A new nanostructured carrier design including oil to enhance the pharmaceutical properties of...

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European Journal of Pharmaceutics and Biopharmaceutics xxx (2014) xxx–xxx

EJPB 11626 No. of Pages 12, Model 5G

10 May 2014

Contents lists available at ScienceDirect

European Journal of Pharmaceutics and Biopharmaceutics

journal homepage: www.elsevier .com/locate /e jpb

Research paper

A new nanostructured carrier design including oil to enhancethe pharmaceutical properties of retinoid therapy and its therapeuticeffects on chemo-resistant ovarian cancer

http://dx.doi.org/10.1016/j.ejpb.2014.04.0140939-6411/� 2014 Published by Elsevier B.V.

Abbreviations: ABC, ATP-binding cassette; ATRA, all-trans-retinoic acid; BSA,bovine serum albumin; DSC, differential scanning calorimetry; EE, encapsulationefficiency; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;PBS, phosphate buffered saline; PEG, polyethylene glycol; PLGA, poly(lactic-co-glycolic) acid; PLGA-np, poly(lactic-co-glycolic) acid nanoparticles; PONC,polymer-oil nanostructured carriers; PVA, polyvinyl alcohol; TEM, transmissionelectron microscope.⇑ Corresponding author. School of Pharmacy, Temple University, 3307 North

Broad Street, Philadelphia, PA 19140, USA. Tel.: +1 215 707 8173; fax: +1 215 7073678.

E-mail addresses: [email protected], [email protected](H.L. Wong).

Please cite this article in press as: M. Narvekar et al., A new nanostructured carrier design including oil to enhance the pharmaceutical propertiesinoid therapy and its therapeutic effects on chemo-resistant ovarian cancer, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2014

Mayuri Narvekar, Hui Yi Xue, Ngoc T. Tranx, Mariam Mikhael, Ho Lun Wong ⇑School of Pharmacy, Temple University, Philadelphia, PA, USA

293031323334353637383940

a r t i c l e i n f o

Article history:Received 30 January 2014Accepted in revised form 28 April 2014Available online xxxx

Keywords:Controlled drug deliveryNanomedicineAll-trans-retinoic acidPoorly-water soluble drugsChemoresistanceOvarian cancer

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a b s t r a c t

All-trans retinoic acid (ATRA) is an appealing alternative drug for the cancers that have failed the conven-tional chemotherapy and become chemo-resistant and more tumorigenic. In this study, we specificallyaddressed two issues commonly associated with ATRA nanotherapeutics: (1) insufficient, unstableentrapment and uncontrolled release of the highly lipophilic ATRA and (2) lack of studies in therapeuti-cally relevant chemo-resistant cancer cell models. A polymer-oil nanostructured carrier (PONC) com-posed of oil and PLGA was designed and studied in an ovarian cancer cell subline SKOV-3PR that couldwithstand up to 300 nM paclitaxel and expressed high levels of multidrug resistance transporter ABCB1and tumorigenic marker CD133. Differential scanning calorimetry of PONC revealed superior polymeramorphosity and dispersion of the entrapped ATRA in a manner comparable to nanostructured lipid car-riers. With this design, the ATRA encapsulation efficiency was increased up to 8.5-fold and a 5-day con-trolled release profile was obtained. ATRA-PONC was able to induce extensive apoptotic cell death andexert substantially higher long-term anti-tumorigenic effects (IC50 of ATRA-PONC: 2 lg/ml versus freeATRA: 17.5 lg/ml; p < 0.05) in SKOV-3PR cells. Mechanistic studies indicated that these enhanced antican-cer effects were likely attributable to higher cell permeation by the well-dispersed drug/oil steadilyreleased from PONC. To conclude, a nanostructured, oil-in-polymer hybrid carrier design has been devel-oped for efficient ATRA delivery and treatment of the chemo-exposed, chemo-resistant sub-population ofovarian cancer, exemplifying a convenient strategy to vastly improve the pharmaceutical and therapeuticproperties of tough-to-deliver lipophilic, poorly water-soluble anticancer compounds.

� 2014 Published by Elsevier B.V.

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1. Introduction

Solid malignant diseases such as ovarian cancer frequentlyacquire chemo-resistance after exposing to the conventional cyto-toxic chemotherapy. 30% Ovarian cancer treated with the standard

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taxane/platinum-based chemotherapy failed to respond, and themajority recurred over time and became refractory to furtherchemo-treatment [1,2]. In view of these issues, researchers inrecent years have begun to focus on the less cytotoxic drugs asalternative therapies for chemo-resistant cancers. All-trans-retinoicacid (ATRA) is one of these alternatives [3,4]. Instead of relyingheavily on inducing cytotoxicity in the cycling cancer cells, ATRAcontrols cancer progression with its unique cell-differentiating,anti-proliferative and apoptosis-inducing activities mediated bythe retinoic acid receptors and retinoid X receptors [5]. This drugis therefore officially approved for the treatment of acute promylo-cytic leukemia, a subtype of blood cancer [5,6]. However, repurpos-ing ATRA for non-blood, solid malignancies remains a dauntingtask. Systemic delivery of ATRA to these cancers is inefficient whichleads to frequent side effects [5,7–9]. There is clearly an unmet clin-ical need for improved delivery of this valuable alternative antican-cer drug.

of ret-.04.014

http://dx.doi.org/10.1016/j.ejpb.2014.04.014

mailto:[email protected]

mailto:[email protected]


http://www.sciencedirect.com/science/journal/09396411

http://www.elsevier.com/locate/ejpb


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Fig. 1. Schemes comparing the designs of the standard polymeric nanoparticles(top) with polymer-oil nanostructured carriers (PONC, bottom). Green/blue colorrefers to polymeric component, yellow color refers to oil component, and the carriersurfaces are coated with polyethylene glycol or targeting moieties. It is hypothe-sized in the scheme that the oil-in-polymer design of PONC will increase theamorphosity of the polymer so the drug molecules can be entrapped in a moreefficient and uniform manner. (For interpretation of the references to color in thisfigure legend, the reader is referred to the web version of this article.)

2 M. Narvekar et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2014) xxx–xxx


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Delivery of ATRA is mainly limited by its poor physico-chemicalproperties (e.g. high lipophilicity: logP = 6.3 and low aqueous sol-ubility: 29 lg/ml) [7,8,10] and unfavorable pharmacokineticbehaviors (e.g. non-specific binding: serum binding > 95%,VD > 100 L; short half-life: t1/2 = 0.5–2 h) [11]. To improve theseaspects, nanosystems of ATRA such as liposomes, micro/nano-emulsion and solid lipid nanoparticles were developed with mostof them demonstrating promising anticancer activities [7,11–13].However, two key issues still need to be addressed. First, thereremain challenges in designing a nanosystem to optimally dealwith the issues related to the high lipophilicity of ATRA molecules.For instance, loading of ATRA in nanosystems is often inadequateand unstable. Many nanosystems reported had ATRA payloads ataround 1–2% by weight or below. Moreover, we noticed in our pre-vious study that even after apparently successful loading, thepoorly-soluble ATRA molecules tended to deposit on or near thesurface of polymeric nanocarriers instead of being actuallyentrapped, and this led to significant burst releases of ATRA oncein aqueous environment [14]. If not adequately solved, these issueswill compromise the effectiveness, efficiency and safety of ATRAnanotherapeutics.

Second, considering the unique anticancer mechanisms ofATRA, it is foreseeable that ATRA nanoformulations will eventuallybe used as a second-line treatment or an adjunct for the patientswho have failed the first-line chemotherapy. The chemo-exposedcancers in these patients have generally developed variouschemo-resistance mechanisms [2,15,16]. For ovarian cancer, whilethe mechanisms of acquired chemoresistance are not completelyknown, many findings indicated that the ATP-binding cassette(ABC) drug efflux transporters such as ABCB1 (also known asP-glycoprotein or MDR1), ABCC2 (known as MRP2) and ABCG2(known as BCRP) play a crucial role [2,15]. The expression of ABCB1is particularly responsive to paclitaxel exposure [2,17,18]. How-ever, in previous studies, ATRA nanosystems were mostly evalu-ated in normal cancer cell lines that are still chemo-sensitive. Inother words, the true therapeutic value of ATRA nanotherapeuticshas not been fully validated. The use of a chemo-exposed, chemo-resistant cell model is thus highly warranted.

The present study focuses on tackling the above two issues.Here we report the use of a new class of hybrid nanocarriers knownas polymer-oil nanostructured carriers (PONC) that are designedfor systemic delivery of ATRA. Fig. 1 presents the proposed oil-in-polymer design of PONC, in which the oil-soluble ATRA iswell-dispersed together with the oil component within the poly-meric matrix. We hypothesize that (1) the oil-in-polymer designof PONC will facilitate more efficient, stable entrapment and con-trolled release of ATRA when compared with the standard ‘‘solidcore’’ polymeric nanoparticles and (2) being in a highly dispersedstate in the presence of oil, the ATRA molecules in PONC can beeasily utilized by the ovarian cancer cells that have been exposedand became resistant to paclitaxel treatment. Our overall goal isto study this original ATRA nanotherapeutic design with a moretherapeutically relevant cell model, and understand the mechanis-tic basis of its enhanced anticancer effects.

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2. Materials and methods

2.1. Materials

Poly(lactic-co-glycolic) acid (PLGA) (50:50, viscosity 0.4 dl/g,molecular weight 44 kDa, ester end cap) and Captex 200 (i.e. pro-pylene glycol dicaprylate/dicaprate) were kindly donated fromPurac (Gorinchem, Netherlands) and Abitec (Janesville, WI, USA),respectively. Polyethylene glycol–PLGA (PEG–PLGA, PEG (2000)–PLGA(4110)) was purchased from Polyscitech (West Lafayette, IN,

Please cite this article in press as: M. Narvekar et al., A new nanostructured cainoid therapy and its therapeutic effects on chemo-resistant ovarian cancer, Eu

USA). MK-571 was purchased from Santa Cruz Biotechnology (Dal-las, TX, USA), CD133 antibody and blocking reagent from MiltenyiBiotec (Auburn, CA, USA), Spectra/Por dialysis membrane fromSpectrum Labs (Gardena, CA, USA), bovine serum albumin (BSA,Fraction V) and polyvinyl alcohol (PVA, molecular weight30–70 kDa, 87–90% hydrolyzed) from Fisher Scientific (Pittsburgh,PA, USA). ATRA, verapamil, Ko-143, amantadine, cytochalasin-Dand other chemicals were purchased from Sigma Aldrich, Inc. (StLouis, MO, USA).

2.2. Cell culture

Human ovarian carcinoma SKOV-3 cells were purchased fromAmerican Type Culture Collection (Manassas, VA). The cells weregrown in RPMI-1640 medium supplemented with 10% fetal bovineserum, 50,000 units penicillin G, and 50,000 lg streptomycin atstandard cell culture conditions (37 �C, humidified atmosphere of5% CO2). Cells were passaged every 4–6 days.

2.3. Preparation of PONC and control nanosystems

PONC were prepared using standard emulsification–solventevaporation technique. In brief, ATRA and oil (Captex 200) weremixed in 0.3, 1.5 or 3.0 to 6.0 w/w ratio. In a typical preparation,the drug–oil phase containing 6 mg oil was diluted with 1 mldichloromethane, and to this organic phase a mixture of 21.6 mgPLGA and 2.4 mg PEG–PLGA was dispersed. The final organic phasewas added dropwise to 5 ml aqueous solution of 1.5%w/v PVA overa period of 2 min under constant stirring. The mixture was vor-texed for 10–15 s and subjected to sonication for 6 min on ice(40 kHz, 120 V, Bransonic 3510, Danbury, CT). The emulsionformed was stirred overnight for solvent evaporation. The nanocar-riers formed were separated from the aqueous phase by centrifu-gation at 15,000 rpm for 25–30 min. Unbound PVA andunencapsulated drug were washed away with 50 ml cold double-distilled water three times. The nanocarriers formed were freshlyused or lyophilized for further experiments.

rrier design including oil to enhance the pharmaceutical properties of ret-r. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2014.04.014


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For comparison, the traditional solid core PLGA nanoparticles(PLGA-np) were prepared using the above procedure except thatthe oil component was not included (i.e. oil-free polymeric nano-carrier). In selected studies, simple oil-in-water nanoemulsion offree ATRA was prepared by sonicating the drug/Captex 200 oilphase in aqueous solution of 2% soy lecithin for 6 min. This servedas the positive control representing free drug that was not encap-sulated in polymeric matrix. The nanoemulsion was used within24 h after preparation.

2.4. Size distribution, zeta potentials and morphology

Particle size and zeta potential values were measured by photoncorrelation spectroscopy using Malvern Zetasizer NanoZS90(Worcestershire, UK). PONC or PLGA-np fresh samples were sus-pended in distilled water and 12 successive cycles were run at25 �C. Size data based on the distribution by intensity werepresented.

The morphology of nanoparticles was determined by transmis-sion electron microscope (TEM) using JEOL, JEM-1400 TEM (Tokyo,Japan) at 120 kV. A drop of nanocarrier suspension (concentration:0.5 mg/ml) was placed on a 400-mesh copper grid (Ultra thin car-bon type A, Ted Pella Inc., Redding, CA). Samples were dried infumehood before examination.

2.5. Differential scanning calorimetry (DSC)

Samples each containing 2–3 mg lyophilized nanoparticleswere weighted in aluminum pans, sealed, and equilibrated at20 �C for 10 min. Analysis was performed using a DSC Q200 differ-ential scanning calorimeter (TA Instruments, New Castle, DE) at aheating rate of 10 �C/min from 20 to 200 �C. Data were collectedand analyzed using software TA Universal Analysis 2000 (version4.4A). DSC thermograms of ATRA-PONC, ATRA loaded PLGA-npand ATRA drug powder were compared.

2.6. ATRA encapsulation efficiency (EE) and release profiles

Nanocarriers were dissolved in DMSO, and the amount of drugentrapped was spectrophotometrically analyzed using SpectramaxM2 microplate reader (Molecular Devices, Sunnyvale, CA) at360 nm for ATRA. The linear calibration curve for ATRA wasobtained in DMSO in the range of 1.25 lg/ml to 20 lg/ml. Blank,drug-free nanoparticles served as the background control and wedid not detect significant absorbance at the selected wavelengths.Experiments were repeated in triplicate.

EE values were calculated according to the following formula:

EE ð%Þ ¼ ðAmount of drug encapsulated=Amount of drug addedÞ� 100%

The stability of the drug encapsulation in PONC was evaluatedby measuring the EE values of lyophilized ATRA-PONC samplesafter 6-month storage at 4 �C or �20 �C.

The amount of drug released in the medium was determinedindirectly by measuring the residual drug content in the nanopar-ticles. Each nanoparticle sample was dispersed in 1 ml of PBS andtransferred to cellulose ester dialysis bags (MWCO = 10 kDa).Sealed bags containing the samples were placed in 1 l of releasemedium (PBS at pH 7.4) at 37 �C under magnetic stirring at120 rpm. The volume of medium used has exceeded the requiredamount for maintaining the sink conditions (assuming that all0.12 mg ATRA was released, the highest concentration was0.12 lg/ml, far below the aqueous solubility of ATRA, i.e. 29 lg/ml). After 24 h, the entire medium was changed with freshmedium. At each selected time point, the content of a bag was


withdrawn and the residual drug content in the nanoparticleswas spectrophotometrically determined as described in the previ-ous section. ATRA oil-in-water nanoemulsion (free drug dissolvedin oil and not encapsulated in polymer) was used as positive con-trol. Experiments were repeated in triplicate.

2.7. Dispersion and biological stability in serum-enriched medium

To measure the dispersion stability, the nanocarriers (3 mg/ml)were incubated in PBS (pH 7.4) enriched with 10%w/v BSA (i.e.serum enriched PBS) at 37 �C with mild agitation using a bath sha-ker. At predetermined time points aliquots of samples were with-drawn and the nanoparticle size distribution was measured byphoton correlation spectroscopy as described in the previoussection.

To evaluate the biological stability of the encapsulated ATRA,ATRA-PONC was incubated in serum-enriched PBS at 37 �C. At pre-determined time points samples were withdrawn and centrifugeddown, and ATRA-PONC dissolved in DMSO as described in previoussection. The ATRA concentration was spectrophotometrically mea-sured and adjusted to 17.5 lg/ml with cell culture buffer. Colonyformation assay was performed on SKOV-3PR with these samples(please see Section 2.11 for detailed procedure). Free ATRA(17.5 lg/ml) in DMSO served as positive control. Drug-free med-ium and blank PONC served as negative controls. The effects oncolony growth were compared.

2.8. Development and maintenance of highly chemo-resistant cell line(SKOV-3PR)

Chemo-exposed, drug-resistant SKOV-3PR subline was devel-oped by exposing the parental cell line SKOV-3 to increasing con-centrations of paclitaxel in stepwise manner. SKOV-3 cells grownunder standard cell culture conditions were initially exposed tothe lowest paclitaxel concentration (10 nM) for 2 h. After drugexposure the cells were allowed to grow in a drug free medium tillthe next passage. The drug concentration was gradually increasedevery week till 300 nM paclitaxel concentration was reached. Thecells were maintained at paclitaxel containing medium to preservethe acquired chemo-resistance and used within 15 passages.

2.9. Confirmation of acquired drug resistance and tumorigenicity

ABCB1 (or P-glycoprotein) expression is one of the best indica-tors to monitor the progress of chemoresistance developmentupon paclitaxel exposure [18]. Western blotting analysis was per-formed for this purpose. The cells were harvested with m-PERMammalian Protein Extraction Reagent (Pierce, Rockford, IL) sup-plemented with 0.2%v/v protease inhibitor cocktail P8340(Sigma–Aldrich, St. Louis, MO). Aliquots of cell lysate were resolvedon an 8%w/v sodium dodecyl sulfate–polyacrylamide gel (25 lgprotein loading per lane) and electrotransferred onto a polyvinyli-dene difluoride membrane (Immobilon-P 0.45 lm, Millipore, Bille-rica, MA). Membranes were blocked with Tris-buffered salinecontaining 0.1% Tween 20 and 5% dry skim milk powder. ABCB1and b-actin were detected with goat MDR-1 monoclonal antibody(1:400) and b-actin antibody AC-40 (1:3000), respectively, fol-lowed by horse-radish peroxidase conjugated anti-goat IgG(1:7000 for ABCB1, 1:5000 for b-actin) as secondary antibody. Pro-teins were visualized using enhanced chemiluminescence accord-ing to the manufacturers’ instructions (Pierce, Rockford, IL), andband intensities analyzed using Odyssey Imaging System (Li-CorBiosciences, Lincoln, NE).

Functional assays were performed as previously reported toevaluate the drug efflux functions of the cancer cells afterchemo-exposure [19]. Sub-confluent cells were plated onto



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24-well plate under standard cell culture conditions. The attachedcells were incubated with 50 lM of ABCB1 substrate rhodamine-123 for 0–4 h at 37 �C. At predetermined time points, cells werewashed with cold PBS to remove the rhodamine not taken up bythe cells. This was followed by cell lysis with 2%w/v sodium dode-cyl sulfate in PBS. Fluorescence of rhodamine-123 in the cell lysatewas measured at 510 nm excitation and 540 nm emission.

Cellular CD133 is a key marker of tumor-initiating cells of ovar-ian cancer [20]. Its expression was evaluated by immunofluores-cent staining and imaging following the manufacturer’s protocol(AC133, Miltenyi Biotec). Cancer cells were blocked with Fc recep-tor blocking reagent and stained with CD133/1 antibody (1:10) inthe dark at 4 �C. Cells were washed three times, transferred to acover glass and viewed under Axiostar Plus epifluorescence micro-scope (Carl Zeiss, Oberkochen, Germany). Images were capturedwith Insight camera model 8.0 and image analysis conducted usingSpots Advanced imaging software (v.4.6, Diagnostic Instrument,Sterling Heights, MI).

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2.10. Short-term anticancer effects: MTT assays and cell morphology

MTT assays were performed to evaluate the short-term antican-cer activity of ATRA-PONC. Sub-confluent cells were seeded onto96-well microplates and allowed to attach under standard cell cul-ture conditions. After 24 h, the cells were treated with ATRA loadednanocarriers or free drug solution (equivalent to 2.5–50 lg/mldrug). Blank PONC were included as a negative control. After5 days, the cells were incubated with 5 mg/ml 3-(4,5-dimethylthi-azol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (20 llper well) for 2 h. After discarding the medium at the end of incuba-tion, 100 ll of DMSO was added into each well to dissolve the pur-ple formazan dye produced by the metabolically active cells. TheUV absorbance of the colored dye produced was measured at560 nm. Untreated cells served as baseline control. Cell viabilitywas calculated as

Cell viability % ¼ ðAbssample=AbscontrolÞ � 100%

where Abssample and Abscontrol represent the absorbance determinedfor cells treated with nanoparticles and for baseline control(untreated), respectively.

To examine the effects of ATRA-PONC on the cancer cell mor-phology, after 5-day treatment, the cells were washed with PBS,and the floating and adherent cells were collected and incubatedwith the nuclear dye DRAQ5 for 5 min at 37 �C. The stained cellswere imaged as described in the previous section.

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2.11. Colony-forming assay

Colony-forming assay was conducted to evaluate the long-termanti-proliferative effect of ATRA-PONC. 500 Cells were seeded ontoa 35-mm culture dish and allowed to adhere overnight. The cellswere treated for 5 days as this was the time duration for the drugto completely release from the nanocarriers. On the fifth day thedrug-containing medium was replaced with fresh medium andthe cells were allowed to proliferate in a drug-free environment.There was no further medium change because the cell densitywas low and this also avoided disturbance of the cell colonies. Cellsthat were able to proliferate would form viable colonies typicallyin 10–14 days. Cell colonies were fixed with 0.5% solution of meth-ylene blue in methanol. Viable cell colonies, which are defined asthose containing >50 cells per colony, were counted and the resultsnormalized with respect to the untreated control. The experimentwas performed in triplicate.


2.12. Effects of inhibition of drug efflux transport and endocytosis onATRA efficacy

MTT assays were repeated in SKOV-3PR cells in the presence ofABC transporter inhibitors and endocytosis inhibitors. Inhibitorsincluded ABCB1 inhibitor verapamil (10 lM), ABCC2 inhibitorMK-571 (25 lM), ABCG2 inhibitor Ko-143 (0.1 lM), clathrin-medi-ated endocytosis inhibitor amantadine (10 lM) and actin-mediatedendocytosis inhibitor cytochalasin-D (0.1 lM). Inhibitors and nano-carriers containing ATRA (equivalent to 0–10 lg/ml drug) wereadded to the wells with cancer cells and incubated for 5 days at37 �C. Cell viability was evaluated at the end of treatment asdescribed in the MTT section. Untreated cells served as the baseline.Results are presented as% decrease in viability comparing groupswith inhibitors to the corresponding groups without inhibitors.

To confirm that the endocytosis inhibitors were effective inSKOV-3PR at the concentrations used, we prepared FPR648 labeledPONC decorated with folate (folate-PONC) and rhodamine labeledsolid lipid nanoparticles (SLN) as positive controls. Folate-PONCwere prepared similarly to FPR648-PONC as in 2.13 except thatthe carriers included 2%w/w PLGA-mPEG-folate (Polyscitech, WestLafayette, IN, USA). SLN were prepared as in a previous study [19]including 1%w/w 1,2-dipalmitoyl-sn-glycero-3-phosphoethanol-amine-N-(lissamine rhodamine B sulfonyl) (ammonium salt)(Avanti Polar Lipids, Alabaster, Alabama, USA) as fluorescencelabel. SKOV-3PR cells were grown on 96-well plates (20,000 cellsper well) and treated with 5 mg/ml nanoparticles in the presenceof amantadine or cytochalasin-D for 4 h. Cells were washed withcold PBS three times and solubilized with 1% Triton X-100. Cellularuptake of nanoparticles was indicated by the fluorescence mea-sured with spectrophotometer (Spectramax M2, MolecularDevices, Sunnyvale, CA) and results normalized against uninhib-ited control.

PONC were prepared with inclusion of 2% mol ratio folate toform folate-PONC and solid lipid nanoparticles (SLN) were pre-pared as in a previous study [19]. The uptake fluorescence intensi-ties representing nanoparticle uptake were normalized against thecells without exposing to inhibitors or low temperature at 4 �C.

2.13. Nanocarrier uptake study by epifluorescence microscopy

SKOV-3PR cells were allowed to attach on poly-L-lysine pre-treated glass cover slips under normal cell culture conditions.The cells were incubated for 4 h with 5 mg/ml of the nanocarrierformulations at 37 �C. In this time duration the cells were foundto remain viable as they still excluded trypan blue dye. The oilcomponent of nanocarrier was labeled with the oil dye Nile Redand the polymeric component labeled with FPR648-conjugatedPLGA (Polyscitech, West Lafayette, IN, USA). The cells on cover slipswere fixed with 10% buffered formalin and microscopically viewedas described in the previous section.

2.14. Statistical analysis

Results were expressed as means ± SD. The significance of dif-ferences was assessed using Student’s t-test, and p < 0.05 is consid-ered significant unless otherwise specified.

3. Results

3.1. The oil-in-polymer design improved the size and ATRAencapsulation efficiency

Table 1 summarizes the size and zeta potential analysis of theoil-included PONC and the oil-free PLGA-np. All nanocarriers had



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Table 1The size, polydispersity index and zeta potential of ATRA-nanosystems.

ATRA payload (%w/w) Size (nm) PDI Zeta potential (mV)

PLGA-np PONC PLGA-np PONC PLGA-np PONC

0 170.0 ± 11.5 177.7 ± 3.8 0.07 ± 0.02 0.06 ± 0.06 �11.2 ± 2.3 �12.6 ± 1.11 176.7 ± 8.0 171.2 ± 6.1 0.16 ± 0.06* 0.07 ± 0.02 �10.5 ± 1.7 �9.6 ± 0.65 255.4 ± 8.8* 187.6 ± 2.0 0.34 ± 0.02* 0.1 ± 0.02 �11.3 ± 0.6 �11.0 ± 0.8

10 270.8 ± 18.1* 288.0 ± 20.4* 0.28 ± 0.02* 0.38 ± 0.08* �13.0 ± 1.9 �18.5 ± 1.1*

Data expressed as means ± S.D. (N = 3). PDI – polydispersity index; PLGA-np – PLGA nanoparticles; PONC – polymer-oil nanostructured carriers.* p < 0.05 comparing with 0% ATRA payload.



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moderately negative zeta potentials that are typical of PLGA-baseddevices [21]. Regarding the size and polydispersity index (PDI) ofthe nanocarriers, the data showed that they were influenced bytwo factors: ATRA payload and the inclusion of oil into the PLGApolymer. ATRA loading tended to increase the size and PDI values,but this effect was less substantial in PONC than in PLGA-np. At5%w/w ATRA payload, the mean diameter of PONC was below200 nm and its PDI was low (0.10 ± 0.02), both significantly lower(p < 0.05) than PLGA-np (mean diameter = 255.4 nm; PDI = 0.34).The size distribution in Fig. 2A (left panel) indicates broad, biomo-dal size distribution of PLGA-np with 5% payload, suggesting con-siderable nanocarrier aggregation or drug precipitation on thePLGA-np surface. The result shows that PONC could successfullyaccommodate higher ATRA payload up to 5%w/w as compared tothe traditional solid core PLGA-np.

Fig. 2B presents representative TEM images of samples of ATRA-PONC and ATRA-loaded PLGA-np. The PONC has typical uniformsolid nanosphere morphology instead of a vesicle-like structureas in the cases of nanocapsules or liposomes.

As shown in Fig. 2C, PONC (red) showed significantly higherencapsulation efficiency (EE) values than PLGA-np (black) at allthree ATRA payloads (1%, 5%, 10%, all p < 0.05). The EE of PONCexceeded 50% in all three payloads and was higher than PLGA-npto 8.5-fold at 10%w/w ATRA payload. The EE values declined ingeneral as the ATRA payload increased. This decline was moregradual for PONC (73% EE at 1%w/w loading down to 50% EE at10%w/w loading) as compared to PLGA-np.

Table 2 lists the ATRA EE values of ATRA-PONC samples afterlyophilization and storage. The data show that ATRA remained sta-bly entrapped after lyophilization and 6-month storage indicatingno significant drug leakage from PONC (p > 0.05 comparing EE val-ues before and after storage).

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3.2. Oil inclusion increased amorphosity of polymer and dispersion ofentrapped ATRA

Fig. 2D presents the DSC thermograms which reveal informa-tion about the physical states of nanomaterials and entrappeddrugs. ATRA-PONC (bottom, in red) was compared with PLGA-np(top, in green) and ATRA drug powder (middle, in black, as control).The peak pointing downward in the thermogram indicated netenergy absorption by the sample for phase transition. For the poly-mer, when compared with PLGA-np, PONC demonstrated a lessintense peak of melting comparing with the PLGA peak (see bluearrows, peak area ratio of PONC versus PLGA-np � 1:1.8), as wellas depression in the PLGA melting temperature (to �41 �C from47 �C). These findings indicated that the inclusion of oil in PONChas increased the amorphosity of the PLGA polymer matrix inthe nanocarrier, so less energy was needed for the phase transition.For the drug, the melting peak of ATRA control in our measurementwas around 181–186 �C, consistent with the Product informationfrom the supplier (180–182 �C, Sigma Inc.). The ATRA peak was


moderate in PLGA-np and barely detectable in PONC (see redarrows), indicating that the entrapped ATRA molecules in PONCwere in a highly dispersed state so very little energy was neededfor the state change.

3.3. Improved ATRA release profiles and stability in aqueous medium

The ATRA release profiles from PONC (red diamonds), PLGA-np(black dots) and free ATRA in oil nanoemulsion (blue dot line, aspositive control representing unencapsulated drug) are presentedin Fig. 3A. PLGA-np released 69.9% of the ATRA payload in the first30 min and 95.7% in 24 h. This quick release profile was similar tothe unencapsulated ATRA. In comparison, only 7.6% of ATRA pay-load was released in the first 30 min and 48.2% in 24 h from PONC.It continued to gradually release the encapsulated drug up to 88.0%of the payload at the end of the fifth day. Overall, the inclusion ofoil in PONC reduced the burst release of ATRA and resulted in amore controlled, sustained release profile when compared withthe standard PLGA-np and simple nanoemulsion of ATRA. Similarrelease behaviors were observed in release buffer supplementedwith 10% BSA (data not shown in Fig. 3A). The quicker release ofPLGA-np was probably caused by two factors. First, it indicatedthat most of the ATRA of PLGA-np was on or near the nanocarriersurface because this drug was expelled by the tighter structure ofthe PLGA polymer. In comparison, the oil provides the room forthe drug to stay within the polymeric core. Second, the higherPDI of PLGA-np also indicated that there was many smaller parti-cles (also shown in the TEM images in Fig. 2B). The drug couldbe released at a much faster rate from these smaller size carriers.

To evaluate the dispersion stability of PONC especially in thepresence of high serum content (10%w/v BSA), PONC was incubatedin serum-enriched PBS at 37 �C to evaluate their physical stability(Fig. 3B). We did not notice a significant increase in the size of PONCwhich indicate particle aggregation or swelling after 24 h (p > 0.05).There was an increase in the polydispersity index (the numbersabove the bars), but the highest number still remained smaller than0.25, indicating some degree of aggregation but it remained moder-ate. Overall, the result confirmed the dispersion stability of PONC inaqueous environment with high serum protein level.

Fig. 3C summarizes the anticancer activities of the ATRAextracted from ATRA-PONC incubated in serum-enriched PBS at37 �C up to 72 h. The goal of this experiment is to verify that theATRA remained therapeutically active after encapsulation byPONC. The ATRA concentrations in all groups were thus spectro-photometrically (at 360 nm wavelength) adjusted to the same con-centration (17.5 lg/ml) to compare the therapeutic activities. Thehigher the activities the fewer colonies were formed. It was shownthat no significant loss (p > 0.05) in the anticancer activities wasobserved when compared with the freshly prepared ATRA solution.The data show that the anticancer activities of the ATRA remainedunchanged after encapsulation in PONC and exposed to serum-enriched buffer for 72 h.



Fig. 2. Physico-chemical characterization of ATRA-loaded nanocarriers. (A) Size distribution of PLGA nanoparticles (PLGA-np) and polymer-oil nanostructured carriers(PONC) with 5%w/w ATRA payload. (B) Transmission electron microscope (TEM) images of PONC and PLGA-np. The top row shows the low magnification images (5000–6000�) and bottom row shows the high magnification images (15,000–20,000�). Nanoparticles contain 5%w/w ATRA. Scale bar indicates 200 nm for the low magnificationimages and 100 nm for the high magnification images, respectively. (C) Comparing the ATRA encapsulation efficiency between PLGA-np and PONC at different expected drugpayload. �p < 0.05. (D) Differential scanning calorimetry (DSC) thermograms of ATRA-PONC, ATRA loaded PLGA-np and ATRA drug powder are compared. (For interpretationof the references to color in this figure legend, the reader is referred to the web version of this article.)



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Please cite this article in press as: M. Narvekar et al., A new nanostructured carrier design including oil to enhance the pharmaceutical properties of ret-inoid therapy and its therapeutic effects on chemo-resistant ovarian cancer, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2014.04.014


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Table 2Effects of storage on ATRA encapsulation.

Conditions Encapsulation efficiency (%)

Before lyophilizationa storage 73.5 ± 3.7b

6 Months at 4 �C 69.9 ± 1.56 Months at �20 �C 70.2 ± 0.9

a �2% (w/v) sucrose as a lyoprotectant.b Means ± S.D., N = 3.

Fig. 3. Evaluation of the behaviors of ATRA-PONC in aqueous environment.Nanocarriers were incubated in phosphate buffered saline enriched with 10%bovine serum albumin at 37 �C. (A) Drug release profiles in stirred, sinkcondition. ATRA-PONC (red diamond), ATRA loaded PLGA-np (black filled circle)and ATRA in simple oil nanoemulsion (blue dotted line) are compared. Data areexpressed as mean ± S.D. (N = 3) (B) Dispersion stability: the effects on the sizeof ATRA-PONC are shown. Data are reported as mean ± S.D. (N P 3). Nostatistical significance observed comparing various time-points to nanocarrierbefore incubation (i.e. 0 h). The number above each bar is the correspondingpolydispersity index. (C) Biological stability: the effects on anticancer activity ofATRA were evaluated with colony formation assay. The ATRA extracted fromATRA-PONC using DMSO was compared to fresh ATRA solution in DMSO. Datashown are normalized against cells treated with drug free DMSO and expressedas% viable colonies. Data are reported as mean ± S.D. (N = 3). (For interpretationof the references to color in this figure legend, the reader is referred to the webversion of this article.)



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3.4. Development of SKOV-3PR ovarian cancer subline andconfirmation of acquired multidrug resistance phenotype andtumorigenic marker expression

As shown in Fig. 4A, ABCB1 (P-glycoprotein) expression inSKOV-3PR cells exposed up to 300 nM paclitaxel was substantiallyelevated when compared with the chemo-naive cell line SKOV-3and cells exposed to lower paclitaxel level (30 nM). To confirm ifthis translated into increased drug efflux, a functional assay withthe rhodamine 123 as drug transporter substrate as describedwas performed [22]. Significantly less fluorescent drug wasretained by SKOV-3PR cells than the chemo-naive SKOV-3 cells(Fig. 4B), confirming the elevated drug efflux activity in thechemo-exposed SKOV-3PR subline.

Fig. 4C shows the representative images comparing the tumor-initiating cell marker CD133 expression between SKOV-3 (leftpanel) and SKOV-3PR (right panel). CD133 was shown in greenand the cell nuclei in blue. Noticeably higher expression wasobserved in SKOV-3PR cells, indicating their increased tumorigenicpotential after repeated paclitaxel exposure.

3.5. Enhanced ATRA anticancer efficacy after PONC encapsulation

The short-term effects of PONC were compared with free ATRAin SKOV-3 and SKOV-3PR cells using MTT assays. As shown inFig. 5A, ATRA-PONC were significantly more toxic than free ATRAin all drug concentrations tested in both chemo-naive andchemo-exposed cells (p < 0.05). The activities of ATRA-PONC wereapparently not affected by whether the cells were chemo-exposedor not (p > 0.05 comparing the two ATRA-PONC curves). BlankPONC showed no significant cell toxicity in low concentrationrange and only modest cell toxicity (>80% viability) in high concen-trations, indicating that the substantial enhancement of the ATRA’santicancer activities after encapsulation in PONC was not causedby the inherent carrier toxicity, but more likely derived from thenanostructured oil-in-polymer design of PONC.

Representative images of the cell morphology after treatmentwere shown in Fig. 5B. The DNA was stained with DNA-specificdye DRAQ5 (shown in blue) for visualization. With ATRA-PONCtreatment, the majority of SKOV-3PR cells showed signs of apopto-sis such as chromatin condensation and nuclear fragmentation(indicated by red arrows). No such activities were observed inblank PONC.

Fig. 5C presents the result of colony formation assay performedon SKOV-3PR. In all ATRA concentrations except 0.25 lg/ml, ATRA-PONC was significantly more effective than free ATRA (top panel).The IC50 of ATRA-PONC was around 2 lg/ml and that of free ATRAwas 17.5 lg/ml, nearly an 8-fold reduction. At higher ATRA con-centrations (17.5 lg/ml), no cell colony could be formed from pro-liferation of individual cells in ATRA-PONC group, while numeroussmall but visible cell colonies were slowly formed in the free ATRAgroup (bottom panel).

3.6. Pharmacological inhibition study of ATRA-PONC

Inhibition study was performed to understand what caused theobserved improvements of ATRA-PONC over free ATRA. It shouldbe noted that the concentrations of all inhibitors used did notcause significant reduction in cell viability (indicated by MTT assayand by dye exclusion) or visible changes in cell morphology whenused alone. Fig. 6A and B present the changes in anticancer activity(as% decrease in cancer cell viability) in SKOV-3PR after concurrentinhibition of various drug efflux pathways (Fig. 6A) or endocytosispathways (Fig. 6B). A decrease indicates enhancement of antican-cer effectiveness by blocking the tested pathway. Fig. 6A showsthat out of the three drug efflux inhibitors (verapamil for ABCB1,



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Fig. 4. Chemoresistance and tumorigenic marker expression in chemo-exposed SKOV-3PR cells. (A) Western blot analysis of ABCB1 (P-glycoprotein) expression in SKOV-3control (lane 1), SKOV-3PR after 30 nM of paclitaxel treatment (lane 2), SKOV-3PR after 300 nM of paclitaxel treatment (lane 3). b-Actin was included as internal control. (B)Functional assay. The increased drug efflux from SKOV-3 PR is shown as lower rhodamine-123 fluorescence intensity. Results are expressed as mean ± S.D. (N P 3). (C)Increased cellular expression of CD133 (green) after paclitaxel exposure. Cell nuclei were shown in blue. (For interpretation of the references to color in this figure legend, thereader is referred to the web version of this article.)



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MK-571 for ABCC2 and Ko-143 for ABCG2), only MK-571 moder-ately but significantly enhanced the ATRA effectiveness, but theenhancements were comparable between the free ATRA groupand the ATRA-PONC group.

Fig. 6B shows that the two endocytosis inhibitors (amantadinefor clathrin-dependent endocytosis, cytochalasin-D for actin-dependent endocytosis) both did not significantly affect the activ-ities of all groups, and the differences between the free ATRA groupand ATRA-PONC were minimal. To verify that the two endocytosisinhibitors were effective in blocking the endocytosis pathways inSKOV-3PR at the concentrations used, we prepared FPR648 labeledPONC decorated with folate and rhodamine labeled solid lipidnanoparticles as positive controls. Previous studies have shownthat folate decorated nanoparticles and solid lipid nanoparticleswere taken up by clathrin-mediated endocytosis and actin-medi-ated endocytosis, respectively [19,23]. Our data (Fig. 6C) show thatthe endocytosis of these two nanoparticles into SKOV-3PR cells wassignificantly blocked (comparable to uptake at 4 �C), showing thatthe SKOV-3PR cells were sensitive to the two inhibitors. Hence, theresults indicated that the tested drug efflux and endocytosis path-ways were not the key factors that contributed to the enhancedanticancer activities of ATRA-PONC over free ATRA.

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3.7. Increased uptake of the drug/oil components of ATRA-PONC

Fig. 7A and B show the distribution of PLGA (with FPR-648) andoil (with lipid labeling agent Nile Red), respectively, in SKOV-3PR

cells. Only low intracellular PLGA level was observed in the viablecells treated with ATRA-PONC, indicating weak internalization ofthe nanocarriers (Fig. 7A). In contrast, high Nile Red/oil level wasobserved within the ATRA-PONC treated cells (Fig. 7B). In compar-ison, the cells treated with oil nanoemulsion carrying same quan-tity of the oil labeling agent showed a visibly lower intracellularfluorescence. The efficiency of dye/oil uptake was evidently higherwhen delivered by the PONC system as compared to the simple oilnanoemulsion.

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4. Discussion

ATRA offers a highly promising alternative option for cancertherapy considering its unique anti-proliferative, anti-differentia-tion and apoptotic activities in various cancer cell sub-popula-tions including tumor-initiating cells [24,25]. The key challengeto its clinical translation lies in the design of an efficient and safemeans to solubilize and deliver this highly lipophilic drug(logP = 6.3). The present study first focuses on solving the issuesderived from the high lipophilicity of ATRA, specifically the lowpayload and stability of ATRA entrapment in a nanosystem, using


the proposed oil-in-polymer nanostructured design (Fig. 1). Thesecond objective is to validate this nanosystem design in a moretherapeutically relevant cell model, specifically, the chemo-exposed, chemo-resistant and tumorigenic ovarian cancer cellsub-population that is supposed to be the main target of ATRAtherapy. Overall, our data show that these two goals have beenaccomplished.

Not only the absolute ATRA payload, in our previous studieswith standard PLGA-np, we showed that even after successfulapparent entrapment, the ATRA molecules tended to distributenear the nanoparticle surface which led to nanoparticle aggrega-tion, size growth, and drug burst releases and precipitation [14].These issues were not observed in the current PONC system. Thesize of PONC did not significantly increase up to 5% ATRA payload(Fig. 2C), and the ATRA EE of PONC was higher than the standardPLGA-np up to over 8-fold at 10% loading. According to the DSCthermograms (Fig. 2D), the oil inclusion has evidently increasedthe amorphosity of the polymeric matrix. The ATRA thus becamemore uniformly distributed and more stably entrapped as reflectedin the storage data in Table 2. This oil-in-polymer design is compa-rable to the nanostructured lipid carriers, which use oil to intro-duce similar ‘‘nanostructure’’ in the nanoparticle core made insolid lipids instead of polymer. Both designs similarly resulted inimproved drug encapsulation and dispersion within the nanosys-tem [26].

As ATRA-PONC is intended for systemic cancer therapy, it isimportant to know if the more efficient and stable ATRA entrap-ment could translate into good stability in a serum protein-enriched aqueous environment. This working hypothesis wassupported by various findings. Controlled, sustained release profileof ATRA from PONC as compared to PLGA-np was obtained. PONCalso demonstrated good dispersion stability (Fig. 3B) and biologicalstability (Fig. 3C) after 72 h incubation. These improvements arecrucial considering the high toxicity and low stability of free ATRA[27]. ATRA is associated with a high incidence of systemic toxicitiessuch as mucositis, myositis, hyperbilirubinemia, as well as otherpotentially lethal symptoms [6,28,29]. It is also easily metabolized(e.g. by CYP26) or oxidized [27,30]. The release profile and stabilityof ATRA-PONC is desirable for lowering the risks of these events.

ATRA-PONC was studied in paclitaxel-exposed and paclitaxel-resistant ovarian cancer cells as paclitaxel is the current first-lineoption for ovarian cancer treatment [1,31]. The SKOV-3PR sublinedeveloped was able to withstand paclitaxel level as high as300 nM and has developed the classical multidrug resistance phe-notype as indicated by increased ABCB1 expression (Fig. 3A) andefflux of the standard ABCB1 substrate rhodamine-123 (Fig. 3B).As it was reported that ATRA is effective against cancer cells withhigh tumorigenicity, we also evaluated the expression of CD133,which has been identified as a defining marker of the tumor-initi-



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Fig. 5. Anticancer effects of ATRA-PONC. (A) Evaluation of cytotoxic effects in SKOV-3 (left) and SKOV-3PR cells (right) using MTT assay. Assay was performed 5 days afterinitiation of treatment. ATRA-PONC was compared to free ATRA (in simple oil nanoemulsion as positive control) and blank PONC. Cell viability was normalized againstuntreated cells and expressed as% cell viability (as means ± S.D., N = 3). (B) Cell morphology after treatment with blank PONC (left) or ATRA-PONC (right). SKOV-3PR cells weretreated and examined 5 days later. DNA was stained with DRAQ5 for microscope examination (shown in blue). Red arrows indicate chromatin condensation and nucleifragmentation which are key features of apoptosis. (C) Evaluation of anti-tumorigenic activity using colony-formation assay. 500 treated SKOV-3PR cells were seeded onculture dishes to determine if the cells maintained the tumorigenic capability to grow into viable colonies. Results are presented as mean + S.D. (N P 3, in top panel).� indicates p < 0.05. Bottom panel shows the representative image of the stained cell colonies. (For interpretation of the references to color in this figure legend, the reader isreferred to the web version of this article.)



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ating cell sub-population in ovarian cancer [20,32]. Fig. 3C shows anoticeable increase in its expression after paclitaxel treatment,which agrees with a recent report on another ovarian cancer cell


line A2780 similarly exposed to paclitaxel [31]. The resultingSKOV-3PR subline thus provides a valuable tool for more accurateassessment of the actual efficacy of ATRA-PONC.



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Fig. 6. Effects of inhibitors of (A) ABC drug efflux transporters, (B) endocytosis on anticancer activities and (C) verification of endocytosis activities. The viabilities of SKOV-3PR

cells with or without concurrent use of the inhibitors are compared and expressed as% decrease in cell viability after inhibition. For (C), the uptake fluorescence intensitiesrepresenting nanoparticle uptake in the presence of endocytosis inhibitors (amantandine or cytochalasin-D) or at low temperature at 4 �C were normalized against the cellswithout exposing to inhibitors. All results are presented as mean ± S.D. (N = 3). (For interpretation of the references to color in this figure legend, the reader is referred to theweb version of this article.)

Fig. 7. Images showing uptake of PLGA and oil into SKOV-3PR. (A) Cellular uptake of PLGA. Cells were treated with ATRA-PONC containing fluorescent FPR648-conjugatedPLGA (in green, right). Non-fluorescent PONC (left) image is included to show the baseline fluorescence. (B) Cellular uptake of oil stained with fluorescent Nile Red dye (inred). Cells treated with labeled ATRA-PONC (right) were compared with those treated with blank PONC + dye/drug in simple oil nanoemulsion (left). (For interpretation of thereferences to color in this figure legend, the reader is referred to the web version of this article.)



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Using this cell model, we demonstrated significantly enhancedefficacy of ATRA when delivered by PONC as compared to the freedrug (Fig. 5A). The enhancement in anticancer effects was observedin both SKOV-3 and SKOV-3PR cells, proving that ATRA-PONC canbe effective in both chemo-naive and chemo-exposed forms ofovarian cancers. The primary form of cytotoxicity induced byATRA-PONC was apoptosis as indicated by the cell morphology inFig. 5B. This is consistent with the documented ATRA anticancermechanisms [5].

Besides apoptosis, ATRA is particularly known for its anti-tumorigenic effects for reducing the risk of cancer cell repopulationafter chemotherapy [31]. This aspect was studied by performingcolony formation assays which measure the capability of individ-ual SKOV-3PR cells proliferating into viable colonies. Fig. 5C showsthat ATRA-PONC was nearly nine times more potent than the ATRAfree drug (IC50: 2 lg/ml versus 17.5 lg/ml) against SKOV-3PR cells.


To sum up, by packaging ATRA in PONC system, it is possible tosubstantially enhance both of the short-term apoptotic effectsand long-term anti-tumorigenic effects against the cancer cellsub-population that are practically refractory to standardchemotherapy.

The next logical question is what led to the observed enhance-ment of ATRA anticancer effects by PONC. Nanocarriers can over-come drug resistance by various mechanisms such assuppressing the cellular drug efflux, increasing endocytosis, orenhancing passive drug permeation into the cancer cells [33].Understanding this aspect may allow better optimization of theanticancer efficacy of a nanotherapeutic. Our data in Fig. 6A showno significant differences comparing ATRA-PONC with the corre-sponding free ATRA groups receiving different drug efflux inhibi-tors. Hence, the higher anticancer effects of ATRA-PONC were notcaused by inhibiting the drug efflux transporters like many other



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Fig. 8. Proposed cellular mechanism leading to enhanced anticancer effects of ATRAwhen delivered by PONC. (For interpretation of the references to color in this figurelegend, the reader is referred to the web version of this article.)



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nanosystems, e.g. block polymer micelles [34]. Inhibiting the clath-rin-mediated (amantadine) and actin-mediated endocytosis (cyto-chalasin-D) also did not affect the efficacy of ATRA-PONC (Fig. 6B).It can be inferred that PONC did not potentiate the ATRA activitiesby promoting endocytosis-mediated uptake.

We then used fluorescent-labeled polymeric (PLGA) and the oil(Captex) components to investigate how SKOV-3PR cells handleATRA-PONC. It was found that only a low level of PLGA could enterthe cytoplasm (Fig. 7A), which agrees with the endocytosis inhibi-tion data. Meanwhile, strong uptake of the oil-labeling agent fromPONC was observed (Fig. 7B). The findings suggest that PONC pro-moted drug uptake into the chemo-resistant cells by increasing thepermeation of the released drug molecules without inducing sig-nificant endocytosis. Meanwhile, much lower cellular dye/oil levelwas observed in the simple oil nanoemulsion group. The loadeddye was likely released too quickly without control (as in therelease profile in Fig. 3) and this was prone to precipitation in anon-sink condition. Moreover, we also noticed that without beingstabilized by the polymer, the oil droplets tended to coalescenceinto larger ones during incubation which may further compromisethe drug uptake efficiency.

Based on the findings, we propose the model in Fig. 8. With thehelp of the oil-in-polymer nanostructured design of PONC, theencapsulated lipophilic molecules such as ATRA or Nile Red are sta-bilized in a highly dispersed (possibly solubilized) state within theamorphous polymeric matrix. These entrapped drug molecules(which may be associated with oil) can be gradually released fromthe PONC core in a controlled, sustained manner with low risk ofdrug precipitation. After release, the drug/oil are in finely dividedform which facilitate efficient permeation across the lipid bilayermembrane of the cancer cells to achieve high intracellular drugconcentration. Questions still remain about how the drug/oil cantransverse the distance from the carrier to the cells. It is possiblethat drug/oil droplets or molecules were transferred from thePONC to the cells when the PONC were in contact with the cell sur-faces. More extensive studies of PONC loading other lipophilicdrugs will help validate this mechanism. Another limitation aboutthis study is that the works are focused in cell cultures. An in vivostudy is in progress to provide further proof of concept for thispromising nanotherapy.

5. Conclusions

The observed drug entrapment and uptake enhancement mech-anisms of PONC are apparently not drug-specific and limited to


ATRA. PONC thus may serve as a promising generic nanodeliverydevice for other lipophilic, poorly water-soluble anticancer drugs.In fact, our group has been studying the use of PONC for docetaxeldelivery and equally encouraging results were obtained (early datapresented in [35]). Also, this mechanism should be able to be com-plemented by the more cell-specific active mechanism. We are inthe progress to include active-targeting moieties to furtherenhance the cancer specificity of the ATRA-PONC therapy. At thisstage, it is safe to conclude that a new nanosystem design for effi-cient, stable entrapment and controlled delivery of ATRA has beendeveloped, and the resulting ATRA-PONC has demonstratedencouraging potential to tackle the chemo-exposed, chemo-resis-tant and tumorigenic sub-population of ovarian cancer, one ofthe most lethal and poorly responsive malignant diseases. In amore general sense, this study also exemplifies the use of the oil-in-polymer hybrid strategy to conveniently improve the pharma-ceutical and therapeutic properties of tough-to-deliver lipophilicanticancer compounds. This is significant as the majority of therecently developed anticancer drug molecules are similarly over-lipophilic.

Acknowledgements

The authors thank Purac (Gorinchem, Netherlands) and Abitec(Janesville, WI, USA) for the chemicals they donated to our lab.We also thank Dr. M. Illies at Temple University School of Phar-macy for granting us the access to the instruments for the DSCexperiments. The study was supported by Drug Discovery ResearchInitiative, Temple University, Philadelphia.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.ejpb.2014.04.014.

References

[1] R.F. Ozols, Systemic therapy for ovarian cancer: current status and newtreatments, Semin. Oncol. 33 (2006) S3–S11.

[2] C. Tian, C.B. Ambrosone, K.M. Darcy, T.C. Krivak, D.K. Armstrong, M.A.Bookman, W. Davis, H. Zhao, K. Moysich, H. Gallion, J.A. DeLoia, Commonvariants in ABCB1, ABCC2 and ABCG2 genes and clinical outcomes amongwomen with advanced stage ovarian cancer treated with platinum and taxane-based chemotherapy: a gynecologic oncology group study, Gynecol. Oncol. 124(2012) 575–581.

[3] G.Y. Hong, Y.I. Jeong, S.J. Lee, E. Lee, J.S. Oh, H.C. Lee, Combination of paclitaxel-and retinoic acid-incorporated nanoparticles for the treatment of CT-26 coloncarcinoma, Arch. Pharm. Res. 34 (2011) 407–417.

[4] F. Ravandi, Acute promyelocytic leukemia can be treated successfully withoutcytotoxic chemotherapy, Oncology 25 (2011) 741–743.

[5] Siddikuzzaman, C. Guruvayoorappan, V.M. Berlin Grace, All trans retinoic acidand cancer, Immunopharmacol. Immunotoxicol. 33 (2011) 241–249.

[6] R. Masetti, F. Vendemini, D. Zama, C. Biagi, P. Gasperini, A. Pession, All-transretinoic acid in the treatment of pediatric acute promyelocytic leukemia,Expert Rev. Anticancer Ther. 12 (2012) 1191–1204.

[7] A. Chinsriwongkul, P. Chareanputtakhun, T. Ngawhirunpat, T. Rojanarata, W.Sila-on, U. Ruktanonchai, P. Opanasopit, Nanostructured lipid carriers (NLC) forparenteral delivery of an anticancer drug, AAPS PharmSciTech 13 (2012) 150–158.

[8] Y. Li, X.R. Qi, Y. Maitani, T. Nagai, PEG–PLA diblock copolymer micelle-likenanoparticles as all-trans-retinoic acid carrier: in vitro and in vivocharacterizations, Nanotechnology 20 (2009) 055106.

[9] S.J. Um, S.Y. Lee, E.J. Kim, H.S. Han, Y.M. Koh, K.J. Hong, H.S. Sin, J.S. Park,Antiproliferative mechanism of retinoid derivatives in ovarian cancer cells,Cancer Lett. 174 (2001) 127–134.

[10] Chinsriwongkul, P. Opanasopit, T. Ngawhirunpat, N. Chareansriwilaiwat, W.Sila-On, U. Ruktanonchai, Physicochemical properties of lipid emulsionsformulated with high-load all-trans-retinoic acid, PDA J. Pharm. Sci. Technol.61 (2007) 461–471.

[11] J. Su, N. Zhang, P.C. Ho, Evaluation of the pharmacokinetics of All-Trans-Retinoic Acid (ATRA) in wistar rats after intravenous administration of ATRAloaded into tributyrin submicron emulsion and its cellular activity on caco-2and HepG2 cell lines, J. Pharm. Sci. 97 (2008) 2844–2853.




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[12] S. Kawakami, P. Opanasopit, M. Yokoyama, N. Chansri, T. Yamamoto, T. Okano,F. Yamashita, M. Hashida, Biodistribution characteristics of all-trans retinoicacid incorporated in liposomes and polymeric micelles following intravenousadministration, J. Pharm. Sci. 94 (2005) 2606–2615.

[13] A. Chinsriwongkul, P. Opanasopit, T. Ngawhirunpat, T. Rojanarata, W. Sila-On,U. Ruktanonchai, Oleic acid enhances all-trans retinoic acid loading in nano-lipid emulsions, PDA J. Pharm. Sci. Technol. 64 (2010) 113–123.

[14] M. Narvekar, H.Y. Xue, H.L. Wong, A novel hybrid delivery system: polymer-oilnanostructured carrier for controlled delivery of highly lipophilic drug all-trans-retinoic acid (ATRA), Int. J. Pharm. 436 (2012) 721–731.

[15] B. Thibault, M. Castells, J.P. Delord, B. Couderc, Ovarian cancermicroenvironment: implications for cancer dissemination andchemoresistance acquisition, Cancer Metastasis Rev. (2013) (Epub ahead ofprint).

[16] A. Alisi, W.C. Cho, F. Locatelli, D. Fruci, Multidrug resistance and cancer stemcells in neuroblastoma and hepatoblastoma, Int. J. Mol. Sci. 14 (2013) 24706–24725.

[17] R. Karki, B.L. Seagle, W. Nieves-Neira, S. Shahabi, Taxanes in combination withbiologic agents for ovarian and breast cancers, Anticancer Drugs (2013) (Epubahead of print).

[18] S. Hille, D.T. Rein, M. Riffelmann, R. Neumann, J. Sartorius, A. Pfützner, C.M.Kurbacher, T. Schöndorf, M. Breidenbach, Anticancer drugs induce MDR1 geneexpression in recurrent ovarian cancer, Anticancer Drugs 17 (2006) 1041–1044.

[19] H.L. Wong, R. Bendayan, A.M. Rauth, H.Y. Xue, K. Babakhanian, X.Y. Wu, Amechanistic study of enhanced doxorubicin uptake and retention in multidrugresistant breast cancer cells using a polymer–lipid hybrid nanoparticle system,J. Pharmacol. Exp. Ther. 317 (2006) 1372–1381.

[20] M.D. Curley, V.A. Therrien, C.L. Cummings, P.A. Sergent, C.R. Koulouris, A.M.Friel, D.J. Roberts, M.V. Seiden, D.T. Scadden, B.R. Rueda, R. Foster, CD133expression defines a tumor initiating cell population in primary humanovarian cancer, Stem Cells 27 (2009) 2875–2883.

[21] F. Danhier, E. Ansorena, J.M. Silva, A. Le Breton, V. Préat, PLGA-basednanoparticles: an overview of biomedical applications, J. Control. Release161 (2012) 505–522.

[22] Z. Sulová, D. Macejová, M. Seres, J. Sedlák, J. Brtko, A. Breier, Combinedtreatment of P-gp-positive L1210/VCR cells by verapamil and all-trans retinoicacid induces down-regulation of P-glycoprotein expression and transportactivity, Toxicol. In Vitro 22 (2008) 96–105.

[23] W.L. Suen, Y. Chau, Size-dependent internalisation of folate-decoratednanoparticles via the pathways of clathrin and caveolae-mediatedendocytosis in ARPE-19 cells, J. Pharm. Pharmacol. 66 (2014) 564–573.

[24] E.E. Oldridge, H.F. Walker, M.J. Stower, M.S. Simms, V.M. Mann, A.T. Collins, D.Pellacani, N.J. Maitland, Retinoic acid represses invasion and stem cell


phenotype by induction of the metastasis suppressors RARRES1 and LXN,Oncogenesis 2 (2013) e45.

[25] L.C. Chee, J. Hendy, L.E. Purton, G.A. McArthur, ATRA and the specific RARaagonist, NRX195183, have opposing effects on the clonogenicity of pre-leukemic murine AML1-ETO bone marrow cells, Leukemia 27 (2013) 1369–1380.

[26] M.A. Iqbal, S. Md, J.K. Sahni, S. Baboota, S. Dang, J. Ali, Nanostructured lipidcarriers system: recent advances in drug delivery, J. Drug Target. 20 (2012)813–830.

[27] G. Clemens, K.R. Flower, P. Gardner, A.P. Henderson, J.P. Knowles, T.B. Marder,A. Whiting, S. Przyborski, Design and biological evaluation of syntheticretinoids: probing length vs. stability vs. activity, Mol. BioSyst. 9 (2012)3124–3134.

[28] W. Yu, C.M. Burns, All-trans retinoic acid-induced focal myositis, synovitis, andmononeuritis, J. Clin. Rheumatol. 15 (2009) 358–360.

[29] B.C. de-Medeiros, E. Strapasson, R. Pasquini, C.R. de-Medeiros, Effect of all-trans retinoic acid on newly diagnosed acute promyelocytic leukemiapatients: results of a Brazilian center, Braz. J. Med. Biol. Res. 31 (1998)1537–1543.

[30] J.L. Armstrong, G.A. Taylor, H.D. Thomas, A.V. Boddy, C.P.F. Redfern, G.J. Veal,Molecular targeting of retinoic acid metabolism in neuroblastoma: the role ofthe CYP26 inhibitor R116010 in vitro and in vivo, Br. J. Cancer 96 (2007) 1675–1683.

[31] X. Han, F. Du, L. Jiang, Y. Zhu, Z. Chen, Y. Liu, T. Hong, T. Wang, Y. Mao, X. Wu,I.C. Bruce, J. Jin, X. Ma, D. Hua, A2780 human ovarian cancer cells withacquired paclitaxel resistance display cancer stem cell properties, Oncol. Lett.6 (2013) 1295–1298.

[32] R. Strauss, Z.Y. Li, Y. Liu, I. Beyer, J. Persson, P. Sova, T. Moller, S. Pesonen, A.Hemminki, P. Hamerlik, C. Drescher, N. Urban, J. Bartek, A. Lieber, Analysis ofepithelial and mesenchymal markers in ovarian cancer reveals phenotypicheterogeneity and plasticity, PLoS ONE (2011), http://dx.doi.org/10.1371/journal.pone.0016186.

[33] H.L. Wong, R. Bendayan, A.M. Rauth, Y. Li, X.Y. Wu, Chemotherapy withanticancer drugs entrapped into solid lipid nanoparticles, Adv. Drug Deliv. Rev.59 (2007) 491–504.

[34] A. Cambón, J. Brea, M.I. Loza, C. Alvarez-Lorenzo, A. Concheiro, S. Barbosa, P.Taboada, V. Mosquera, Cytocompatibility and P-glycoprotein inhibition ofblock copolymers: structure–activity relationship, Mol. Pharm. 10 (2013)3232–3241.

[35] N.T. Tran, H.Y. Xue, M. Narvekar, S. Liu, H.L. Wong, Development andevaluation of a new nanostructured carrier for docetaxel delivery toadvanced stage cancers. AAPS Meeting 2013, W4005. <http://abstracts.aaps.org/published/ContentInfo.aspx?conID=44489>.


http://dx.doi.org/10.1371/journal.pone.0016186

http://dx.doi.org/10.1371/journal.pone.0016186

http://www.abstracts.aaps.org/published/ContentInfo.aspx?conID=44489

http://www.abstracts.aaps.org/published/ContentInfo.aspx?conID=44489


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