Inhibition of ectopic glioma tumor growth by a potent ferrocenyl drug loaded into stealth lipid nanocapsules

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Nanomedicine: Nanotechnology, Biology, and Medicinexx (2014) xxx–xxx

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Inhibition of ectopic glioma tumor growth by a potent ferrocenyl drugloaded into stealth lipid nanocapsules

Anne-Laure Lainea,b, Anne Clavreulb,c, Audrey Rousseaud, Clément Tétaudb,c,Anne Vessierese, Emmanuel Garciona,b, Gerard Jaouene, Léo Aubert f, Matthieu Guilbert f,

Jean-Pierre Benoita,b, Robert-Alain Toillonf, Catherine Passirania,b,⁎aLUNAM Université – Micro et Nanomédecines Biomimétiques, Angers, France

bInserm U1066, IBS-CHU, Angers, FrancecDépartement de Neurochirurgie – CHU, Angers, France

dDépartement de Pathologie Cellulaire et Tissulaire – CHU, Angers, FranceeCNRS, UMR 7576, ENSCP, Paris, France

fInserm U908, Université Lille 1, Villeneuve d'Ascq, France

Received 12 September 2013; accepted 3 May 2014

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Abstract

In this work, a novel ferrocenyl complex (ansa-FcdiOH) was assessed for brain tumor therapy through stealth lipid nanocapsules (LNCs).Stealth LNCs, prepared according to a one-step process, showed rapid uptake by cancer cells and extended blood circulation time. Theferrocenyl complex was successfully encapsulated into these LNCs measuring 40 nm with a high loading capacity (6.4%). In vitro studiesshowed a potent anticancer effect of ansa-FcdiOH on 9L cells with a low IC50 value (0.1 μM) associated with an oxidative stress and a dose-dependent alteration of the cell cycle. Repeated intravenous injections of stealth ansa-FcdiOH LNCs in ectopic glioma bearing rats induced asignificant tumor growth inhibition, supported by a reduced number of proliferative cells in tumors compared to control group. Additionally,no liver damage manifestation was observed in treated animals. These results indicated that stealth ansa-FcdiOH LNCs might be consideredas a potential new approach for cancer chemotherapy.

RKey words: ansa-FcdiOH; Nanomedicine; Cell cycle; EPR effect

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RIntroduction

Malignant brain tumor remains one of the main medicalchallenges for scientists as no life-saving treatment has beenfound to date despite aggressive therapy modalities (Stuppprotocol).1 Consequently, alternative strategies are needed totackle this dismal tumor.

Over the past few years, Pr. Jaouen and his team havestruggled to develop potent bioorganometalic complexes foranticancer application. Their chemistry is based on the couplingbetween a ferrocene moiety and a tamoxifen-like skeleton, and

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The authors state no competing interests.This work was supported by the ANR Blanc Program Mecaferrol.⁎Corresponding author at: Inserm U1066, IBS-CHU Angers, 4 rue

Larrey, 49933 Angers Cedex 9.E-mail address: [email protected] (C. Passirani).

http://dx.doi.org/10.1016/j.nano.2014.05.0021549-9634/© 2014 Published by Elsevier Inc.

Please cite this article as: Laine A.-L., et al., Inhibition of ectopic glioma tumorNanomedicine: NBM 2014;xx:1-11, http://dx.doi.org/10.1016/j.nano.2014.05.0

the resulting complex belongs to the ferrocifen family.2,3 Severalmolecules from their collection have already showed strongantiproliferative activities on experimental glioma and breastcancer models.4–7 Recently, a new ferrocenyl complex, ansa-FcdiOH (Figure 1), has emerged and turned out to be the mosteffective agent according to in vitro antiproliferative assays.Interestingly, ansa-FcdiOH was tested on 60 cancer cell lines ofthe National Cancer Institute (NCI) and showed to be stronglyactive on CNS (central nervous system) cancer cells. In addition,its cytotoxic behavior did not match with any of the 171 referencemolecules from the NCI database (COMPARE analysis).2 So far,its mechanism of action is not fully understood although the roleplayed by its redox properties has been already underlined.8

Therefore, ansa-FcdiOH was selected in this work to further studyits anticancer activity in vitro and in vivo on 9L brain tumors.

Due to the hydrophobic characteristic of this molecule, theuse of a dosage form was required for its in vivo evaluation.

growth by a potent ferrocenyl drug loaded into stealth lipid nanocapsules.02

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Currently, nanocarriers represent the most promising and mostinvestigated delivery systems for various unmet medicalneeds9,10 including cancer therapy.11 The discovery of theenhanced permeability and retention (EPR) phenomenon hasprompted galenists to develop long circulating nanocarriers (alsocalled stealth nanocarriers) to promote the EPR effect andultimately to achieve an enhanced drug accumulation intotumors.12 A widely used technique to obtain such nanocarriersconsists in coating nanoparticles with a polyethylene glycol(PEG) based molecule.13,14

Our laboratory have developed and patented a nanodeliverysystem based on a phase inversion temperature method andcalled lipid nanocapsules (LNCs).15 Standard PEGylation ofLNCs is achieved via the post-insertion (PI) technique16 ofDSPE-mPEG 2000 (1,2-Distearoyl-sn-glycero-3-phosphoetha-nolamine-N-[methoxy-(polyethyleneglycol)-2000]) involving aprior LNC formulation step and a subsequent incubation step asperformed on liposomes.17 The main drawback with this methodconcerns the additional 2 to 4 h incubation step extending thecomplete formulation process time. An alternative PEGylationmethod was approached by Hoarau et al consisting in incorporat-ing the DSPE-PEG in the initial LNC mixture,18 but had not beensubjected to further evaluation so far. Because this latter techniqueprovides a simple while efficient PEGylation process, we designedfor this study “one-step” (OS) stealth drug-loaded LNCs. In anattempt to determine their pharmacological behavior, 9L celluptake, blood circulation profile and accelerated blood clearance(ABC) were assessed through fluorescent stealth OS LNCs.

The anticancer activity of ansa-FcdiOH was investigatedthereafter on 9L rat glioma model, firstly in vitro with cell cycleanalyses and, secondly, in vivo through stealth ansa-FcdiOH-LNC injections in ectopic glioma bearing rats. To furtherdecipher the ferrocenyl drug mechanism of action, cancer cellproliferation and intratumoral immune reaction were evaluatedon tumor sections.

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Stealth ansa-FcdiOH-LNC formulation

Stealth LNCswere prepared according to the “one-step process”based on a phase inversion temperature method15,18 Briefly, thepreparation process consisted in mixing all the excipients [SolutolHS15 (15.9% w/w), Lipoid (1.4% w/w), Labrafac (19.4% w/w),NaCl (3.0% w/w), water (55.8% w/w), the drug as powder(2.6% w/w) and DSPE-mPEG 2000 (1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy-(polyethyleneglycol)-2000],1.9% w/w)] under magnetic stirring. Three cycles of progressiveheating and cooling between 90 °C and 50 °C were thenperformed. At the last cooling step, the ansa-FcdiOH LNCsuspension was diluted with water (71.4% w/w). The resultingsuspension was passed through a 0.2 μM filter to remove thenon-entrapped drug. The final ansa-FcdiOH concentration was8 mg/mL (19 mM).

Fluorescent stealth LNCs were obtained by labeling the LNCswith the fluorescent DiI (1,10-dioctadecyl-3,3,30,30-tetramethylindocarbocyanine perchlorate) probe at 4 mmol/LLabrafac or with

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DiD (1,1'-Dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanineperchlorate) at 2.5 mmol/LLabrafac.

LNC characterization

The determination of the drug loading was achieved afterfiltering the LNC step through a 0.2 μMfilter and dissolving 30 μLof LNCs into 5 mL of methanol (Fisher chemical). As the drugsolubility in water is very low, it was assumed that the drug fractionunentrapped during the LNC formulation is in a precipitated state.Then, the filtering step allowed the free drug to be separated fromthe drug loaded into LNC. The drug loading in the filtered LNCsuspension is subsequently measured by spectrophotometry (UV2600, Schimadzu, Champs surMarne, France) against a calibrationcurve generated from the drug solubilized in methanol.

The average hydrodynamic diameter and the zeta potential ofnanocapsules were determined at 25 °C, in triplicate, using aMalvern Zetasizer (Nano Series DTS 1060, Malvern InstrumentsS.A., Worcestershire, UK). For the measurement, the LNCs werediluted in MilliQ water (50 μL LNC in 2.95 mL water).

Osmolarity measurement was performed in triplicate on aVapro Osmometer (ELITECH group, Signes, France).

Tumor cell line and culture

Rat 9L gliosarcoma cells were obtained from the EuropeanCollection of Cell Culture (Sigma, Saint-Quentin Fallavier,France). The cells were cultured at 37 °C in a humidifiedatmosphere containing 5% CO2 in Eagle's minimal essentialmedium (EMEM) (Lonza, Verviers, Belgium) supplied with 1%non essential amino acids (Lonza), 10% fetal calf serum (FCS)(Lonza) and 1% antibiotic and antimycotic solution (Sigma).

In vitro cell uptake

9L cells were seeded in 6-well plates for 72 h. Cells were thentreated with DiD-LNCs, OS stealth DiD-LNCs and PI stealth DID,diluted at 1/500 in a serum-free culturemedium, for 2 h at 37 °C or4 °C. At the end of the incubation period, cells were fixed in 2%formaldehyde and analyzed using a FACScan flow cytometer withCellQuest Software (BD Biosciences). All experiments wereperformed in triplicate and presented as mean ± SD.

In vitro cell viability

A suspension of 9L cells (1.9 × 104 cells/mL) was put in eachwell of 24-well plates for 48 h. On day 2, the culture medium wasremoved and cells were treated with increasing concentrations(0.01-10 μmol/L) of various formulations. After 72 h of incuba-tion at 37 °C, the medium containing samples was replaced byfresh medium. Cell survival percentage was estimated by theMTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) survival assay according to thesupplier's instructions (Promega, Lyon, France).

In vitro reactive oxygen species (ROS) generation

In order to assess the ROS involvement in the ansa-FcdiOHmechanism of action, cells were co-treated with ansa-FcdiOH atthe IC50 concentration and an antioxidant agent (ascorbic acid(Sigma) at 200 μM). The test was performed through the MTS

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Figure 1. The ansa-FcdiOH molecule.

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assay in the same conditions (seeding, treatment schedule,measurement) as described above. As controls, cells were treatedwith ascorbic acid alone. This experiment was performed intriplicate and presented as mean ± SD.

Cell cycle analysis

Cell cycle analysis was performed as previously described.4

Briefly, cells were treated with free ansa-FcdiOH at increasingconcentration (0.05-0.5 μM) for 24, 48 and 72 h of treatment. Atthe end of the experiment, cells were trypsinized, washed twicewith PBS and fixed (ice cold EtOH 70% v/v, 1 h, −20 °C). Cellswere subsequently incubated in PBS containing RNAse A(50 μg/mL, 15 min, 20 °C) and then in PBS containingpropidium iodide (20 μg/mL, 4 h, 20 °C, in the dark). Cellcycle was analyzed with Cyan LX9 cytometer (BeckmanCoulter, France) and data were processed by a multicyclesoftware (Phoenix Flow Systems, San Diego, USA).

Determination of apoptotic cells

One hundred thousand cells were seeded in 35-mm dishes andtreated for 24 h with ansa-FcdiOH. Cells were then fixed in coldmethanol (−20 °C, 10 min) and stained with 1 μg/mL Hoechst33258 (30 min, 20 °C, in the dark). Apoptotic cells exhibitingcondensed and fragmented nuclei were counted under a NikonEclipse fluorescence microscope. At least, 200 cells in randomlyselected fields were examined.

Animals

In vivo efficacy experiments were carried out on 10 to11-week-old Syngeneic Fischer F344 female rats (Charles RiverLaboratories France, L'Arbresle, France), weighing 160-180 g.Animal care was performed in strict accordance with FrenchMinistry of Agriculture regulations. The animal procedures wereapproved by the Animal Experimentation Ethic Committee ofPays de la Loire, protocol number CEEA.2012.32.

For accelerated blood clearance experiments, 10 to 11-week-old female Sprague rats (SCAHU, Service Commund'Animalerie Hospitalo-Universitaire, Angers, France) wereused weighing 260-300 g.

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ABC phenomenon evaluation

Female Sprague rats were randomly divided into 2 groups.The ABC phenomenon was investigated after a second injectionof LNCs. The initial injection used stealth LNCs andconventional LNCs for comparison and was administered viathe tail vein (400 μL/rats). For the second injection, stealth DiI-labeled LNCs and DiI-LNCs were injected intravenously(400 μL/rats) at day 7.

Blood samples were collected by cardiac puncture atdesignated time intervals (5 min, 30 min, 1 h, 2 h, 4 h and6 h, 8 rats per group) in a blood collection heparin tube(Microtube, 1.3 mL, Sarstedt AG & Co., Nümbrecht, Germany).DiI fluorescence contained in plasma was measured at theemission wavelength of 544 nm with an excitation wavelengthof 590 nm by a Fluoroscan (Ascent FL, Thermo FisherScientific, Cergy-Pontoise, France). One hundred percent offluorescence was considered as the value counted at 5 min post-injection. The blood circulation profiles after the secondinjection were compared to the original profiles obtained througha distinct experiment with one single injection.

In vivo antitumor efficacy

In vivo anticancer activity was evaluated against 9L tumor-bearing fisher rats. Animals were manipulated under isoflur-ane–oxygen anaesthesia. After shaving and disinfection, ratswere intradermically implanted with 1.5 × 106 9L cells on theright flank. At day 7 post tumor implantation, animals werehomogeneously divided into three groups: physiologicalsaline solution (0.9% NaCl), unloaded stealth LNCs andstealth ansa-FcdiOH-LNCs (ansa-FcdiOH dose: 20 mg/kg),with 9 rats per group. The treatments, administered via thelateral tail vein, were started at day 7 and had beenadministered daily for 2 weeks with a mid-term break of2 days (10 injections in total). Tumour growth was followedby regularly measuring the length and width of tumours with acalliper. The tumour volume (V) was estimated by themathematical ellipsoid formula:

V ¼ π=6ð Þ � widthð Þ2 � lengthð Þ

At day 21 post-tumor inoculation, rats were sacrificed in aCO2 chamber, and tumors and livers were excised andimmediately fixed in 4% formaldehyde solution to be embeddedin paraffin.

Tumor and liver slide histology

The embedded tissues were sectioned (4 μM) along thelongitudinal axis of the tumor. The liver and tumor sections werestained with hematoxylin and eosin (H&E) for histologicalanalysis and were observed by an anatomopathologist.

Tumor immunostaining

Tumor tissues were subjected to immunohistochemicalstaining against OX6 and CD3 to analyze macrophage and Tlymphocyte infiltration, respectively. Cell proliferation was alsoassessed through PCNA immunofluorescence. Briefly, tumor

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Figure 2. PEG coating density influence on 9L cell uptake. The percentage of positive cells and the Geo mean are indicated on the figure. DiD-LNCs: no PEGcoating, stealth DiD-LNCs (OS): low PEG coating, stealth DiD-LNCs (PI): high PEG coating.

Table 1t1:1

Physico-chemical characteristics of unloaded stealth LNCs and stealth ansa-FcdiOH-LNCs.t1:2

t1:3 Hydrodynamic diameter (nm) PdI Zeta potential (mV) Osmolarity (mOsm) Drug payload (% w/w)

t1:4 Stealth LNCs 60.3 ± 0.8 0.08 −18.8 ± 0.7 327 ± 4 –t1:5 Stealth ansa-FcdiOH-LNCs 40.1 ± 0.1 0.03 −14.1 ± 1.1 370 ± 3 6.4

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slides were deparaffinized and subsequently subjected to antigenretrieval using a citrate buffer pH 6 for 40 min at 90 °C. Inorder to block nonspecific binding, sections were incubated inPBS containing 4% BSA and 10% normal goat serum.Incubations with primary antibodies (anti-rat OX6 (1/50),anti-rat CD3 (1/50) and anti-rat PCNA (1/1000)) wereperformed overnight at 4 °C. Anti-OX6 and anti-CD3 primaryantibodies were detected using a biotinylated secondaryantibody followed by the avidin–biotin complex methodaccording to the supplier's directions (Vector). Sections wererevealed with 3,3-diaminobenzidine (Sigma) counterstainedwith hematoxylin and permanently mounted. Anti-PCNAprimary antibody was detected using a biotinylated secondaryantibody amplified with streptavidin–FITC (Dako). Nucleiwere counterstained with DAPI (Sigma). Sections of three ratsof each group (control and stealth ansa-FcdiOH-LNCs) wereanalyzed under a microscope (Axioscope 2 optical, Zein, LePecq, Germany). PCNA+ cell counting was performed in threetumor sections per rat using the Metaview computerized image-analysis system (Roper scientific, Evry, France). Six fields persections taken at ×200 magnification were randomly chosen in

the tumor. Results were expressed as the mean number ofPCNA+ cells/mm2 for each group ± SEM.

Statistical analysis

For in vitro experiments, the results were expressed as themean ± SD and Student t test was performed between treatedand control groups.

Statistical significance between control and treated groups forin vivo experiments was evaluated using Mann and Withney testand was considered as significant with P b 0.05. Results wereexpressed as a mean ± SEM.

Results

Formulation and characterization of the stealthansa-FcdiOH-loaded LNCs

Stealth ansa-FcdiOH loaded LNCs were successfully pre-pared at 8 mg/mL (6.4% w/w) (Table 1). The direct incorpora-tion of DSPE-PEG into the LNC mixture slightly increased thecarrier size in comparison to conventional LNCs (50 nm) in

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Figure 3. Antiproliferative activity of ansa-FcdiOH. (A) 9L cell survival after 72 h exposure to various treatments. (B) Co-treatment with ascorbic acid (AA).*P b 0.05, **P b 0.01.

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encapsulation markedly decreased the diameter compared tostealth LNCs, reaching a size of 40 nm. The zeta potentialmeasurement revealed similar negatively charged surfaces forboth stealth LNCs, indicative of the DSPE-PEG sheathing. Asthe formulations were intended for intravenous delivery, theosmolarity was settled at approximately 300 mOsm.

In vitro cell uptake

The glioma cell uptake of stealth DiD-labeled LNCs obtainedvia the OS process were measured and compared to uncoatedLNCs at an early time point (2 h) and at 4 °C and 37 °C(Figure 2). Additionally, in order to further assess the PEGcorona influence on the cell internalization, LNCs with higherDSPE-PEG coating density were analyzed as a control with thetwo other formulations. These LNCs were prepared through thePI technique, as it allowed a higher DSPE-PEG coating than withthe OS one, as previously described.16 Accordingly, the DSPE-PEG content for PI DiD-LNCs (9% w/w) was almost twice asmuch as that of OS DiD-LNCs (4.8% w/w).

High DSPE-PEG coating density hampered the LNC celluptake (Geo mean = 33.5) in comparison to low PEG density,whose uptake was similar to conventional LNCs (Geo mean =66.5 and 63.2, respectively). Four degrees celsius condition,which usually permits to turn off cell metabolic activitiesincluding active cell internalization, allowed the assessment of

passive uptake. For each LNC batch, a weaker internalizationwas observed at 4 °C in comparison to 37 °C, revealing a majorcontribution from the active uptake. In addition, DSPE-PEGcoating tended to inhibit the passive LNC internalization with thestrongest effect observed for PI LNCs.

In vitro antiproliferative assay

After 72 h cell exposure, ansa-FcdiOH was able to reach anIC50 value of 0.1 μM (Figure 3, A). Similar profiles and IC50values were obtained for free and encapsulated drug whichproved that ansa-FcdiOH was still pharmacologically activedespite its entrapment in LNCs. Moreover, the co-treatment withan antioxidant agent partially inhibited the activity of ansa-FcdiOH (Figure 3, B).

Cell cycle analysis

Two major dose-dependent effects on the cell cycledistribution could be distinguished (Figure 4). At low dose(50 nM), ansa-FcdiOH treatment induced an increase of 9L cellsin S phase fraction (Figure 4, A). This effect was detectable from24 h treatment, subsequently leading to a prolonged S phaseblockade up to 72 h with a slight increase of sub G0/G1population at 0.1 μM. For higher concentrations (0.5 μM), cellswere rapidly stacked in G0/G1 phase and cell cycles were nolonger analyzed because of a massive increase of sub G0/G1population at 48 and 72 h (Figure 4, B). Such sub G0/G1 cell

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Figure 4. Cell cycle distribution and apoptosis counting. (A) Concentration dependent effects. (B) Time dependent effects. (C) Percentage of Hoechst-stainedapoptotic cells after 24 h exposure to increasing ansa-FcdiOH dose.

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Figure 5. In vivo study. (A) ABC phenomenon evaluation (1st inj.: first injection, 2nd inj.: second injection). (B) In vivo antitumor efficacy. Treatment days areindicated by red arrows. The pictures represent an overview of the tumor mass at D14 for each group of animal. n = number of animals per group. *P b 0.05.

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population revealed a degradation of DNA within the treatedcells which could occur by necrosis or apoptosis. Therefore,Hoechst staining determination of apoptosis was performed after24 h treatment. As shown in Figure 4, C, apoptotic nucleiincreased in a dose dependent manner under ansa-FcdiOHtreatment indicating that sub G0/G1 fraction observed by flowcytometry was due to apoptosis induction.

Accelerated blood clearance (ABC) phenomenon investigation

ABC phenomenon was investigated at the second stealth DiILNC injection administered 7 days after pretreatment withunloaded stealth LNCs. The blood circulation profiles of thestealth LNCs are displayed in Figure 5, A, and compared toconventional LNCs (without DSPE-mPEG coating) as shortcirculating LNC control. A slight decrease in stealth LNC bloodcirculation time was observed at the second injection. However,the whole plasmatic profile remained extended compared touncoated LNCs.

In vivo tumor growth inhibition study

Interestingly, stealth ansa-FcdiOH-LNC treatment sloweddown the tumor growth from day 11 compared to the two controlgroups (Figure 5, B). From day 14, the tumor volume curveplateaued meaning that ansa-FcdiOH inhibited the tumor growth.

To determine whether ansa-FcdiOH affects 9L tumor growthat the level of cell proliferation, a PCNA immunofluorescentanalysis was performed (Figure 6, A). The number of positivelyresponding cells was estimated at 1553 ± 105 PCNA+ cells/mm2

for the drug treated group, which was significantly lowercomparatively to that of the saline group with 2025 ± 123PCNA+ cells/mm2.

Anti-tumor immune response analysis

In an attempt to evaluate whether the tumor growth inhibitioncould stem from an intratumoral immune response, macrophageand T lymphocyte infiltrations were immunohistologicallystained (Figure 6, B). Both tumor sections of stealth ansa-FcdiOH-LNCs and saline groups displayed similar macrophageand lymphocyte infiltration.

Liver histology

Considering the aggressive protocol involving repeatedinjections of stealth ansa-FcdiOH LNCs, it seems necessary totake an interest in the rat livers. Specimen sections of the controlgroups and ansa-FcdiOH treated groups (Figure 6, B) showednormal histological appearance of the organ meaning that nodamage manifestation was detected within ansa-FcdiOH groups.

Discussion

Despite intensive research efforts, the diagnosis of amalignant brain cancer remains associated with a grim prognosis.Current available drug therapies are insufficiently effective totreat this aggressive disease, mainly attributed to tumor-cellresistance, and ultimately leading to ineluctable recurrence.

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Figure 6. Immunohistological analyses. (A) PCNA-stained proliferating cells in tissue sections. Green: PCNA+ cells, blue: nuclei stained by DAPI. **P b 0.01(scale bar = 100 μM). (B) Effect of stealth ansa-FcdiOH-LNCs treatment on liver (b) and on tumor (d, f) compared to saline treatment (a and c, e respectively)(scale bar = 50 μM).

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Consequently, innovative treatments are urgently needed toimprove the outcome for patients.

The drug of interest in this work is a ferrocenyl molecule(ansa-FcdiOH) which can be described as a dyphenol metallo-complex. Polyphenol are currently under considerable investi-gation due to their cytotoxic effect on cancer cells. Among them,curcumin is subject to great interest in particular in glioblastomatreatment and has already shown promising outcome.19,20

Besides a potential polyphenolic activity, ansa-FcdiOH canalso take advantage of its metallic part which may confer aunique and alternative activity over organic compound.

Considering the poor water soluble property of this ferrocenylcomplex, a drug delivery system was required for furtherevaluation. As LNCs have already proved to be a suitable tumor-delivery nanosystem for other ferrocenyl compounds,4,5 weobviously selected this carrier for the development of injectableansa-FcdiOH dosage form. As above-mentioned, the in vivodrug delivery performance of nanoparticles is commonly boostedthrough the addition of PEG onto their surface.

While conventional LNC PEGylation is achieved through apost-insertion technique, we designed for this study alternativestealth ansa-FcdiOH-LNCs via a one-step process intended tofacilitate a future transposition in clinic. In a study conducted inparallel, the influence of the quantity of PEGylated phospho-lipids (DSPE-mPEG) added into the LNC formulation wasassessed.21 It has been shown that the formation of PEGylatedLNCs through a unique step was possible up to a DSPE-PEGconcentration of 6% w/w resulting in a gradual increase in LNCdiameter. In the present study, the DSPE-mPEG concentrationwas set at 4.8% (w/w), which was shown to provide longcirculation property while maintaining a satisfactory size. Thedrug loading capacity of these OS-LNCs was therefore evaluatedthrough the encapsulation of ansa-FcdiOH, achieving samemaximal payload as that of the uncoated ones (data not shown).Accordingly, stealth ansa-FcdiOH-LNCs were successfullyprepared by the one-step phase inversion temperature methodwith a satisfactory size of 40 nm, high drug loading and stealthfeatures, making them suitable for intravenous delivery.

It could be noticed that drug loaded stealth LNCs showedsmaller hydrodynamic size than unloaded stealth LNCs. Thisphenomenon may be induced by the ansa-FcdiOH entrapmentinto the LNC shell structure as previously reported with theencapsulation of another polyphenol molecule, the quercetin.22

As a matter of fact, polyphenols are able to interact withmembrane bilayers and can bind to the lipid–water interface.23

Based on these considerations, the size alteration is assumed toresult from the drug arrangement between the HS-PEG chains atthe LNC interface.

Proper PEGylated phospholipid anchorage onto the LNCsurface was evidenced by the high negative surface chargestemming from the PEG dipoles4 as well as from the negativelycharged phosphate groups of DSPE-mPEG. Additionally,the stealth ansa-FcdiOH-LNCs displayed a narrow sizedistribution (PdI = 0.05), which is a critical parameter controllingthe biodistribution.

As nanocarrier stability is an important factor to consider, astability study at 37 °C was performed on stealth ansa-FcdiOH-LNCs in comparison to uncoated ansa-FcdiOH LNCs. Surpris-

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ingly, stealth ansa-FcdiOH-LNCs exhibited enhanced physicalstability over regular ansa-FcdiOH-LNCs. The latter displayedindeed a dramatic increase in size probably originating from theinterface destabilization by the drug (data not shown). This resultemphasized the capacity of PEGylation to ensure the physicalLNC stability. Additionally, no precipitation phenomenon wasobserved either visually or via particle size measurement duringthe stability assay, meaning that no burst release of the drugseemed to occur. Furthermore, we recently assessed the releasekinetics from the LNCs through FRET (Förster resonance energytransfer) monitoring. This fluorescence technique based on theco-loading of a donor/acceptor probe pair permitted to easilymonitor the carrier stability of encapsulation. Although themolecules entrapped were fluorescent probes, stealth LNCsshowed great stability of encapsulation. More interestingly, longPEG chain grafting seemed to prevent from drug leakingobserved with uncoated LNCs.21

It was previously demonstrated that conventional LNCs wererapidly internalized by glioma cells through endocyticpathway.24 However, the influence of DSPE-PEG coating onLNC uptake had not been evaluated so far. Although PEGcoating create a hydrophilic corona able to prevent LNCs fromthe opsonization phenomenon, it might also hinder nanoparticleinternalization by cells. Accordingly, we assessed the LNCuptake by 9L glioma cells depending on the level of PEG coatingdensity. Highly PEG coated-LNCs obtained through the PItechnique showed reduced cell uptake compared to LNCs with alower OS PEG coating. Interestingly, the later displayed similarinternalization to uncoated LNCs. This study highlighted the factthat hydrophilic PEG corona could reduce cell interactions,likely owing to steric repulsions and lack of affinity. Theseobservations further supported our choice of a simple OS processfor the design of stealth LNCs, achieving a low while adequatePEG coating over the PI technique. Additionally, these OSstealth LNCs exhibited the capacity to deliver their loading at theintracellular level mostly through active uptake.

In vitro antiproliferative assay demonstrated strong ansa-FcdiOH effect against 9L glioma cells achieving an IC50 of0.1 μM and the encapsulation in stealth LNCs did not alter itspotency. In comparison to FcdiOH, an analogous drug to ansa-FcdiOH which has already shown promising activity on braintumor,5 ansa-FcdiOH displayed a lower IC50 value on 9L cellline demonstrating its hopeful potent activity. Remarkably, ansa-FcdiOH showed also a lower IC50 value against glioma cell linesin comparison to curcumin with 25 μM19 or to gold compoundsreaching 3 μM.25 The partial inhibition of the drug activity whenassociated with an antioxidant supported a ROS-mediated celldeath. This is commonly encountered with metallodrugs such asruthenium compounds26 and gold-based compounds25 andpolyphenol drugs.27,28

In order to get a deeper insight into the ansa-FcdiOHmechanismof action, cell cycle analyses were performed. As a result, ansa-FcdiOH altered the 9L cell cycle in a dose dependent manner withthe arrest in S-phase at low dose (50 nM) and in G0/G1 phase athigher dose associated with apoptosis induction. In a previouswork, the impact of LNCs in the drug effect on cell cycleprogression was investigated. Cell cycle analyses were conductedafter incubation of MDA-MB-231 breast cancer cells with

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FcOHTam, a compound belonging to the same ferrocenyl family asansa-FcdiOH.4 Interestingly, it has been shown that the drugdisplayed similar activity on the cell cycle regardless of theformulation (free drug, drug loaded-LNCs, drug loaded stealthLNCs). Additionally, the unloaded vehicles did not affect thecell cycle.

Based on clinical settings, we planned for the in vivo efficacystudy a treatment protocol composed of multiple doses. Repeatedinjections indeed aimed to increase the drug concentration intumor tissue as demonstrated in the literature with multipleinjections of chitosan-based nanoparticles.29 However, it hasbeen reported that multiple injections of PEGylated nanocarriersinduced an immune reaction with M immunoglobulin productionagainst PEG which, as consequence, led to an accelerated bloodclearance phenomenon as soon as the second injection ofPEGylated nanocarriers was given.30 Additionally, the seconddose was shown to preferentially end up in Kupffer cells of theliver.31 This consideration is a matter of concern, as, besidesaffecting the passive targeting ability of the cargo, it also cancause severe liver damage in the case of toxic anticancer drugs.Consequently, ABC phenomenon was investigated at the secondstealth DiI LNC injection administered 7 days after pretreatmentwith unloaded stealth LNCs. As reported in the literature, this7 day interval corresponds to the maximum magnitude of ABCeffect.30 Comfortingly, no dramatic decrease in LNC bloodcirculation time was observed in comparison to PEG liposomeswhich lose their entire stealth properties at the second dose.30

Additionally, the extended circulation time for stealth LNCs overconventional LNCs was confirmed. This ABC effect seems to behighly dependent on the type of nanocarrier used and, to date, noparameter can help in predicting the potential induction ofantibodies upon injections of PEGylated nanosystems.

In vivo antitumor studies showed that repeated injection ofstealth ansa-FcdiOH-LNCs could significantly inhibit 9L tumorgrowth compared to controls. This observed anticancer activitywas further evidenced by a significant reduced number ofintratumoral proliferating cells upon ansa-FcdiOH treatment.This could be due to the cell blockade in G0/G1 phase but also inS-phase since prolonged S-phase arrest is associated withdecrease of S phase related cell cycle protein like PCNA.32,33

It must be pointed out that 9L tumor has been shown to behighly immunogenic34 which might mislead about the drug-induced tumor treatment. However, macrophage and T lympho-cyte infiltration staining suggested that ansa-FcdiOH activity didnot seem to be related to a distinct activation of an intratumoralimmune response.

Hepatotoxicity is one of the major issues encountered withpatients undergoing chemotherapy.35 Interestingly, the repeatedinjections of stealth ansa-FcdiOH-LNCs did not induce apparentliver damage. This is intriguing since metal-based drugsespecially cisplatin36 often induced hepatic adverse effects.This non-toxicity to liver may also be ascribed to the drugconfinement into the LNCs protecting healthy tissue which is oneof the crucial advantages of nanocarriers permitting to overcomedose-limiting toxicity encountered with several drugs.37

Taken all together, these results have proved that stealth ansa-FcdiOH-LNCs may represent an alternative approach to treatcancer and may comply with the current need in oncology.

Moreover, nanoparticles seem to be a promising tool capable ofovercoming many of the major obstacles encountered in braintumor treatment.38 Accordingly, further studies have beenplanned in order to assess the performance of these stealthansa-FcdiOH-LNCs in an orthotopic glioma model.

Acknowledgments

The authors would like to thank Pascal Pigeon (ENSCP) foransa-FcdiOH synthesis.

OFAppendix A. Supplementary data

Supplementary data to this article can be found online athttp://dx.doi.org/10.1016/j.nano.2014.05.002.

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1 Graphical Abstract

2 Nanomedicine: Nanotechnology, Biology, and Medicine xxx (2014) xxx

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5 Inhibition of ectopic glioma tumor growth by a potent ferrocenyl6 drug loaded into stealth lipid nanocapsules

7

8 Anne-Laure Laine a,b, Anne Clavreul b,c, Audrey Rousseau d,9 Clément Tétaud b,c, Anne Vessieres e, Emmanuel Garcion a,b,10 Gerard Jaouen e, Léo Aubert f, Matthieu Guilbert f, Jean-Pierre Benoit a,b,11 Robert-Alain Toillon f, Catherine Passirani a,b,⁎

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aLUNAM Université – Micro et Nanomédecines Biomimétiques, Angers, France14

bInserm U1066, IBS-CHU, Angers, France15

cDépartement de Neurochirurgie – CHU, Angers, France16

dDépartement de Pathologie Cellulaire et Tissulaire – CHU, Angers, France17

eCNRS, UMR 7576, ENSCP, Paris, France18

fInserm U908, Université Lille 1, Villeneuve d'Ascq, France

1920 Daily intravenous injections of stealth LNCs loaded with a novel ferrocenyl21 compound (ansa-FcdiOH) induced a significant tumor growth inhibition22 compared to controls.

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