Biocompatible Lipid Nanoparticles as Carriers To ... - ACS Publications

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Biocompatible Lipid Nanoparticles as Carriers To Improve Curcumin Efficacy in Ovarian Cancer Treatment Maria Luisa Bondì,*,† Maria Rita Emma,‡ Chiara Botto,§ Giuseppa Augello,‡ Antonina Azzolina,‡ Francesca Di Gaudio,∥ Emanuela Fabiola Craparo,§ Gennara Cavallaro,§ Dimcho Bachvarov,⊥,# and Melchiorre Cervello‡ †

Istituto per lo Studio dei Materiali Nanostrutturati (ISMN), CNR, U.O.S. Palermo, via Ugo La Malfa, 153, 90146 Palermo, Italy Istituto di Biomedicina ed Immunologia Molecolare (IBIM) “Alberto Monroy”, CNR, via Ugo La Malfa 153, 90146 Palermo, Italy § Laboratorio di Polimeri Biocompatibili, Dipartimento di Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche (STEBICEF), Via Archirafi 32, 90123 Palermo, Italy ∥ Dipartimento di Biopatologia e Biotecnologie Mediche (DIBIMED), Scuola di Medicina e Chirurgia, via Del Vespro 129, 90127 Palermo, Italy ⊥ Cancer Research Centre, Hôpital L’Hotel-Dieu de Québec, Centre Hospitalier Universitaire de Québec, Quebec City, Quebec, Canada # Department of Molecular Medicine, Faculty of Medicine, Laval University, Quebec City, Quebec, Canada ‡

ABSTRACT: Curcumin is a natural molecule with proved anticancer efficacy on several human cancer cell lines. However, its clinical application has been limited due to its poor bioavailability. Nanocarrier-based drug delivery approaches could make curcumin dispersible in aqueous media, thus overtaking the limits of its low solubility. The aim of this study was to increase the bioavailability and the antitumoral activity of curcumin, by entrapping it into nanostructured lipid carriers (NLCs). For this purpose here we describe the preparation and characterization of three kinds of curcumin-loaded NLCs. The nanosystems allowed the achievement of a controlled release of curcumin, the amounts of curcumin released after 24 h from Compritol− Captex, Compritol−Miglyol, and Compritol NLCs being, respectively, equal to 33, 28, and 18% w/w on the total entrapped curcumin. Considering the slower curcumin release profile, Compritol NLCs were chosen to perform successive in vitro studies on ovarian cancer cell lines. The results show that curcumin-loaded NLCs maintain anticancer activity, and reduce cell colony survival more effectively than free curcumin. As an example, the ability of A2780S cells to form colonies was decreased after treatment with 5 μM free curcumin by 50% ± 6, whereas, at the same concentration, the delivery of curcumin with NLC significantly (p < 0.05) inhibited colony formation to approximately 88% ± 1, therefore potentiating the activity of curcumin to inhibit A2780S cell growth. The obtained results clearly suggest that the entrapment of curcumin into NLCs increases curcumin efficacy in vitro, indicating the potential use of NLCs as curcumin delivery systems. KEYWORDS: nanostructured lipid carriers, curcumin, drug release, cancer, epithelial ovarian cells

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hydrolysis that occurs rapidly above neutral pH.17 Moreover, one of the major limits related to curcumin studies involves very low serum levels, due to poor intestinal absorption, rapid metabolism, and rapid systemic elimination following oral use. In fact, the curcumin is first biotransformed to dihydrocurcumin and tetrahydrocurcumin, and subsequently converted to monoglucuronide conjugates. After oral administration curcumin is metabolized primarily by reduction and conjugation. Reduction can already occur in the gut by the NADPHdependent reductase. The reduced metabolites, especially tetraand hexahydrocurcumin, represent the largest portion of curcumin metabolites and exist almost exclusively as conjugates with glucuronic acid and sulfate in plasma. With few exceptions their biological activities are strongly reduced compared to

INTRODUCTION

Recent studies on the molecular basis of epithelial ovarian cancer (EOC) development and progression create new opportunities to develop anticancer molecules that would affect specific metabolic pathways and decrease side effects of conventional treatments. In this context, the delivery of anticancer molecules into cancer cells or tissues by nanocarriers may provide a new paradigm in cancer treatment.1−5 Curcumin, a natural compound, extracted from the rhizome of Curcuma longa, possesses various pharmacological activities such as antiamyloid, antioxidant, anti-inflammatory, and anticancer properties. In the literature it is widely reported that curcumin induces apoptosis and/or cell cycle arrest in different human cancer cell lines (colorectal, breast, lung, prostate, and hepatocellular carcinomas, among others).6−10 Though curcumin has good therapeutic efficacy, its clinical application has been limited due to its low solubility and stability in aqueous solution.3,9,11−16 Researchers have shown that the curcumin degradation in water solution is linked to © 2017 American Chemical Society

Received: Revised: Accepted: Published: 1342

October 3, 2016 January 18, 2017 January 23, 2017 January 23, 2017 DOI: 10.1021/acs.jafc.6b04409 J. Agric. Food Chem. 2017, 65, 1342−1352

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Journal of Agricultural and Food Chemistry

Figure 1. Mean size of empty and curcumin-loaded NLCs in twice-distilled water, PBS at pH 7.4 and NaCl 0.9% (w/w) .

Figure 2. PDI of empty and curcumin-loaded NLCs in twice-distilled water, PBS at pH 7.4 and NaCl 0.9% (w/w).

those of curcumin.18−21 Other studies revealed very low plasma levels of curcumin (1.8−11 nM) after oral administration.3 However, the low bioavailability of curcumin decreases its effectiveness in vivo.22 To overcome these problems, it is important to entrap curcumin into nanostructures, which are able to deliver it into cancer cells, thus improving its bioavailability. Therefore, efforts have been made by entrapping curcumin in nano- and microvectors such as liposomes and polymeric lipid nanocarriers.3,12−15,22−27 In particular, lipidbased nanoparticles seem to be the more interesting colloidal carriers.28−32 Moreover, it is strongly reported in the literature that the interaction of curcumin with lipids and liposomes improves its stability.17 Nanostructured lipid carriers (NLCs) are easy to produce avoiding the use of organic solvents, show great stability during long-term storage, and are amenable to both lyophilization and steam sterilization.2,33−37 Moreover, NLCs are composed of safe and biocompatible excipients,

already used for pharmaceutical dosage forms.1 In this context, many authors have analyzed potential of lipid nanocarriers as delivery vectors for lipophilic compounds.3,37,19,38,39 In particular, we demonstrated in a recent work that curcumin entrapped into NLCs was internalized into neuroblastoma LAN5 cells, determining a major efficacy in inhibiting cell viability and inducing the expression of the heat shock protein 70 (Hsp70), compared to free curcumin.3 In this study, we aimed to entrap curcumin in NLCs with high efficiency, to release it in human plasma, and finally to demonstrate the efficacy of curcumin to treat cisplatin-sensitive human epithelial ovarian cancer (EOC) cells (A2780S) and cisplatin-resistant EOC cells (A2780CP). In particular, here we report the preparation and characterization of curcumin-loaded NLCs and the efficiency of curcumin incorporation (drug loading). The release profile was also studied by evaluating the release kinetics of curcumin from NLCs in human plasma. 1343

DOI: 10.1021/acs.jafc.6b04409 J. Agric. Food Chem. 2017, 65, 1342−1352

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Figure 3. Zeta potential values of empty and curcumin-loaded NLCs in twice-distilled water, PBS at pH 7.4 and NaCl 0.9% (w/w). organic solvents and water (tetrahydrofuran, H2O, and acetic acid (60:35:5 v/v), and a flow rate of 0.2 mL/min was used. The curcumin peak was analyzed at a wavelength of 426 nm and quantified by comparison with a standard curve obtained using curcumin solutions in organic solution at known concentrations. The linear regression value was R2 = 0.9948. The linearity of the method was studied in the range 40−200 μg/mL. The detection limit of curcumin was 10 ng/mL. Determination of Curcumin Amount. In order to quantificate curcumin encapsulated into curcumin-loaded NLCs, THF solutions (0.2 mg/mL) were filtered through 0.45 μm filters (PTFE) and determined by liquid chromatography. With the aim to ensure that the curcumin was not retained by the PTFE filter, several curcumin THF solutions at known concentrations were filtered and evaluated by HPLC analysis, determining the concentrations, before and after filtration. No differences in curcumin values were found. The results were reported as loading capacity (LC%), weight % of the entrapped curcumin, and the total sample weight (lipid matrix plus curcumin). Curcumin Release Kinetics in Human Plasma. Curcumin kinetics were analyzed at seven prefixed time intervals. Each suspension (7 batches), containing 2 mg of curcumin-loaded nanoparticles in 3 mL of human plasma, was incubated under mechanical stirring at 37 ± 0.1 °C. At the prefixed times, each batches was ultracentrifuged at 40,000 rpm for 15 min at 4 °C. 4 mL of acetonitrile was added to supernatant, and the dispersion was centrifuged at 11,800 rpm for 15 min at 4 °C. Successively the supernatant was filtered (0.2 μm PTFE filters) and injected to the HPLC. To evaluate the curcumin amount loaded into residual NLC batches, 5 mL of THF

Moreover, we demonstrated that the NLCs loaded with curcumin have been taken up in cell culture models and displayed antiproliferative and proapoptotic activity in EOC cells.

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MATERIALS AND METHODS

Chemicals. Compritol 888 ATO (glycerides (mono-, di-, and tri) of fat acids C22 (15, 50, and 35% w/w)) was a gift from Gattefossè Italia s.r.l. (Milan, Italy); Captex 355 EP/NF (mixture of triglycerides of caprylic and capric acids 50 and 50% w/w) was supplied by Abitec Corporation (Columbus, OH, USA). Miglyol 812 (glycerides of caproic, caprylic, and capric acids 50, 45, and 5% w/w) was supplied by Sasol GmbH (Hamburg, Germany). Epikuron 200 (soya phosphatidylcholine 95%) was a kind gift from Cargill (Milan, Italy). Taurocholate sodium salt was a gift by PCA (Basaluzzo, Italy). Curcumin was obtained from Merck (Milan, Italy). The other reagents and solvents were purchased from Sigma-Aldrich (Milan, Italy). Empty and Curcumin-Loaded NLC Preparation. In this work we described the preparation of empty or curcumin-loaded NLCs by precipitation technique. Briefly, the lipid or mixture was heated to 5−10 °C above its melting point. Epikuron 200 (soya phosphatidylcholine 95%) in ethanol was added, under stirring, to the hot lipid. Lecithin-based surfactants, used for the particle stabilization, can also prevent their opsonization, thus increasing drug circulation time in the bloodstream significantly.40 For obtaining loaded nanoparticles, curcumin was added under to the melted lipid phase before the addition of the ethanolic Epikuron solution. To obtain NLCs, the hot lipid phase was poured into 100 mL of Milli-Q water containing taurocholate sodium salt at 2−3 °C (used as anionic cosurfactant, in order to obtain stable nanoparticles with negative surface charge) and homogenized by using an homogenizer (Ultra Turrax T25, IKA, D-Staufen) at 13,500 rpm for 10 min. NLCs were dialyzed (Visking Tubing, cutoff 12000−14000 Da) and lyophilized (FreeZone freezedryer, Labconco, USA). The purification process allowed complete removal of the ethanol used during preparation. Dimensional Analysis. The mean size and polydispersity of each nanoparticle suspension were determined by DLS measurements (Zetasizer Nano ZS, Malvern Instrument, U.K.) that utilize the photon correlation spectroscopy (PCS) technique. The measurements were obtained in backscattering (fixed angle of 173°) and at 25 °C on samples dispersed in Milli-Q water, in saline aqueous solutions such as 0.9% NaCl and phosphate buffer (PBS) at pH 7.4. All the samples were analyzed in triplicate. Surface Charge Determination. The surface charge is determined by zeta potential measurements with the same instrument used for the dimensional analysis, exploiting the principles of electrophoretic mobility. Empty and curcumin-loaded NLCs were dispersed in Milli-Q water, NaCl 0.9%, and PBS at pH 7.4. Each sample was analyzed in triplicate. Curcumin HPLC Analysis. The HPLC system (Shimadzu Instrument, Japan) consisted of two pumps LC-20 AD, an SPD-20 AV UV−vis detector, a C18 column (3 μm, 150 × 4.6 mm i.d.), and an autosampler SIL-20A HT. The mobile phase consisted of a mixture of

Table 1. Loading Capacity (LC %) and Entrapment Efficiency (EE %) sample

LC %

EE %

NLCs Compritol−Captex NLCs Compritol−Miglyol NLCs Compritol

38.7 17.4 15.5

100.0 44.9 40.0

Figure 4. Release profiles of curcumin in human plasma at 37 ± 0.1 °C from NLCs. Each value is the mean of three experiments. 1344


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Figure 5. FTIR spectra for curcumin, empty NLCs, and curcumin-loaded NLCs.

Figure 6. Hemolysis results of free curcumin and empty and drug-loaded NLCs (at drug concentrations of 5, 15, and 30 μM) after incubation with red blood cells for 1 h at 37 °C. Free curcumin and empty and drug-loaded nanoparticles showed no significant lysis of red blood cells. Data represent mean ± SD for three separate experiments. was added to the pellet after centrifugation, filtered with 0.2 μm (PTFE filters), and injected to the HPLC. Fourier Transform Infrared (FTIR) Spectroscopy. The FTIR spectra of curcumin, Compritol NLCs, and curcumin-loaded Compritol NLCs were recorded in the range of 4000−400 cm−1 using an FTIR spectrophotometer (PerkinElmer 1720 Fourier transform). Hemolytic Test. Human erythrocytes were recovered by centrifugation at 2,200 rpm for 10 min at 4 °C, after isolation from fresh K3 EDTA-treated blood. The erythrocytes were washed (8 times) with

PBS at pH 7.4, recovered by centrifugation, and suspended again in the same saline solution. Successively, a suspension of erythrocytes in PBS at pH 7.4 (final concentration 4%) was prepared. This dispersion was freshly prepared and used within a few hours. Curcumin free, empty, or curcumin-loaded NLCs (with a curcumin concentration 5−30 μM) were incubated into the erythrocyte suspension for 1 h at 37 °C under constant shaking. The release of hemoglobin was determined, after centrifugation, by UV analysis of the supernatant at 540 nm (UV-1800 UV−vis, Shimadzu). Controls for zero hemolysis (blank) and 100% hemolysis consisted of erythrocytes suspended in 1345


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Cell Viability and Clonogenic Assays. To evaluate and compare the effect of free curcumin and curcumin-loaded NLCs on cell viability, A2780S and A2780CP cells were plated in plates (96-well) at concentration 5 × 103 cells/well. After 24 h, the different doses of free curcumin and curcumin-loaded NLCs were added to the cells. NLCs without curcumin were also tested on the two cell lines grown at the same concentrations corresponding to that of curcumin-loaded NLCs. The effects on cell viability were evaluated after 72 h of treatment. MTS assay was performed as previously reported.41 Furthermore, the effects of free curcumin, curcumin-loaded NLCs, and empty NLCs on cell growth were evaluated by performing a clonogenic assay. Cells were plated in six-well plates at a concentration of 200 cells/well. After 24 h, cells were exposed to different doses of free curcumin, curcumin-loaded NLCs, and empty NLCs for 48 h. Then, cells were washed with medium and allowed to grow for 7 days. Finally, the colonies were treated as previously reported.28 All the experiments were performed in triplicate and repeated three times. Western Blot Analysis. 3 × 105 cells were seeded in 6-well plates and, after 24 h, the vehicle alone (DMSO), the free curcumin (30 μM), the curcumin-loaded NLCs (30 μM), and the empty-NLCs were added. Whole cell lysates were obtained 24 h after treatment using RIPA Buffer (Cell Signaling Technologies Inc., Danvers, MA). Equal amounts of cell lysates were used to perform Western blot analyses. After transfer, membranes (nitrocellulose) were placed in blocking buffer (OBB, LI-COR) diluted in Tris-buffered saline and incubated for 1 h at room temperature. Primary antibodies were diluted in OBB. Secondary goat anti-mouse antibodies conjugated to IRDye 800CW (LI-COR) or goat anti-rabbit Alexa Fluor 680 (Molecular Probes, Invitrogen, USA) were diluted in OBB. Membranes were scanned and analyzed with an Odyssey IR scanner by Odyssey 3.0 imaging software. The signals of antibodies were determined as integrated intensities of regions defined around the bands of interest in either channel, with primary mouse antibodies raised against β-actin (Sigma) and β-catenin and primary rabbit antibodies raised against PARP, phospho-p38, phospho-JNK, Bcl-2, Survivin, Caspase-3, Mcl-1, and IL-6 (Santa Cruz, Dallas, TX, USA). Statistical Analysis. Statistical analysis was determined using Student’s t test. The criterion for statistical significance was p < 0.05.

Figure 7. Cisplatin-sensitive (A2780S) and cisplatin-resistant (A2780CP) human EOC cells were cultured in the presence of the vehicle alone (DMSO) or free curcumin (30 μM), empty NLCs, and curcumin-loaded NLCs (30 μM) for 24 h. Curcumin treatment alters the morphology of cells. Cell monolayers were examined under an inverted light microscope and photographed. Images are representative of three independent experiments.

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PBS and 1% Triton X-100 w/v, respectively. Each experiment was performed in triplicate and repeated twice. The erythrocyte lysis percentage was calculated using the following formula:

% hemolysis =

(Abssample − Abs blank ) (Abs100%lysis − Abs blank )

RESULTS AND DISCUSSION In this study, in the aim to show the good potential of lipid nanocarriers as vectors for lipophilic molecules such as curcumin to treat ovarian cancer, the preparation and the characterization of empty and curcumin-loaded nanostructured lipid carriers (NLCs) are reported. Curcumin, as previously reported in the Introduction, possesses several pharmacological properties.42 However, free curcumin has low bioavailability and its encapsulation into a nanovector, such as NLCs, could offer several advantages such as the potential of administration in cancer therapy and the passing of tumor cell resistance.3,43−46 In this work, empty and curcumin-loaded NLCs with different matrix composition were obtained by precipitation technique and Epikuron 200 and taurocholate sodium salt as surfactants were used. In particular, three matrices were obtained by using solid lipid (Compritol) alone or in mixture with liquid lipid (Miglyol 812 or Captex 355 EP/NF) at a weight ratio equal to 2:1 in order to evaluate the effect of the matrix composition on the chemical−physical properties of NLCs and loading capacity (LC%). In particular, to prepare curcumin-loaded NLCs, a hot ethanolic solution of Epikuron 200 was poured into the melted lipid matrix containing curcumin at 85 °C. NLCs were obtained by dispersing the obtained hot solution in an aqueous solution of taurocholate sodium salt (3−4 °C), under mechanical stirring. After preparation, curcumin-loaded NLCs had a bright

× 100

where Abs sample is the absorbance value of the hemoglobin released from erythrocytes treated with curcumin free, empty, or curcuminloaded NLCs; Absblank is the absorbance of the hemoglobin of erythrocytes treated with PBS buffer; and Abs100%lysis is the absorbance of the hemoglobin of erythrocytes treated with a solution of surfactant (1% Triton X-100). Reagents and Cell Culture. Free curcumin was dissolved in organic solution (dimethyl sulfoxide). Empty NLCs and curcuminloaded NLCs were resuspended in RPMI and sonicated for 5 min before their use in all biological assays. Cisplatin-sensitive human EOC cells A2780S were kept in DMEM/F12 (10% FBS); cisplatin-resistant human EOC cells A2780CP were kept in DMEM/F12 (10% FBS) supplemented with 1 μg/mL cisplatin and moved to DMEM/F12 without cisplatin 72 h before performing biological assays. Both cell lines were used at low passage number. Cell Morphology, Curcumin Uptake, and Hoechst Staining. Cells were seeded in 8-well chamber slides at a concentration of 5 × 103 cells/well. After 24 h, cells were exposed to free curcumin, curcumin-loaded NLCs, and empty NLCs at a concentration of 30 μM. Dimethyl sulfoxide was used as control of free curcumin treated cells. After 24 h, cells were fixed using 3.7% paraformaldehyde, permeabilized, and stained using Hoechst 33258 (Sigma) at final concentration of 2.5 μg/mL for 30 min at 37 °C. Upon three washings with PBS, slides were mounted using Vectashield mounting medium and images were acquired by Zeiss fluorescence microscope. 1346


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Figure 8. Increase in fluorescence was observed by fluorescent microscopy in A2780S and A2780CP cells incubated for 24 h with free curcumin (30 μM) and curcumin-loaded NLCs (30 μM) as compared to vehicle-treated (DMSO) or empty NLC-treated cells.

Compritol and Captex 355 EP/NF. The higher loading capacity of Compritol−Captex NLCs could be due to the higher ability of Captex 355 EP/NF to retain curcumin, within the matrix. This liquid lipid is in fact composed of a mixture of triglycerides containing fatty acids with carbon chains longer than the ones contained in Miglyol 812 triglycerides, that could better solubilize highly hydrophobic molecules as curcumin. The stability of the NLC dispersions is one of the major concerns for the final product characteristics. Being heterogeneous systems, NLCs show a significant tendency to aggregate and lose their chemical−physical properties during storage. Thus, long-term stability (3 months at room temperature) was evaluated for all obtained curcumin-loaded NLCs as lyophilized powder, and no differences were observed after redispersion in twice-distilled water in terms of particle size, PDI, zeta potential, and loaded curcumin as compared with fresh samples (data not shown). These systems, considering the good chemical−physical characteristics, could be proposed for intravenous administration. In order to evaluate the curcumin release kinetics, curcumin-loaded nanoparticles were subjected to release studies in human plasma. The results are reported in Figure 4. As can be seen, the release profiles showed an initial burst effect and successively a controlled curcumin release, the amounts of released curcumin after 24 h incubation from NLCs based on Compritol−Captex, Compritol−Miglyol, and Compritol being, respectively, equal to 33, 28, and 18% w/w on the total entrapped curcumin. In Compritol−Captex and Compritol−Miglyol NLCs, the presence of liquid lipids could facilitate the diffusion of curcumin throughout the matrix, compared to Compritol NLCs, consisting of a solid matrix.

yellow opalescent aspect. For obtaining empty NLC samples the same process was followed step by step, avoiding the addition of curcumin to the melted lipid. After preparation and purification, the formulations showed, in twice-distilled water, a mean size between 100 and 160 nm (Figure 1), rather low polydispersity index (PDI) values (Figure 2), and negative zeta potential (Figure 3). Analysis by DLS confirmed the presence of particles only at the nanoscale and a low polydispersity index (data not shown). An increase until doubling of the particle size in isotonic PBS at pH 7.4 and NaCl 0.9% aqueous saline medium occurs, and this effect could be due to the charge shielding effect of the solution ions, which determines a reduction (in absolute value) of the particles’ surface charge (as shown in Figure 3), and lowers the repulsive forces between the particles, thus allowing their aggregation. This effect is confirmed by the increase in PDI values of measurement done in the same medium (as shown in Figure 2). No significant differences were found between each empty and curcumin-loaded system in terms of mean size and PDI, indicating that the curcumin entrapment does not affect the production process, but a slight decrease of zeta potential in the presence of curcumin was evidenced, probably due to the deposition of a small amount of curcumin on the particles’ surface. In Table 1, the LC % (expressed as % weight ratio of loaded entrapped curcumin and the total dried batch weight) and the EE % (expressed a s% weight ratio between entrapped curcumin and total curcumin amount used to prepare the nanocarriers) of curcumin loaded-NLCs are also reported. The best results in terms of LC % and EE %, determined by chromatographic analysis on each curcumin loaded system, were obtained when the lipid matrix was composed of 1347


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Journal of Agricultural and Food Chemistry Moreover, it was also evaluated that the unreleased curcumin was still inside the NLCs (data not reported). The presence of a high amount of unreleased curcumin is important as curcumin-loaded NLCs could arrive to target organ in which curcumin could be locally released in a controlled and sustained way. Considering the slower curcumin release profile, the NLCs composed only of Compritol were chosen to perform successive studies. FTIR spectroscopy was employed to obtain conformational information about the lipid molecules, and it is used to investigate the interactions between lipid and curcumin. From the FTIR spectra of free curcumin and the optimized formulation (Compritol NLCs) it was confirmed that there were no interactions between lipid and curcumin (Figure 5). Hemocompatibility is a fundamental property for a target delivery system for parenteral administration, the bloodstream being the main path for these carriers to reach and target the tumor cells. Therefore, the potential curcumin-loaded NLC interactions with erythrocyte membranes and their hemolytic effects were studied by hemolysis assays, incubating free curcumin and empty and curcumin-loaded NLCs with erythrocytes and determining the amount of hemoglobin released. Figure 6 shows that, under the chosen conditions, empty and curcumin-loaded NLCs displayed no significant hemolytic effects, indicative for no significative interactions with erythrocyte membranes. Moreover, in vitro cytotoxicity assays were carried out on human EOC cells. We studied the potential toxicity of curcuminloaded NLCs on human EOC cell lines, the cisplatin-sensitive A2780 cells and the cisplatin-resistant A2780CP cells. The obtained results were compared with free curcumin. Microscopic examination of A2780S and A2780CP cells treated with free curcumin (30 μM), empty NLCs, and curcuminloaded NLCs (30 μM) revealed that only the treatment with free curcumin and curcumin-loaded NLCs reduces the cell number and alters cell morphology (Figure 7). Taking the advantage of green intrinsic fluorescence of curcumin, free curcumin and curcumin-loaded NLC uptake by A2780S and A2780CP cells was also investigated. We found that cells treated with curcumin-loaded NLCs had an intracellular fluorescence comparable to cells treated with free curcumin (Figure 8). Furthermore, the cytotoxicity of free curcumin, empty NLCs, and curcumin-loaded NLCs was monitored using cell viability assay. As far as cancer is concerned, in vitro studies have demonstrated that cancer cells do not die unless they are exposed to curcumin concentrations of 5−50 μM for several hours.47 Free curcumin and curcumin-loaded NLCs significantly inhibited the viability of A2780 and A2780CP cells. The cytotoxic effect of curcumin-loaded NLCs was comparable to that of free curcumin in A2780S cells with an IC50 of 21.2 μM ± 3.5 and 22.2 μM ± 1.8, respectively (Figure 9). However, A2780CP cells were more sensitive to curcumin than A2780S cells, displaying IC50 values of 20.2 ± 2.5 and 19.0 μM ± 1.4 with free curcumin or curcumin-loaded NLCs, respectively (Figure 9). To evaluate the effect of curcumin-loaded NLCs on the capacity to form colonies, A2780S and A2780CP cells were subjected to long-term clonogenic assay, a cell survival and proliferation assay based on the capability of a single cell to grow into a colony. The ability of A2780S and A2780CP cells to form colonies was inhibited upon treatment with free curcumin up to a concentration of 5 μM, whereas curcumin

Figure 9. Effects of free curcumin (◆), empty NLCs (▲), and curcumin-loaded NLCs (■) on the viability A2780S and A2780CP cells. Cells were grown and treated for 72 h; thereafter cell viability was assessed by the MTS assay. Data are expressed as the percentage of control cells and are the means ± SD of three separate experiments, each of which was performed in triplicate.

entrapped into NLCs inhibited colony formation at 2.5 μM (Figure 10). The obtained data show that curcumin-loaded NLCs possess anticancer activity and reduce cell colony survival more effectively than free curcumin. As an example, the ability of A2780S cells to form colonies was inhibited after treatment with 5 μM free curcumin by 50% ± 6, whereas, at the same concentration, the delivery of curcumin with NLCs significantly (p < 0.05) inhibited colony formation to approximately 88% ± 1, therefore increasing the activity of curcumin to inhibit A2780S cell growth. To elucidate whether the mechanisms of action of curcuminloaded NLCs mirror that of free-curcumin, we performed nucleic acid staining with Hoechst 33258 to analyze apoptosis induction. In both cell lines, nuclear condensation and fragmentation, which are hallmarks of apoptosis, were revealed by Hoechst staining only in cells treated with free curcumin and curcumin-loaded NLCs (Figure 11). We further confirmed the induction of apoptosis by curcumin-loaded NLCs in A2780S and A2780CP cells by immunoblotting analysis. Cleavage of PARP and reduction of procaspase-3 (both representing classical markers of apoptosis) were observed in both cell lines after treatment with curcuminloaded NLCs (Figure 12). In addition, we found that curcumin-loaded NLCs induced reduction of some antiapoptotic proteins, such as Bcl-2, Mcl-1, 1348


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Figure 10. Curcumin-loaded nanoparticles inhibit the clonogenic potential of ovarian cancer cells. Colony assays were performed comparing the effects of free and curcumin-loaded nanoparticles in inhibiting the clonogenicity of A2780S and A2780CP cells. Representative plates are illustrated for cells treated for 7 days with vehicle alone (DMSO), indicating concentrations of free-curcumin and equivalent dosage of empty NLCs and curcumin-loaded NLCs. Surviving colonies were stained (upper panel) and counted (lower panel). Data are expressed as a percentage of colonies in control cells and are the means ± SD of three experiments, each of which was performed in triplicate.

Figure 11. A2780S and A2780CP cells were cultured in the presence of vehicle alone (DMSO), free-curcumin (30 μM), empty NLCs, and curcumin-loaded NLCs (30 μM). Cells were then fixed, stained with DNA-specific Hoechst 33258 dye, and visualized by fluorescence microscopy at 400× magnification. Arrowheads indicate cells showing nuclear fragmentation.

and curcumin-loaded NLCs. As shown in Figure 11, free and curcumin-loaded NLCs equally downregulated the levels of IL-6 in both cell lines. Finally, we also analyzed the expression of β-catenin, a key molecule involved in the Wnt signaling pathway, which plays an important role in EOC tumorigenesis and chemoresistance.51−53 Treatment with free and curcuminloaded NLCs inhibited the expression of β-catenin in both cell lines, thus suggesting that β-catenin might represent a newly identified curcumin target. All together, these results demonstrated

and survivin, and activated the p38 mitogen-activated protein kinase (MAPK) proapoptotic pathway. Numerous data indicate that curcumin inhibits NF-κB, a transcription factor involved in inflammatory response and in cancer development. In particular, the proinflammatory cytokine IL-6 (a recognized NF-κB target) and its pathway display highly oncogenic functions in ovarian cancer, and it is a well-recognized EOC therapeutic target.48−50Therefore, we analyzed the expression of IL-6 after treatment with free 1349


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fragmentation), PARP cleavage, procaspase-3,Bcl-2, Mcl-1, and survivin reduction, in addition to activation of the MAPK proapoptotic pathway. Moreover, free and curcumin-loaded NLCs equally downregulated the steady state levels of the cytokine IL-6 (a recognized NF-κB gene target), and also inhibited the expression of the oncoprotein β-catenin (a component of Wnt signaling, representing a newly identified curcumin target). Our data suggest that the dietary administration of curcumin entrapped into the lipid nanoparticles could improve its bioavailability and could encourage future in vivo studies for application in cancer and other diseases that might benefit from the effects of this compound.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +39 091 6809367. Fax: +39 091 6809399. E-mail: [email protected]. ORCID

Maria Luisa Bondì: 0000-0002-2674-1654 Francesca Di Gaudio: 0000-0002-5910-5750 Notes

The authors declare no competing financial interest.

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REFERENCES

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Figure 12. A2780S and A2780CP cells were grown and treated for 24 h with vehicle alone (DMSO), free curcumin (30 μM), empty NLCs, and curcumin-loaded NLCs (30 μM). After treatment, cells were harvested and lysed, and equal amounts of extracted protein were analyzed for PARP, caspase-3, Mcl-1, Bcl-2, survivin, phospo-p38, β-catenin, and IL-6 by Western blotting. The data represent two independent experiments with comparable outcomes. Arrowheads indicate the cleaved 85 kDa form of PARP.

an improved therapeutic efficacy of curcumin-loaded NLCs compared to free curcumin and suggested that lipid nanoparticles could have a great potential as curcumin drug delivery systems (DDSs). Moreover, our data encourage further in vivo studies for cancer and other pathologies that might benefit from the effects of curcumin entrapped into NLCs. In conclusion, the potential of NLCs as carriers for curcumin for the treatment of ovarian cancer, a well-known chemopreventive agent, was demonstrated. NLCs, obtained from biocompatible lipids, were prepared by the precipitation technique as empty or curcumin-loaded systems, and showed average diameters in the colloidal size range, good loading capacity, and prolonged release in human plasma. In vitro experiments on cisplatin sensitive (A2780S) and resistant (A2780CP) EOC cells showed that curcumin loaded into NLCs has in vitro therapeutic efficacy comparable to that of free curcumin against A2780S and A2780CP cells, as assessed by cell viability. However, in both cell lines, curcumin-loaded NLCs showed improved anticancer potential in clonogenic assays, as compared to free curcumin. Further, the mechanisms of action of curcumin-loaded NLCs mirrored that of free curcumin, including the induction of cellular apoptosis (as measured by nuclear condensation and 1350


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NOTE ADDED AFTER ASAP PUBLICATION This article published February 7, 2017 with an incorrect reference 41. The reference was modified and the article reposted.

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