology. Thirty-five thousandMDA-MB-231 cells/well were incubated for 4 h at 37 °C with F3-targetedliposomal DXR (p[F3]SL) or DXR:C6-Cer combination at amolar ratio of 1:1 or 1:2 (p[F3]DC11 and p[F3]DC12, respectively), or with the non-targeted counterparts (pSL, pDC11 or pDC12,respectively) at 2 μM DXR. The experiment was prolonged up to 92 h. Figures represent the frequency distributions of nuclear area, determined by DAPI staining analysis, for untreatedcells (A), liposomal doxorubicin (B), liposomes encapsulating the DXR:C6-Cer, either at 1:1 (C) or 1:2 molar (D), respectively. (E) Represents the mean nuclear area analysis upon incu-bation with each of the mentioned samples (Bars represent mean area ± SEM; Kruskal–Wallis non-parametric test p b 0.0001; ***p b 0.001 comparing F3-targeted vs non-targeted for-mulations by Dunn's multicomparison test). Data were collected from a representative experiment.


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However, to take full advantage of such combination, one has todevelop strategies to ensure a synchronized temporal and spatialdelivery of the individual drugs. Several lipid-based formulationscontaining ceramides have been described. Some examples includeceramides formulated either as single agent in non-targeted ortransferrin-targeted nanoparticles [35,36] or in combination withpaclitaxel, doxorubicin or tamoxifen [37–39]. Others have demon-strated that combination of liposomal ceramide with sorafenib in-creases the effectiveness of the latter [40]. Nonetheless, despiteincreased cytotoxicity of those combinations, lack of an appropriatemathematical analysis may render the selected drug ratios not nec-essarily synergistic. Furthermore, EPR-driven tumor accumulationof the mentioned drug combinations, though presenting higher effi-cacy than free drugs, could be limited by the absence of specific intra-cellular delivery to essential (and more accessible) cell populationswithin the tumor microenvironment [4,41]. Considering the impor-tance of targeting different components of the tumor microenviron-ment, as previously emphasized [4,16,17], we have engineered theprevious F3-peptide pH sensitive liposomes developed in ourgroup, to accommodate the selected DXR:C6-Cer synergistic combi-nation. The F3 peptide is a moiety that specifically targets nucleolin,a cell membrane protein overexpressed in cancer cells and endothe-lial cells of angiogenic blood vessels [22,42].

Using calcein-loaded liposomes, we have shown that incorporationof C6-ceramide in the liposomal bilayer did not impair delivery norpromoted significant instability (Fig. 3, non-targeted liposomes ver-sus free calcein). Such result correlates with the one generated withthe 35 mol.% C6-ceramide-incorporating liposomes by Khazanovet al. (maximum load), well above the 18.5 mol.% we used [37]. Theabsence of impaired delivery led us to engineer a F3-targeted formu-lation containing the synergistic DXR:C6-Cer 1:2 molar ratio (Fig. 2).DXR:C6-Cer 1:1 molar ratio was also loaded into liposomes to use asa control for an additive/antagonist effect (Fig. 2).

Characterization data demonstrated that C6-ceramide had mini-mal impact in measured physical parameters (including drug


Eretention) as compared to F3-targeted (p[F3]SL) or non-targeted(pSL) liposomes containing only doxorubicin (Fig. 4). DSPC and cho-lesterol are known to increase drug retention in pH sensitive lipo-somes [43]. Their removal to accommodate the incorporation ofC6-ceramide might have led to increased defects in membrane bilay-er promoted by the sphingolipid [37]. This was likely the reason be-hind the differences observed on drug retention (in HBS) betweendoxorubicin- and combination-encapsulating liposomes. Nonethe-less, the same data demonstrated that the F3 ligand stabilized the bi-layer of C6-ceramide loaded liposomes, to the same extent ofliposomes without C6-ceramide (Fig. 4D). This might be explainedby the additional hydration layer on the liposomal surface, arisingfrom the charged F3 peptide. This effect seems to improve drug re-tention (Fig. 4D) and minimize aggregation (Fig. 4C). Furthermore,the differences observed in the different media, might be related totheir differences in ionic and/or protein composition. Those, inturn, may promote different degrees of lipid headgroup hydration,especially of CHEMS, upon providing several counterions, which ren-der unequal levels of membrane stabilization [44,45]. This fact couldexplain the increased drug retention of pDC11 and pDC12 formula-tions in cell culture medium when compared to HBS buffer. Other-wise, in serum, all tested formulations exhibited lower drugretention than in other media, notwithstanding the fact thatcombination-containing liposomes have marginally lower drug re-tention, relative to the counterparts without ceramide (Fig. 4F).That could be due to protein/lipid interaction known to destabilizethe lipid bilayers allowing doxorubicin to escape and bind to serumproteins [46,47].

The incorporation of pro-apoptotic ceramide in liposome bilayer,by itself, has shown to render any formulation more cytotoxic eitherby passive internalization of the intact liposome or by simple lipidtranslocation onto the cell membrane [37]. The free drug combina-tion studies clearly indicated that the 1:2 DXR:C6-cer molar ratiowas synergistic, whereas the 1:1 molar ratio was mildly additive orantagonistic (Fig. 2). Surprisingly, F3-targeted liposomes



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encapsulating the DXR:C6-Cer combination at 1:1 molar ratio (p[F3]DC11) enabled 90% of cell death for an incubation period as short as4 h (Fig. 5 and Table 1). This effect was unattainable even by targetedliposomes containing only doxorubicin or C6-ceramide, after 24 h ofincubation (Fig. S1, Supplemental data). Overall, p[F3]DC11 performedsimilarly to p[F3]DC12 nanoparticle against triple negative MDA-MB-231 breast cancer cells, regardless the incubation time (Fig. 5 andTable 1). This unexpected result suggested that the intracellulardelivery of both drugs, at the selected ratio, may change the interactionnature of the combinations, rendering them synergistic (Fig. S2,Supplemental data). This result is, in some extent, different from theone reported by Tardi et al., where encapsulation (in non-targeted lipo-somes) of antagonist 3:1 ratio of irinotecan:cisplatin rendered a lowerefficacy, despite increased irinotecan loading, compared to the synergis-tic CPX-571 formulation [48]. This thus indicated that the interactionbetween drugs might also be dependent on the mechanism of drug de-livery, particularly on the ability to delivery intracellularly a specificdrug ratio. Nonetheless, it has been demonstrated that the encapsula-tion of the combination at 1:2 molar ratio (p[F3]DC12) enabled in-creased liposomal citotoxicity using half of the drug loading utilized toprepare the standard targeted formulation (p[F3]SL). Ultimately, thiscould prove useful to further increase the safety associated with theuse of liposomal doxorubicin.

Despite the increased cytotoxic effect, FACS analysis indicated simi-lar mechanism of cell death for all formulations, with predominance oflate apoptotic/necrotic population and higher levels of apoptotic cellsover necrotic cells (Fig. S4, Supplemental data). Doxorubicin has beendescribed to induce apoptosis (caspase-dependent cell death) or necro-sis (caspase-independent cell death) after mitotic catastrophe [49,50],in a dose-dependent manner. On the other hand, ceramides have beendescribed to mediate apoptosis through several mechanisms includingp38 MAPK, Akt-mediated induction of p27(kip1) and channel-opening in the mitochondria [9,10,51–53]. In this context, whereasDXR depends on cell cycle to promote cell death even at low doses,the apoptosis-induced effect by C6-ceramide is highly dependent onthe dose and time of exposure [9,49]. Consistently, microscopy analysisof nuclear geometry, revealed an increase in the nuclei size, indicative ofnucleus swelling, and cell swelling upon incubation with F3-targetednanoparticles containing DXR, when compared to the non-targetedcounterparts, which is coherent with cell induced-necrosis (Fig. 6)[34]. Therefore, at the tested concentration, observed results demon-strated mainly the contribution of doxorubicin for cell death, despitethe fact that free ceramide can indeed induce apoptotic cell death, asmeasured by us with annexin V (Fig. S3, Supplemental data) and consis-tent with literature [10].

5. Conclusion

In the present work, it has been demonstrated that the DXR:C6-cercombination developed a synergistic interaction at the specific 1:2molar ratio, enabling a DXR dose reduction of, at least, 4-fold de-pending on the cellular model. Furthermore, we established the de-velopment of a novel triggered-release nanoparticle targeted bythe F3 peptide, capable of retaining and intracellularly deliver thesynergistic DXR:C6-cer combination, thus increasing the cytotoxicpotential. Additionally, our study suggested that the strategy of de-livery may alter the nature of drug interaction, since a F3-targetedformulation encapsulating an additive/mildly antagonistic DXR:C6-Cer, demonstrated singularly a cytotoxic effect above 90%, for an in-cubation period as short as 4 h. It has been further validated that theencapsulation of the synergistic DXR:C6-Cer 1:2 molar ratio enableda cytotoxic effect above 90% after 24 h of incubation, unattainable bythe F3-targeted liposomal doxorubicin encapsulating twice theamount. The characteristics of the developed nanoparticle, alongwith the demonstrated tropism of the F3 peptide towards breast can-cer cells and the tumor microenvironment [22,42], could enable an


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increase in the therapeutic efficacy, overcoming drug resistancewhile simultaneously decreasing the severe side effects of doxorubi-cin. Overall, the generated data suggest that additive/antagonistic inter-action of free combinationsmay be potentially shifted by specific activeintracellular delivery.

Acknowledgments

Nuno A. Fonseca was a recipient of the fellowship SFRH/BD/64243/2009 from the Portuguese Foundation for Science and Technology(FCT). The work was supported by the grants InovC/UC (2013) andQREN/FEDER/COMPETE (Ref. 30248/IN0617).

Appendix A. Supplementary data

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jconrel.2014.09.024.

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