2.1 Materials
Hydrogenated soya phosphatidylcholine (HSPC), distearoyl phosphatidyl ethanolamine (DSPE), and cholesterol (Chol) were purchased from Lipoid. Triolein was purchased from Sigma Aldrich, USA. Transferrin (Human, low endotoxin) was purchased from Calbiochem, USA. Curcumin was a kind gift from Indsaaf Inc., Batala, India. 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) and coomassie brilliant blue G dye were purchased from Himedia, Mumbai, India. AnnexinV-FITC and propidium iodide were purchased from Biolegend Europe BV, Netherlands. 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA), tetramethyl rhodamine methyl ester were purchased from Sigma Aldrich, Germany. Growth medium RPMI 1640 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 curcumin-loaded solid lipid nanoparticles
The SLN were prepared by a method as described previously (Gupta et al. 2007; Mulik et al. 2010). In brief, HSPC/DSPE/Chol/triolein (1.5:1:1.2:1; w/w) were melted at 80 °C. Curcumin was dissolved in ethanol separately and added to lipid melt phase at 80 °C. Melt phase was injected rapidly to the aqueous phase (0.1 % w/v poloxamer 188, 80 °C) using Ultraturrax high-speed homogenizer at 25,000 rpm speed and homogenized for 15 min. The obtained hot premix was then passed through high pressure homogenizer at 800 bar pressure for 10 cycles at 80 °C. The suspension was immediately cooled to 4–8 °C after homogenization and filtered through 0.2 μm polyethersulfone membrane filter. The pellets of nanoparticles were formed using ultracentrifugation at 25,000×g for 30 min at 4 °C and resuspended in distilled water for lyophilization (LyoPro 3000, Heto-Holten A/S, Allerod, Denmark) using mannitol as a cryoprotectant.
2.2.2 Conjugation of Tf to C-SLN
Covalent coupling of Tf by its carboxyl group to the amino group of DSPE present on the surface of curcumin-loaded solid lipid nanoparticles (C-SLN) was carried out using EDC coupling reaction through amide bond formation as described previously (Gupta et al. 2007; Mulik et al. 2010). Unbound Tf was removed from Tf-C-SLN by centrifugation using Avanti J-301 centrifuge (Beckman Coulter, USA) at 25,000×g for 10 min.
2.2.3 Characterization of SLN
Optimization of preparation of SLN
The preparation of SLN was optimized using 32 factorial design. The variables were determined from the preliminary experiments. The weight ratio of lipids and amount of stabilizer were taken as independent variables and their effect on dependent variables such as, particle size, zeta potential, and percent drug entrapment (PDE) was investigated for optimization (Padamwar and Pokharkar 2006). The particle size of C-SLN and Tf-C-SLN was determined using dynamic light scattering with Malvern Hydro 2000 SM particle size analyzer (Malvern Instruments, UK). For the measurement, the laser obscuration range was maintained between 2 and 5 % (Page-Clisson et al. 1998). Homogeneous size distribution was confirmed from the polydispersity index (PdI). The zeta potential was measured by Laser Doppler Velocimetry using Zetasizer 3000, Malvern Instruments, Malvern, UK at 25 °C. The entrapment efficiency of SLN was determined using the ultracentrifugation method at 18,000×g for 30 min (Beckmann TL-100, MN) (Jenning et al. 2000). In brief, the pellets of SLN were obtained by ultracentrifugation at 18,000×g for 30 min, supernatant was decanted, and curcumin was extracted from the pellets using ethanol. The lyses solution was further diluted with phosphate-buffered saline (PBS) at pH 7.4 and analyzed for curcumin content using ultraviolet–visible (UV–vis) spectrophotometer (V-530, Jasco, Japan) at 424 nm.
Characterization of optimized C-SLN and Tf-C-SLN
Bradford assay using coomassie blue G (Bradford 1976) was used for the quantification of conjugation of Tf on the surface of the C-SLN as per our published research (Mulik et al. 2010). Conjugation efficiency is expressed as milligrams of Tf per millimolar of phospholipids. Transmission electron microscopy (TEM) was performed using Hitachi S-7500 transmission electron microscope (Hitachi, Japan) to determine morphological characteristics and particle size of both the SLN (Mulik et al. 2010).
Stability study
The stability of curcumin in curcumin-solubilized surfactant solution (CSSS) and C-SLN was studied for 6 months at 40 °C temperature and 75 % relative humidity in the presence of light by determining the drug content using HPLC method coupled with UV–vis detector as previously described (Mulik et al. 2010). Change in particle size and zeta potential of SLN was also observed over a period of 6 months to determine physical stability of SLN.
2.3 1H NMR study
Nuclear magnetic resonance (NMR) spectra of CSSS, blank solid lipid nanoparticles (B-SLN), and C-SLN were obtained using Varian (Mercury YH-300 MHz) NMR instrument using CDCl3 (Merck, Germany) as a solvent and tetramethylsilane as an internal standard. The samples dissolved in CDCl3 were spinned at 20 RPS at a power level of −5 db. The pulse length was 5 μs with a delay time of 10 s. NMR chemical shifts (δ) are reported in parts per million (Mulik et al. 2009; Reddy and Murthy 2004).
2.4 Differential scanning colorimetry of nanoparticles
Thermal analysis of CSSS, B-SLN, and C-SLN was used to provide additional information on the lipid–drug relationship and the nature of formed nanoparticles (Jenning et al. 2000 and Tiyaboonchaia et al. 2007). A Mettler Toledo DSC 821e equipped with an intracooler (Mettler Toledo, Switzerland) was used for characterization with a 5 mg sample in hermetically sealed aluminium pans heated from 25 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.
2.5 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 % fetal 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.
2.6 MTT assay
SH-SY5Y Cells (2 × 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 CSSS, C-SLN and Tf-C-SLN (2, 4, 8, 16, 32, and 64 μM curcumin/well) using surfactant solution (SS) and B-SLN as the respective controls and incubated for 24 h at 37 °C in CO2 incubator. After the treatment, medium was removed, cells were washed three times with PBS and fresh medium was added. Then, 25 μl of MTT (5 mg/ml in PBS) was added to the cells and incubated for 3 h at 37 °C in CO2 incubator. MTT gets reduced to purple formazan in living cells by mitochondrial reductase. This reduction takes place only when reductase enzymes are active, and therefore this conversion is used as a measure of viable (living) cells. Then, the cells were lysed and the dark blue crystals were solubilized with 125 μl of a lysis solution (50 % (v/v) N,N,dimethylformamide and 20 % (w/v) sodium dodecylsulphate, pH 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 defined as the relative absorbance of treated cells vs. respective controls. Results were expressed as percent viability vs. dose.
Receptor blocking experiment was carried out to confirm receptor-mediated endocytosis of Tf-C-SLN. Cell surface receptors were blocked by incubating the cells with an excess amount of free Tf for 1 h prior to incubation with Tf-C-SLN, and the effect on cell viability was determined after 24 h treatment (Sahoo and Labhasetwar 2005). For this experiment, 4 μM dose of curcumin was used.
The effect on cell viability with treatment of different time intervals was also assessed. In brief, the cells (1 × 105/well) were seeded in 24-well plate and allowed to attach for 24 h. Then, the medium was replaced with fresh medium and treated with CSSS, C-SLN and Tf-C-SLN (4 μM curcumin/well) using SS and B-SLN 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 percent cell viability vs. time.
2.7 Cell uptake study
Intracelllar uptake of curcumin by SH-SY5Y cells treated with CSSS, C-SLN, and Tf-C-SLN was evaluated using fluorescence microscopy (Weir et al. 2007) and spectrophotometry (Kunwar et al. 2008). For quantitative cell uptake, the cells were treated as described above (10 μM curcumin/well) for different time intervals (3, 6, 12, 24, and 48 h). At each time point, the cells were trypsinized and centrifuged for 3 min at 3,000 rpm. Cell pellets were resuspended in 1 ml methanol and vortexed for 5 min to extract curcumin in methanol fraction. The lysate was then centrifuged at 5,000 rpm for 5 min and absorbance of supernatant containing methanolic curcumin was measured at 428 nm using UV–vis spectrophotometer (V-530, Jasco, Japan). From the calibration plot of methanolic curcumin at 428 nm, the amount of curcumin loading into the cells was determined (Kunwar et al. 2008). The effect on Tf receptor blocking on cell uptake of Tf-C-SLN was also assessed after 24 h treatment with 10 μM curcumin. The uptake of curcumin was expressed as μg of curcumin per 105 cells.
For qualitative estimation of uptake of curcumin by cells, the autofluorescence of curcumin was observed using fluorescence microscopy. Cells (1 × 105/well) were seeded in 24-well plate and allowed to attach for 24 h. By replacing the medium with fresh medium, cells were treated with CSSS, C-SLN and Tf-C-SLN (20 μM curcumin/well) for different time intervals (6, 12, 24, and 48 h). After each time interval, fresh medium was added by prewashing the cells thrice with PBS and fluorescence images of curcumin were captured using Nikon Eclipse TE300 fluorescence microscope with Nikon F601 camera (Nikon, Japan).
2.8 Measurement of ROS
The formation of ROS was evaluated by means of the probe 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA) as described previously (Wang and Joseph 1999). Briefly, SH-SY5Y cells (2 × 104/well) were seeded into 96-well plates and allowed to attach for 24 h. After 24 h, fresh medium was added and cells were treated for 24 h at 37 °C in CO2 incubator with different concentrations of CSSS, C-SLN, and Tf-C-SLN (2, 4, 8, 16, 32, and 64 μM curcumin/well) using SS and B-SLN as the respective controls. After treatment, cells were washed thrice with PBS; H2DCF-DA was added (10 μM) and incubated for 2 h at 37 °C. H2DCF-DA passively diffuses into cells to get converted to H2DCF by intracellular esterases and gets entrapped within the cell. H2DCF get rapidly oxidized to the highly fluorescent 2′,7′-dichlorofluorescein in presence of intracellular ROS. Fluorescence intensity was measured at an excitation wavelength of 502 nm and an emission wavelength of 520 nm using Envision 2104 Multilabel Reader (PerkinElmer Life Sci, USA).
In receptor blocking experiment, cell surface Tf receptors were blocked by incubating cells with an excess amount of free Tf for 1 h prior to incubation with Tf-C-SLN and the effect on ROS generation was determined after 24 h treatment (Sahoo and Labhasetwar 2005). For this experiment, 4 μM dose of curcumin was used.
The effect of time of treatment on the generation of ROS was also evaluated. In brief, the cells (1 × 105/well) were seeded in 48-well plate and allowed to attach for 24 h. Fresh medium was added to the cells and treated with CSSS, C-SLN, and Tf-C-SLN (4 μM curcumin/well) using SS and B-SLN as the respective controls for 3, 6, 12, 24, and 48 h at 37 °C in CO2 incubator. Generation of ROS at different time intervals was determined as described above. Results were expressed as relative fluorescence unit vs. time.
2.9 Cell death analysis
In early apoptotic stage phosphatidylserine (PS) makes way to the outer plasma membrane leaflet. Annexin V-FITC has the ability to bind specifically to PS with high affinity while propidium iodide (PI) conjugates to necrotic cells (Ganta and Amiji 2009). Hence, double staining with annexin V-FITC and PI to detect apoptotic and necrotic cells was performed. SH-SY5Y cells (1 × 106/75 cm2 flask) were seeded and allowed to attach for 24 h. Fresh medium was added and cells were treated with CSSS, C-SLN, and Tf-C-SLN (2 and 4 μM curcumin/well) using SS and B-SLN as the respective controls for 24 and 48 h at 37 °C in CO2 incubator. Cells were collected by trypsinization after treatment, suspended in a fresh medium, and washed with PBS two to three times. Finally, cell pellets were resuspended in Annexin V binding buffer (1 × 106/ml); 100 μl of this cell suspension was stained with 5 μl AnnexinV-FITC and 10 μl PI and incubated for 15 min in dark at room temperature. Finally, 400 μl of Annexin V binding buffer was added. Treated unstained cells (1 × 106/ml) were used as a positive control to detect the possible autofluorescence of curcumin in green (FL1) channel. Approximately 10,000 cells were analyzed by using the flow cytometer (FACSCantoII, BD Biosciences) in FL1 (535 nm) and FL2 channels (>550 nm) for FITC and PI, respectively. Four distinct cell populations were clearly distinguishable from the quadrant gating viz viable (lower left quadrant, AnnexinV-FITC−PI−), early apoptotic (lower right quadrant, AnnexinV-FITC+PI−), late apoptotic and early necrotic (upper right quadrant, AnnexinV-FITC+PI+), and late necrotic (upper left quadrant, AnnexinV-FITC−PI+). The results were analyzed using FACSDiva software. Tf-R-mediated endocytosis of Tf-C-SLN was confirmed from the receptor blocking experiment where cell surface Tf-R were blocked by treatment of cells with excess amount of free Tf for 1 h prior to the treatment with Tf-C-SLN (4 μM for 24 h).
2.10 Cell cycle analysis
Degradation of DNA is an important parameter of detection of apoptosis (Weir et al. 2007). Cell phase distribution was assayed by the determination of DNA contents. In brief, cells (1 × 106/75 cm2 flask) were allowed to attach for 24 h and treated with CSSS, C-SLN and Tf-C-SLN (2 and 4 μM curcumin/well) using SS and B-SLN as the respective controls for 24 h at 37 °C in CO2 incubator. Cells were harvested by centrifugation after treatment, washed with PBS two to three times and fixed in 70 % ethanol for 2 h. The cells were then centrifuged, washed and resuspended in 500 μl PBS containing RNase (100 μg/ml) at room temperature for 30 min. Cellular DNA was then stained with PI (50 μg/ml) and kept in dark for 30 min to stain DNA. The cell cycle was analyzed by a flow cytometer (FACSCantoII) flow cytometer (BD Biosciences) in red (FL2) channel at more than 550 nm, from 10,000 cells. The percent of subG1 fraction was determined by using FACSDiva software.
2.11 Detection of caspase 3
Caspase activation is an important marker of detection of apoptosis. Initiator caspases (caspases 2, 8, 9, and 10) produce activated effector caspases from cleavage of inactive pro-forms of effector caspases (caspases 3, 6, and 7) which in turn cleave cellular protein substrates to start apoptosis (Skommer et al. 2006; Vermes et al. 2000). Herein, we have detected the induction of caspase 3 using CaspGLOW Red Active Caspase 3 Staining Kit. Red-DEVD-FMK is cell permeable, nontoxic probe which binds irreversibly to the activated caspase 3 in apoptotic cells. The red fluorescence label allows for direct detection of activated caspase 3 in apoptotic cells by flow cytometry. Briefly, SH-SY5Y cells (1 × 106/75 cm2 flask) were allowed to attach for 24 h and treated with CSSS, C-SLN, and Tf-C-SLN (2 and 4 μM curcumin/well) using SS and B-SLN as the respective controls for 24 h at 37 °C in CO2 incubator. Cells were collected by trypsinization after treatment, washed three times with PBS and resuspended in wash buffer (1 × 106/ml) by centrifugation at 3,000 rpm for 5 min. Three hundred microliters of this cell suspension was stained with 1 μl Red-DEVD-FMK and incubated for 1 h at 37 °C in CO2 incubator. After incubation, cells were centrifuged at 3,000 rpm for 5 min to remove the supernatant and 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 prepared by adding the caspase inhibitor Z-VAD-FMK at 1 μl/ml to inhibit caspase activation and then stained with Red-DEVD-FMK. All experiments were performed in triplicate and results were expressed as mean ± SD (n = 3).
2.12 Statistical analysis
All the experiments were performed in triplicate and results were expressed as mean ± SD (n = 3). Statistical analysis was done by performing Student’s t test. The differences were considered significant for p values of <0.05.