Results

CH 2 CH OHO

4.3. Results

Isolated micelles were redispersed as suspension in tris buffer (pH 7.4) containing 10 mM CaCl₂. Ca⁺² are necessary for stable CM suspension preparation (Gatti et al., 1995). The turbidity (W) of purified CM suspension was determined at 400 and 600 nm with different CM concentrations. We observed a linear increase of W with CM concentration at both the wavelength (Fig 4.2A). The wavelength dependence of turbidity (Į) measurements is shown in Fig 4.2B, where the value of Į remained nearly constant with the change in CM concentrations.

The size of the casein micelles measured by DLS revealed that the mean diameter of the protein particles was 166.3 (± 33.1) nm (Fig.4.3). SEM and AFM analysis showed that the nanoparticles were roughly spherical in shape (Fig. 4.4 and 4.5). SEM and AFM data were in good agreement with the DLS measurement. The morphology and size of CM-Curcumin complex was similar to that of free CM.

The baseline spectrum of CM suspension showed high absorbance values because of the high degree of Rayleigh scattering present in the samples (Fig 4.6A). To get the spectra of curcumin a difference spectrum was generated by subtracting the CM suspension spectrum from CM-curcumin spectrum. The difference spectrum revealed that the curcumin bounded to CM shows absorbance maxima at 424 nm (Fig 4.6B).

The binding constant was estimated from the increase of the fluorescence intensity of a fixed concentration of curcumin in the presence of increasingly added concentration of CM. There was a blue shift of the curcumin emission maximum and significant increase in fluorescence intensity with increasing concentration of CM (Fig.4.7). Fig. 4.8 represents the variations of curcumin fluorescence intensity with increasing CM concentrations. The plot shows that the fluorescence intensity of curcumin increased initially and gradually leveled off in the higher

Figure 4.2: Stability of casein micelle (CM) suspensions in Tris buffer (pH 7.4) containing 10 mM CaCl2. (A) Turbidity (W) of purified CM suspension at wavelength of 400 nm (Ÿ) and 600 nm (Ŷ) showing a linear change with different CM concentrations. (B) Change of D (wavelength dependence of turbidity) with different CM concentrations. The D value remained nearly constant.

A

B

Figure 4.3: Size distribution of CM suspension measured by DLS. The average diameter of the CM particles was found to be 166.3 nm.

Figure 4.4: Observation of CM particles under scanning electron microscope (SEM).

Figure 4.5: Morphology analysis of CM particles by AFM. Particles were almost spherical in shape with average size distribution of 200 nm.

Figure 4.6: (A) Absorption spectra of CM suspension (broken line) and CM-curcumin complex (solid line). Difference spectra (B) revealed that the curcumin bound to CM shows absorption maxima at 424 nm.

Figure 4.7: Fluorescence emission spectra of 5 µM curcumin in buffer solution (pH 7.4) in presence of CM at different concentrations (a) 0 (b) 0.5 (c) 1.0 (d) 1.5 (e) 2.0 (f) 2.5 (g) 3.0 (h) 4.0 (i) 5.0 (j) 10.0 (k) 12.5 (l) 15.0 (m) 17.5 and (n) 20.0 µM respectively. Excitation wavelength was 420 nm.

Figure 4.8: Change of curcumin fluorescence intensity at 500 nm in the presence of increasing concentrations of CM. It showed that 17 µM CM is required for saturation of 5 µM curcumin binding.

Figure 4.9: Double reciprocal plot of [CM] vs change in curcumin fluorescence intensity (ټFI). Binding constant (Kb) was estimated to be 1.48 X 10⁴ M^-1 between curcumin and CM from the plot.

concentration of CM. The saturation concentration of CM required for complete binding of 5PM curcumin was 17 PM. The binding constant was determined by the following equation (Gatti et al., 1995; Liang et al., 2008):

Where, 'FI is the change of curcumin fluorescence intensity in the presence and absence of CM; 'FI^maxis the maximal change of curcumin fluorescence intensity; Kb is the binding constant and [CM] is the concentration of casein micelles.

The intensity data was used to plot the double reciprocal plot, 1/ [CM] vs 1/'FI (Fig.4.9).

The intercept of the double reciprocal plot on the 1/'FIaxis measures 1/'FI^max, which was used to calculate the binding constant from the value of slope in the plot. The binding constant was estimated to be 1.48 X 10⁴ M^-1.

Intrinsic fluorescence of protein has been widely used to investigate the interaction and binding of drug molecules to proteins in solution. At the excitation wavelength of 280 nm, both tryptophan (Trp) and tyrosine (Tyr) residues have fluorescence emission but when the excitation wavelength is 295 nm, only the Trp residue shows a fluorescence emission. CM shows strong fluorescence emission with a peak at 342 nm upon excitation at 280 nm.

Fig.4.10 shows the fluorescence emission spectra of CM suspension in the presence of different concentrations of curcumin with an excitation wavelength of 280 nm. The intensities of fluorescence emission of CM at 342 nm decreased gradually with the increase of drug concentration. When excited at 295 nm the CM solution showed fluorescence maxima at 344 nm. Fig. 4.12 shows the fluorescence quenching spectra of CM before and

max max

1 1 1

[ ] FI FI K

b

FI CM

' ' '

after incubation with different concentrations of curcumin at an excitation of 295 nm. The quenching data were also analyzed according to the Stern-Volmer equation:

Where, F0 and F are the fluorescence intensities in the absence and presence of curcumin respectively, [Q] is the drug concentration and KSV is the Stern-Volmer quenching constant.

The Stern-Volmer plot for CM fluorescence quenching by curcumin was shown in Fig. 4.11

& 4.13. The plot of F0/F versus Q was found to be linear in both the cases i.e. for 280 and 295 nm excitation. The Stern-Volmer quenching constant (K_sv) was found to be 11.3X10⁴ M^-1 (for 280 nm excitation and 342 nm emission) and 8.3X10⁴ M^-1 (for 295 nm excitation and 344 nm emission). The Stern-Volmer quenching constant (Ksv) can be further expressed as:

Where, k_q is the fluorescence quenching rate constant and IJ0 is the lifetime of the fluorophore in the absence of quencher. For, caseins the value of IJ0 is known to be approximately 4 X 10^-9 s (Chakraborty and Basak, 2007). The k_qvalues for CM fluorescence quenching by curcumin was calculated from above equation and shown in Table 4.1.

Table 4.1: Quenching of CM fluorescence by curcumin.

Excitation wavelength

(nm)

KSV (M^-1) kq (M^-1s^-1) 280 11.3 X 10⁴ 4.52 X 10¹⁴ 295 8.3 X 10⁴ 3.32 X 10¹⁴

F

0

1 [ ]

F K

^SV

Q

X

0

SV q

K k W

Figure 4.10: Quenching of CM intrinsic fluorescence by curcumin. Fluorescence emission spectra of CM suspension at excitation wavelengths of 280 nm in presence of (i) 0 (ii) 1 (iii) 1.5 (iv) 2.0 (v) 2.5 (vi) 3.0 (vii) 3.5 (viii) 4.0 (ix) 4.5 and (x) 5.0 µM curcumin respectively.

Figure 4.11: Stern-Volmer plot of protein fluorescence quenching when excitation wavelength was 280 nm.

Figure 4.12: Fluorescence emission spectra of CM suspension at excitation wavelengths of and 295 nm in presence of (i) 0 (ii) 1 (iii) 1.5 (iv) 2.0 (v) 2.5 (vi) 3.0 (vii) 3.5 (viii) 4.0 (ix) 4.5 (x) 5.0 µM curcumin respectively.

Figure 4.13: Stern-Volmer plot of protein fluorescence quenching when excitation wavelength was 295 nm.

To further investigate the binding of curcumin to CM, we studied the curcumin fluorescence with dissociated casein micelles and casein hydrolysate. Casein micelles dissociate into smaller subunits (average size of 10 to 20 nm) called submicelles after removal of CCP by chelating agent (Panouillé et al., 2004) (Scheme 4.2). The fluorescence of curcumin complexed with both dissociated and intact CM solution showed well defined peak at 500 nm (Fig 4.14). However curcumin added to a solution of casein hydrolysate, did not showed significant fluorescence intensity (Fig. 4.14).

Scheme 4.2: Dissociation of casein micelle into submicelles by removal of CCP through calcium chelation.

To compare the cytotoxicity of free and CM bound drug, HeLa cells were exposed to a series of equivalent concentrations of free or CM-curcumin complex for 48 hours, and the percentage of viable cells was quantified using MTT assay. A dose dependent decrease in cell viability was noticed in both cases (Fig 4.15). CM-curcumin complex exerts a comparable cytotoxic effect with respect to free curcumin on HeLa cells at the same dose.

The values of IC₅₀ for CM-curcumin complex and free curcumin were found to be 12.69 and

14.85 µM respectively. This indicates that the curcumin remain active after complexation with CM. The cellular uptake study of free curcumin and CM-curcumin complex shows a concentration dependent increase in uptake (Fig 4.16).

Figure 4.14: Fluorescence emission spectra of curcumin in presence of undissociated CM and dissociated CM showed high fluorescence intensity due to complexation by hydrophobic interactions and the same with casein hydrolysate showed poor fluorescence intensity due to lack of hydrophobic domains.

Figure 4.15: Cytotoxicity assay of free curcumin (Ƒ) and CM-curcumin complex (Ŷ) on in vitro cultured HeLa cells. CM-curcumin complex showed comparable cytotoxic effect to free curcumin.

Figure 4.16: Cellular uptake study of CM-curcumin complex (Ŷ) and free curcumin (Ƒ) on in vitro cultured HeLa cells.

We examined morphological changes in the HeLa cells after treatment with CM-curcumin complex by microscopic observation. Intracellular green fluorescence of curcumin proves that the HeLa cells efficiently took up the CM-curcumin complex. The cells treated with the CM-curcumin complex underwent marked morphologic changes compared with the vehicle control (Fig 4.17). CM-curcumin treated HeLa cells underwent retraction of cellular processes and showed apoptotic characteristics such as cell shrinkage, membrane blebbing, rounding etc. with increase in incubation time (Fig 4.17B). When cells were treated with CM-curcumin complex for 24 h, cells displayed a rounded morphology and detached from the substratum (Fig 4.17B3). Following 48 h exposure, cellular fragmentation was extensive and few cells remained adherent (Fig 4.17B4). In contrast, control cells treated with CM without drug were well spread with flattened morphology shows nontoxicity of CM. We also observed the intracellular green fluorescence of curcumin in case of treated cells. No fluorescence was observed in case of control cells.

Figure 4.17: Microscopic observation of CM-curcumin complex induced morphological changes with time in HeLa cells. (A1-A4) Control cells treated with CM without drug showed no change of morphology with time. (B1-B4) Cells treated with CM-curcumin complex containing 30 µM curcumin exhibited marked morphological changes due to apoptosis. (C1- C4) Intracellular green fluorescence of curcumin revealed that the complex was efficiently internalized into the cells.