CH 2 CH OHO
4.2. Materials and Methods 1 Materials
Curcumin, MTT and casein hydrolysate was purchased from Himedia Laboratory (Mumbai, India). Calcium chloride and Tris-base were purchased from Merck (Mumbai, India). SDS- PAGE molecular weight markers were from Sigma (Bangalore, India). All other reagents used were of analytical grade.
4.2.2 Purification of casein micelles
Casein micelle was purified from fresh cow milk collected from local dairy according to Diaz et al (1996) with slight modifications. The milk was skimmed by centrifugation at 1500 g for 30 minute at 4ºC. The skimmed milk was stored at 4ºC with 0.05% (w/v) sodium azide to prevent microbial growth if not used immediately. Casein micelles were purified from skimmed milk by centrifuging at 25000 g for 30 minute at 20ºC. The pellet containing casein micelle was then redispersed in Tris buffer (10 mM, pH 7.4) containing 10 mM CaCl2. This centrifugation redispersion process was repeated five times to wash out any whey proteins that are loosely adsorbed over the casein micelle surface. The casein content in the solution was measured by Bradford assay (Bradford, 1976) using bovine serum albumin (BSA) as standard.
4.2.3 Gel electrophoresis analysis
SDS-PAGE was carried out as described by Laemmli (1970) using a Bio-Rad mini gel electrophoresis unit. Total milk protein and isolated CM samples were loaded onto a 12%
gel and electrophoresed under constant current (20 mA). Proteins in the gel were stained using coomassie brilliant blue R-250. The gel was photographed using a gel documentation system (BioRad, USA).
4.2.4 Stability of casein micelle suspension
To determine the stability of the CM suspension upon dilution we measured “the wavelength (O) dependence of turbidity (W) of the suspensions”, represented as (Gatti et al., 1999):
Where, D was obtained from the slope of log W vs log O plots in the 400-800 nm wavelength range. Turbidity measurements were done using UV-visible spectrophotometer (Cary-100 Bio, Varian). Turbidity (W) was calculated using the following equation (Pitkowski et al., 2008):
Where, I and I0 were transmitted intensity and incident intensity of light respectively. L was the sample path length. In our case L was 1 cm. The measurements were done with freshly prepared samples.
4.2.5 Physical characterization of CM suspension
The size of CM solution was measured in a dynamic light scattering particle size analyzer (LB-550, Horiba, Japan) equipped with a 650 nm laser light source. Measurements were carried out at 90º scattering angle at room temperature.
log log d d D W
O
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I
01 ln I L W § · ¨ ¸
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Morphology evaluation of the CM was performed by scanning electron microscopy (SEM) and atomic force microscopy (AFM). For SEM analysis a drop of CM solution was placed on foil paper, air dried, coated with gold in a sputter coater (Quorum Technologies, UK) and observed under electron microscope (LEO 1430 VP, UK). For AFM analysis a drop of CM solution was placed over freshly cleaved mica surface and dried under nitrogen stream.
Images were taken in non-contact mode (Picoscan, Molecular Imaging, USA).
4.2.6 Fluorescence Spectroscopy
The binding of curcumin with casein micelles was quantified by fluorescence spectrophotometry. Steady state fluorescence measurements were carried out in a Spex FluoroMax-3 spectrofluorimeter (HORIBA Jobin Yvon Inc, USA). The fluorescence of curcumin was measured by keeping its concentration constant at 5 µM and varying the CM concentration from 0 to 20 µM. The emission spectra were recorded from 450 to 700 nm with an excitation wavelength of 420 nm. The slit widths were 2 and 5 for excitation and emission respectively. CM solutions without curcumin were used as controls for fluorescence measurements.
Protein intrinsic fluorescence was measured at constant CM concentration (10 µM) in the presence of 0, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 and 5 µM curcumin. Emission spectra were recorded from 300 to 450 nm (at an excitation wavelength of 280 nm) and from 315 to 450 nm (at an excitation wavelength of 295 nm) individually. In this case free curcumin solutions without CM were used as control and fluorescence was recorded similarly.
In both cases the fluorescence spectra of controls were subtracted from respective spectra of samples to cancel out any contribution due to Raman peak and other scattering artifacts. For
calculation of binding constant, corrections for inner filter effect was made using the following equation (Gatti et al., 1998):
Where FIcorr is the corrected fluorescence intensity and FIobs is the background subtracted fluorescence intensity of the sample. Aex and Aem are the measured absorbance of the samples at the excitation and emission wavelengths, respectively.
To study the binding of curcumin to the submicelles, casein submicelle solution was prepared by dissociating CM with 0.1M sodium citrate. The dissociation of CM was confirmed by reduction in turbidity of the suspension (Panouillé et al., 2004). Curcumin was added to this solution at a final concentration of 5 µM. The same concentration of curcumin was also added to a solution of casein hydrolysate. The curcumin fluorescence was recorded in both the solutions as above and compared with that of intact CM solution.
4.2.7 Cell Culture and cytotoxicity assay
Human cervical cancer cell line HeLa was a gift from National Centre for Cell Sciences (Pune, India). Cells were maintained in DMEM containing 2 mM L-glutamine, 1.5 g/L sodium bicarbonate, 0.1 mM non essential amino acids and 1.0 mM sodium pyruvate supplemented with 10% FBS (heat inactivated) and 1% antibiotic-antimycotic solution (1000 U/ml penicillin G, 10 mg/ml streptomycin sulfate, 5mg/ml gentamycin and 25 Pg/ml amphotericin B). Cells were cultured at 37°C in a humidified atmosphere supplied with 5%
CO2.
0.5( Aex +Aem )
10
corr obs
FI FI u
The cell viability was assessed by MTT assay, which is based on the reduction of MTT by the mitochondrial dehydrogenase of live cells to a purple formazan product (Mosmann, 1983). HeLa cells (1x104) were seeded in a 96-well plate (CellBind, Corning, USA). After 24 h of growth the medium was exchanged by the medium containing each of the following substances: empty CM, free curcumin and CM-curcumin complex. The curcumin stock solution (5 mM) was prepared in ethanol. From the stock solution, aliquots of curcumin were rapidly added to the culture medium to give the final concentrations of free curcumin.
In case of CM-curcumin complex, the stock curcumin was diluted with CM solution in culture media. After 48 h treatment, media were removed and cells were washed with phosphate buffer saline (PBS). Then 100 µl of MTT (0.5 mg/ml) in culture medium was added to each well and incubated for 4 h at 37 ºC. After incubation the media was removed and 100 µl of DMSO was added in each well to solubilize the formazan crystals. Amount of formazan formed in each well was determined by measuring the absorbance at 570 nm using a multiwell plate reader (Biorad Microplate Reader, Model 680, CA, USA). The cell viability was calculated by following equation:
Where, Atreated was the absorbance of the treated cells and Acontrol was the absorbance of untreated cells. The IC50 was measured as the concentration of drug at which 50% cells were viable in comparison with that of the control.
treated control
Cell Viability (%) = A x 100 A
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4.2.8 Cellular uptake of curcumin
HeLa cells were seeded in a 24 well plate at a seeding density of 1x104 cells per well in 1 ml of growth medium and allowed to attach for 24 h. For the studies of concentration dependent uptake, cells were treated with equivalent dose of free curcumin and CM-curcumin complex and incubated for 4 h. After the incubation, media was removed and cells were washed twice with PBS. To extract curcumin, cells were lysed by adding methanol. The cell lysate was centrifuged at 10000 rpm for 10 minute at 4ºC. Curcumin content in the supernatant was measured using fluorescence spectrophotometer (Oex = 420 nm and Oem = 540 nm).
4.2.9 Microscopic study
The effect of CM-curcumin complex on the morphology of HeLa cells was assessed by microscopy. Cells were seeded onto 35 mm culture plates (CellBind, Corning, USA) and incubated for 24h for attachment and then treated with CM-curcumin complex containing 30 µM curcumin. Control experiments were carried out by treating similarly plated cells with CM alone. The plates were taken out from the incubator at different time intervals and observed under inverted phase contrast microscope (Eclipse TS100, Nikon, USA) with 40X objective. Photographs were taken by a digital camera (Coolpix 5400, Nikon, USA) attached with the microscope. Simultaneously, fluorescence was observed by excitation with blue filter.