International Journal of Pharmaceutics 437 (2012) 29–41
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Pharmaceutical Nanotechnology
ApoE3 mediated polymeric nanoparticles containing curcumin: Apoptosis induced in vitro anticancer activity against neuroblastoma cells Rohit S. Mulik a,c,d,1, Jukka Mönkkönen c, Risto O. Juvonen d, Kakasaheb. R. Mahadik a, Anant R. Paradkar b,∗ a
Department of Pharmaceutics, Poona College of Pharmacy, Bharati Vidyapeeth University, Erandwane, Pune 411038, India Centre for Pharmaceutical Engineering Science, University of Bradford, Bradford BD7 1DP, UK c Department of Biopharmacy, School of Pharmacy, University of Eastern Finland, P.O. Box 1627, FIN-70211 Kuopio, Finland d Department of Toxicology, School of Pharmacy, University of Eastern Finland, P.O. Box 1627, FIN-70211 Kuopio, Finland b
a r t i c l e
i n f o
Article history: Received 20 June 2012 Received in revised form 24 July 2012 Accepted 27 July 2012 Available online 4 August 2012 Keywords: Curcumin Neuroblastoma Flow cytometry Nanoparticles Apolipoprotein E3
a b s t r a c t Curcumin, a natural phytoconstituent, is known to be therapeutically effective in the treatment of various cancers such as, breast cancer, lung cancer, pancreatic cancer, brain cancer, etc. However, low bioavailability and photodegradation of curcumin hampers its overall therapeutic efficacy. Anionic polymerization method was employed for the preparation of apolipoprotein-E3 mediated curcumin loaded poly(butyl)cyanoacrylate nanoparticles (ApoE3-C-PBCA) and characterized for size, zeta potential, entrapment efficiency, photostability, morphology, and in vitro release study. ApoE3-C-PBCA were found to be effective against SH-SY5Y neuroblastoma cells compared to curcumin solution (CSSS) and curcumin loaded PBCA nanoparticles (C-PBCA) from in vitro cell culture investigations. Flow cytometry techniques employed for the detection of anticancer activity revealed enhanced activity of curcumin against SH-SY5Y neuroblastoma cells with ApoE3-C-PBCA compared to CSSS and C-PBCA, and apoptosis being the underlying mechanism. Present study revealed that ApoE3-C-PBCA has tremendous potential to develop into an effective therapeutic treatment modality against brain cancer. © 2012 Elsevier B.V. All rights reserved.
1. Introduction The treatment of brain cancers is limited by the inadequacy in delivering therapeutic agents in such a way that drug molecules reach the desired targets (Chakraborty et al., 2009; JuilleratJeanneret, 2008). In order to achieve efficient treatment of central nervous system (CNS) cancers, it is necessary to transport therapeutic agents across the specialized vascular system of the brain, the blood–brain barrier (BBB). Transport across BBB presents challenges especially in case of brain tumors due to undefined nature of cerebrovascular system associated with cancer progression and development of biomarkers which can be coupled to therapeutic agents for targeted delivery but bypassing the resistance mechanism (Chakraborty et al., 2009; Juillerat-Jeanneret, 2008). A great deal of effort, therefore, is presently focused on improving CNS bioavailability, and tumors thereof, of therapeutic drugs that can be specifically targeted to diseased tissue, improving therapeutic
∗ Corresponding author. Tel.: +44 1274 233900; fax: +44 1274 234679. E-mail addresses:
[email protected] (R.S. Mulik),
[email protected] (A.R. Paradkar). 1 Current Address: Advanced Imaging Research Center, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX, USA 75390. 0378-5173/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2012.07.062
opportunities, efficiency, and patient survival, while decreasing side-effects to normal cells (Juillerat-Jeanneret, 2008). Various pharmacological agents have been used to open the BBB and direct invasive methods can introduce therapeutic agents into the brain substance (Juillerat-Jeanneret, 2008; Gutman et al., 2000). It is important to consider not only the net delivery of the agent to the CNS, but also the ability of the agent to access the relevant target site within the CNS. Curcumin, an antioxidant principle in Asian spice turmeric has been widely studied for its anticancer activities (Aggarwal et al., 2003; Azuine and Bhide, 1992; Ramachandran et al., 2002; Dorai et al., 2001). The mechanisms proposed for anticancer activity of curcumin include signal transduction pathways modulation, cell cycle inhibition or apoptosis (Aggarwal et al., 2003; Joe et al., 2004), generation of reactive oxygen species (Buttke and Sandstrom, 1994; Jacobson, 1996), p53 expression and inhibition of NFkB (Aggarwal et al., 2005; Lantto et al., 2009). A very restricted number of liposoluble small molecules cross the BBB by free diffusion. All other molecules require specialized targeted drug delivery systems (Gutman et al., 2000). The targeting agents may be antibodies, directed toward an antigen residing on the target tissue, or ligands for receptors or transporters. These may be covalently conjugated via an appropriate chemical bond either directly to the drug or to a vector, such as a nanoparticulate device.
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Apolipoprotein E (ApoE) has been used by few workers as a targeting agent for transport of drug across BBB by low density lipoprotein (LDL) receptor (LDL-R) mediated endocytosis (Kim et al., 2009). There are few reports showing the enhanced drug transport into the brain using ApoE linked albumin nanoparticles (Michaelis et al., 2006; Zensi et al., 2009) and highly efficient transport of polybutyl cyanoacrylate nanoparticles across BBB using ApoE as a ligand (Kreuter et al., 2002). These studies were carried out using mixture of various isoforms of ApoE. Hence, we are proposing a novel drug delivery system containing curcumin using apolipoprotein E3 as a ligand for enhanced uptake and activity against SH-SY5Y neuroblastoma cells, overexpression of LDL-R on the surface of SH-SY5Y cells being well documented. Among three isoforms of ApoE, ApoE3 and ApoE4 have good affinity to LDL-receptor compared to ApoE2 (Lund-Katz et al., 2001; Yamamoto et al., 2008). However, since ApoE4 is associated with the increased risk of coronary heart disease and alzheimer’s disease, ApoE3 was selected as a ligand for proposed drug delivery system (Lund-Katz et al., 2001).
system containing 0.1% w/v poloxamer 188 without curcumin was prepared as a blank system and termed as surfactant solution (SS).
2. Materials and methods
2.2.4. Differential scanning colorimetry (DSC) Thermal characteristics of blank PBCA nanoparticles (B-PBCA), C-PBCA and pure curcumin were investigated using DSC analysis to determine polymer–drug interaction (Mulik et al., 2009; Reddy and Murthy, 2004). Mettler Toledo DSC 821e equipped with an intracooler (Mettler Toledo, Switzerland) was used with sample in hermetically sealed aluminium pans heated from 25 ◦ C to 300 ◦ C at a constant rate of 10 ◦ C/min. Inert atmosphere was maintained by nitrogen purging at a flow rate of 20 ml/min (Mulik et al., 2009).
2.1. Materials n-Butyl cyanoacrylate was a kind gift from Evotite Corporation, China. Poloxamer 188 and Sodium Sulphate were purchased from Merck, Germany. Curcumin was a kind gift from Indsaaf Inc., Batala, India. Coomassie brilliant blue G dye was purchased from Himedia, Mumbai, India. Apolipoprotein E3, human was purchased from Calbiochem, Europe. AnnexinV-Fluorescein isothiocyanate (AnnexinV-FITC) and propidium iodide were purchased from Biolegend Europe BV, Netherlands. 2 ,7 -dichlorodihydrofluorescein diacetate (H2DCF-DA) was purchased from Sigma–Aldrich, USA. CaspGLOW red active caspase-3 staining kit was purchased from Biovision Research Products, USA. Tetramethylrhodamine methyl ester (TMRM) was purchased from Sigma–Aldrich, Germany. Growth medium, DMEM (BE 12-604F) was purchased from BioWhittaker Inc., Lonza, Belgium. 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) was purchased from Sigma–Aldrich, Germany. All aqueous solutions were prepared with distilled and deionized water. All other reagents and chemicals used were of analytical grade. 2.2. Methods 2.2.1. Preparation of apolipoprotein E3 conjugated polybutyl cyanoacrylate nanoparticles containing curcumin (ApoE3-C-PBCA) Nanoparticles were prepared by method previously described (Mulik et al., 2010a,b). In brief, Poloxamer 188 and sodium sulphate were dissolved in 1:1 v/v mixture of 0.1 N HCl and Ethanol. Curcumin (0.1% w/v) was dissolved under stirring. Finally, the monomer, i.e. n-butyl cyanoacrylate was added drop wise under continuous stirring and stirred for 4 h. pH of the suspension was adjusted to 6.0 ± 0.5 at the end of 4 h using 1 N NaOH and stirring was continued for an additional 1 h to complete the reaction. Coating of tween 80 (0.1% w/v) on the prepared nanoparticles was carried out by stirring for 30 min at 200 rpm. The ApoE3 (20 g/ml) was finally conjugated to the prepared nanoparticles by stirring for 1 h at room temperature. 2.2.2. Preparation of curcumin solubilized surfactant solution (CSSS) Curcumin was dissolved in an aqueous system containing 0.1% w/v poloxamer 188 by stirring for 15 min at 400 rpm. Aqueous
2.2.3. Characterization of nanoparticles The particle size analysis was carried out by dynamic light scattering using Malvern Hydro 2000 SM particle size analyzer (Malvern instruments, UK) (Mulik et al., 2009, 2010a,b). The uniformity of the size distribution was determined from the polydispersity index (PdI). Laser Doppler Velocimetry (Zetasizer 3000, Malvern Instruments, UK) at 25 ◦ C was used for the measurement of zeta () potential (Mulik et al., 2009, 2010a,b). The entrapment efficiency of C-PBCA was determined using ultracentrifugation method (Beckmann TL-100, MN, USA) (Mulik et al., 2009, 2010a,b). Quantification of ApoE3 conjugation with C-PBCA was performed by bradford assay using coomassie brilliant blue G dye (Gupta et al., 2007; Bradford, 1976). The photostability of curcumin encapsulated in nanoparticles was determined by previously described method using HPLC. All the values are presented as mean ± S.D. from three replicate samples.
2.2.5. X-ray diffraction study (XRD) analysis The encapsulation of curcumin inside the nanoparticles was confirmed by X-ray diffraction measurements carried out with X-ray diffractometer (PW 1729, Philips, Netherlands) in the diffraction range of 5–50◦ . A Cu-Ka radiation source was used, and the scanning rate (2/min) was 5 ◦ C/min. The nanoparticles were analyzed after vacuum freeze drying (Lee et al., 2007). 2.2.6. Cell culture study SH-SY5Y human neuroblastoma cells were obtained from American Type Culture Collection (ATCC, Manassas, VA). The cells were grown in DMEM medium supplemented with 10% foetal bovine serum and 1% penicillin G-streptomycin (Gibco BRL, Grand Island, NY) at 37 ◦ C in a humidified, 5% CO2 atmosphere in a CO2 incubator. 2.2.6.1. Antiproliferative assay. MTT gets reduced to purple formazan by mitochondrial reductase in living cells. This reduction takes place in presence of active reductase enzymes, and hence, this conversion is used as a measure of viable (living) cells (Mulik et al., 2010a,b). SH-SY5Y Cells (1 × 104 /well) were seeded in a 96-well plate and allowed to attach for 24 h. Then the medium was replaced with fresh medium and the cells were treated with different concentrations of curcumin solubilized surfactant solution, C-PBCA and ApoE3-C-PBCA (0.5, 1, 2, 4, 8, 16, 32, 64 M curcumin/well) using surfactant solution (SS) and B-PBCA as the respective controls and incubated for 24 h at 37 ◦ C in CO2 incubator. Medium was removed after the treatment, cells were washed three times with PBS and fresh medium was added. 25 l of 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (5 mg/ml in PBS) was added to the cells and incubated for 3 h at 37 ◦ C in CO2 incubator. After the incubation, the cells were lysed and the dark blue crystals were solubilized with 125 l of a lysis solution (50% (v/v) N,N-dimethylformamide, 20% (w/v) sodium dodecylsulphate, with an adjusted pH of 4.5). The optical density of each well was measured with a Victor 1420 Multilabel Counter (PerkinElmer Life Sci., USA) equipped with a 570 nm filter. Percent of cell survival was
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defined as the relative absorbance of treated cells versus respective controls. Results were expressed as % cell viability versus dose. To confirm the LDL-R mediated uptake of ApoE3-C-PBCA, LDLR on SH-SY5Y cell surface were blocked by incubating the cells with an excess amount of free ApoE3 for 1 h prior to incubation with ApoE3-C-PBCA in a separate experiment and the effect on cell viability was determined after 24 h treatment (Sahoo and Labhasetwar, 2005). For this experiment 4 M dose of curcumin was used. The effect of treatment time on the cell viability was determined using the same method. In brief, the cells (1 × 105 /well) were seeded in 24-well plate and allowed to attach for 24 h. The medium was replenished with fresh medium and treated with CSSS, C-PBCA and ApoE3-C-PBCA (4 M curcumin/well) using SS and B-PBCA as the respective controls and incubated at 37 ◦ C in CO2 incubator for 3, 6, 12, 24 and 48 h and the effect on cell viability was determined as described above. Results were expressed as % cell viability versus time. 2.2.6.2. Cell uptake study. Qualitative and quantitative estimation of uptake of curcumin by SH-SY5Y cells treated with CSSS, C-PBCA, ApoE3-C-PBCA was carried out using fluorescence microscopy (Weir et al., 2007) and spectrophotometry (Kunwar et al., 2008) respectively. In the qualitative estimation of curcumin uptake by SH-SY5Y cells, the autofluorescence of curcumin was observed using fluorescence microscopy using green filter. Cells (1 × 106 /well) were seeded using 24-well plate and allowed to adhere for 24 h. The medium was replenished with fresh medium, and the cells were treated with CSSS, C-PBCA and ApoE3-C-PBCA (10 M curcumin/well) for different time points (6, 12, 24 and 48 h). The medium was replaced with fresh medium by washing the cells thrice with PBS 7.4 after each time point, and the images were captured using Nikon Eclipse TE300 fluorescence microscope with Nikon F601 camera (Nikon, Japan). In the quantitative determination of uptake of curcumin by SH-SY5Y cells, the cells were treated as described above (10 M curcumin/well) for different time intervals (3, 6, 12, 24, 48 h). The cells were collected by tripsinization after each time point, and centrifuged for 3 min at 3000 rpm. The supernatant was removed; the pellets were resuspended in 1 ml of methanol, and vortexed for 5 min to extract the curcumin in methanol fraction. The lysate was then centrifuged at 5000 rpm for 5 min, and the absorbance of supernatant containing curcumin was measured at 428 nm using UV–vis spectrophotometer (V-530, Jasco, Japan). The effect on LDL-R blocking on cell uptake of ApoE3-C-PBCA was also observed after 24 h treatment with 10 M dose of curcumin. The uptake of curcumin was expressed as g of curcumin per 106 cells. 2.2.6.3. Measurement of reactive oxygen species (ROS). H2DCF-DA is a non-fluorescent permeant molecule that passively diffuses into cells and, gets converted to H2DCF as acetates get cleaved by intracellular esterases, and gets entrapped within the cell. H2DCF get rapidly oxidized to the highly fluorescent 2 ,7 -dichlorofluorescein (DCF) in the presence of intracellular ROS. The formation of reactive oxygen species was determined by means of the probe 2 ,7 dichlorodihydrofluorescein diacetate (H2DCF-DA) method as per previously described (Wang and Joseph, 1999). Briefly, SH-SY5Y Cells (1 × 104 /well) were seeded in a 96-well plate and allowed to attach for 24 h. Then the medium was replaced with fresh medium and the cells were treated with different concentrations of curcumin solubilized surfactant solution (CSSS), C-PBCA and ApoE3-C-PBCA (2, 4, 8, 16, 32, 64 M curcumin/well) using surfactant solution (SS) and B-PBCA as the respective controls and incubated for 24 h at 37 ◦ C in CO2 incubator. Medium was removed after the treatment, cells were washed three times with
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PBS and fresh medium was added. 2 ,7 -Dichlorodihydrofluorescein diacetate was added (10 M) and incubated for 2 h at 37 ◦ C. After 2 h incubation, fluorescence was monitored at an excitation wavelength of 502 nm and an emission wavelength of 520 nm using Envision 2104 Multilabel Reader (PerkinElmer Life Sci, USA). Results were expressed as Relative Fluorescence Units (RFU) versus dose. To confirm the LDL-R mediated uptake of ApoE3-C-PBCA, LDLR on SH-SY5Y cell surface were blocked by incubating the cells with an excess amount of free ApoE3 for 1 h prior to incubation with ApoE3-C-PBCA in a separate experiment and the effect on ROS generation was determined after 24 h treatment (Sahoo and Labhasetwar, 2005). For this experiment 4 M dose of curcumin was used. The effect of time of treatment on the generation of ROS was also determined using the same method. In brief, the cells (1 × 105 /well) were seeded in 48-well plate and allowed to attach for 24 h. Then, the medium was replaced with fresh medium and treated with CSSS, C-PBCA and ApoE3-C-PBCA (4 M curcumin/well) using SS and B-PBCA as the respective controls and incubated at 37 ◦ C in CO2 incubator for 3, 6, 12, 24 and 48 h. The generation of ROS at different time points was determined by using the method as described above. Results were expressed as Relative Fluorescence Unit versus time. 2.2.6.4. Cell death analysis. Phosphatidylserine (PS) gets exposed to external environment during early apoptosis as it comes out from the inner to the outer plasma membrane leaflet. Annexin V-FITC has the ability to bind to PS with high affinity, and propidium iodide (PI) conjugates necrotic cells (Van et al., 1998; Ganta and Amiji, 2009). The double staining with annexin V-FITC and PI was performed to detect apoptotic and necrotic cells. SH-SY5Y cells (1 × 106 /flask) were seeded in cell culture flask (Nunc, 75 cm2 ) and allowed to attach for 24 h. Medium was replenished with fresh medium, and the cells were treated with CSSS, C-PBCA and ApoE3-C-PBCA (2 and 4 M curcumin/well) using SS, and B-PBCA as the respective controls and incubated for 24 h at 37 ◦ C in CO2 incubator to study dose dependent effect. Time dependent effect was investigated by treating the cells with CSSS, C-PBCA and ApoE3-C-PBCA (2 M curcumin/well) using SS, and B-PBCA as the respective controls for 12, 24 and 48 h at 37 ◦ C in CO2 incubator. The medium was removed after the treatment, cells were detached using trypsin-EDTA solution and suspended in fresh medium. Cells were centrifuged at 3000 rpm for 5 min, supernatant was removed and cell pellets were resuspended in Annexin V binding buffer and centrifugation was repeated again. Finally, the cell pellets were resuspended in Annexin V binding buffer (1 × 106 /ml). The cell suspension (100 l) was transferred to 5 ml FACS tube, AnnexinV-FITC (5 l) and 10 l PI were added, and incubated for 15 min at room temperature in dark. Finally, before the FACS analysis, 400 l of Annexin V binding buffer was added. Cells were analyzed by using the flow cytometer (FACSCantoII, BD Biosciences) in FL1 and FL2 channel for FITC and PI respectively. Green (FITC) fluorescence (FL1) was collected at 535 nm, and red (PI) fluorescence (FL2) at more than 550 nm, from 10,000 cells. The results of dose dependent effect are expressed as dot plot from flow cytometry while time dependent effect is expressed as a bar chart indicating the respective percentages of early apoptotic, and late apoptotic and early necrotic. The % of cells was determined by using FACSDiva software. 2.2.6.5. Cell cycle analysis. Cell cycle analysis is a parameter of paramount importance in the detection of apoptotic cells (Weir et al., 2007; Shi et al., 2006). Cell cycle distribution was assessed by the FACS analysis. The effect of CSSS, C-PBCA and ApoE3-C-PBCA on the cell cycle was studied by treating the cells and analyzed using a flow cytometer. Briefly, cells (1 × 106 /flask) were seeded
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in cell culture flask (Nunc, 75 cm2 ) and allowed to attach for 24 h. The cells were then treated with CSSS, C-PBCA and ApoE3-C-PBCA (2 and 4 M curcumin/well), and incubated for 24 h at 37 ◦ C in CO2 incubator. SS, B-PBCA were used as the respective controls. Cells were harvested by centrifugation after the treatment, washed with PBS and fixed in 70% ethanol for 2 h. The cells were then centrifuged, pellets were resuspended in 500 l PBS 7.4 containing RNase (100 g/ml) and kept at room temperature for 30 min. Then, PI (50 g/ml) was added and kept in dark for 30 min to stain DNA. Analysis was carried out using flow cytometer (FACSCantoII flow cytometer (BD Biosciences) in red (FL2) channel at more than 550 nm, from 10,000 cells. The % of subG1 fraction was determined by using FACSDiva software. 2.2.6.6. Detection of mitochondrial membrane potential loss ( m ). In the late stage of apoptosis, loss of mitochondrial membrane potential ( m ) is the main indicative feature. This was detected using lipophilic, cationic fluorescent redistribution dye tetramethylrhodamine methyl (TMRM) ester (Skommer et al., 2006). Briefly, SH-SY5Y cells (1 × 106 /flask) were cultured and allowed to attach for 24 h. Then, the cells were treated with CSSS, C-PBCA and ApoE3-C-PBCA (2 and 4 M curcumin/well), and incubated for 24 h at 37 ◦ C in CO2 incubator. SS, and B-PBCA were used as the respective controls. Medium was removed after the treatment, cells were detached using trypsin-EDTA solution and suspended in fresh medium. The cells were then centrifuged at 3000 rpm for 5 min, and pellets were resuspended in PBS 7.4. The cell suspension (500 l) was transferred to 5 ml FACS tube, TMRM (100 nM) was added and incubated at 37 ◦ C for 20 min in dark. After incubation, 500 l of PBS was further added, and the intensities of red fluorescence of 10,000 cells were analyzed by flow cytometer (FACSCantoII flow cytometer (BD Biosciences) in FL2 channel at more than 550 nm. The % of these low m cells was determined by using FACSDiva software. 2.2.6.7. Detection of caspase-3. Activation of caspases is one of the central feature of apoptosis. During apoptosis, inactive pro-forms of effector caspases (caspase 3, 6, and 7) get cleaved by initiator caspases (caspase 2, 8, 9 and 10) to produce activated effector caspases. These activated caspases then cleave cellular protein substrates to start apoptosis (Vermes et al., 2000). We have detected caspase-3 induction using CaspGLOW Red Active Caspase-3 Staining Kit. Red-DEVD-FMK is cell permeable, nontoxic probe that binds to the activated caspase-3 in apoptotic cells in an irreversible manner. The red fluorescence label allows for direct detection of activated caspase-3 in apoptotic cells by flow cytometry. SH-SY5Y cells (1 × 106 /flask) were seeded in cell culture flask (Nunc, 75 cm2 ) and allowed to adhere for 24 h. Medium was replenished with fresh medium, and the cells were treated with CSSS, C-PBCA, and ApoE3-C-PBCA (2 M and 4 M curcumin/well) for 24 h at 37 ◦ C in CO2 incubator. Cells were detached using trypsin-EDTA solution after the treatment, and fresh medium was added. The cells were separated by centrifugation at 3000 rpm for 5 min and pellets were resuspended in wash buffer (1 × 106 /ml). The cell suspension (300 l) was stained with Red-DEVD-FMK (1 l), and incubated for 1 h at 37 ◦ C in CO2 incubator. The cells were centrifuged at 3000 rpm for 5 min after incubation to remove the supernatant and the cells were resuspended in 0.3 ml wash buffer. Cells were analyzed using flow cytometer (FACSCantoII, BD Biosciences) in FL2 channel. Red fluorescence (FL2) was measured at more than 550 nm, for 10,000 cells. Untreated cells were taken as negative control. An additional control was taken by adding the caspase inhibitor ZVAD-FMK (1 l/ml) to inhibit caspase activation, and then stained with Red-DEVD-FMK. All experiments were performed in triplicate and results were expressed as Mean ± S.D. (n = 3).
2.2.7. Statistical analysis All the experiments were performed in triplicate and results were expressed as Mean ± S.D. (n = 3). Statistical analysis was done by performing Student’s t test. The differences were considered significant for p values of
