CH 2 CH OHO
3.2 Materials and Methods .1 Materials
Pluronic F68 and F127 were purchased from Aldrich (Bangalore, India). Curcumin was procured from Himedia (Mumbai, India). MTT was from SRL (Mumbail, India). All the salts and solvents used in the study were purchased from Merck (Mumbai, India). Cell culture plates were purchased from Corning (USA). All the cell culture media and reagents used in the study were purchased from Sigma (USA). HeLa, cancer cell line was obtained as gift from National Center for Cell Science (Pune, India). All other reagents and buffer solution components were analytical grade preparations. Distilled and deionized water was used in all experiments.
3.2.2 Preparation of drug loaded Pluronic micelles
Curcumin loaded Pluronic micelles was prepared by thin film rehydration method. Stock solutions of curcumin (1 mg/ml) and Pluronic copolymers (10 mg/ml) were prepared in methanol and chloroform respectively. Required amount of stock solutions were transferred to separate tubes to obtain different drug to polymer ratio and drug containing polymer films were prepared by evaporating organic solvent under vacuum. The films were rehydrated in PBS (0.01M, pH 7.4) by extensive vortexing to prepare drug encapsulated micelles. Amount of curcumin incorporated in micelles was estimated spectrophotometrically as reported earlier in Chapter 2. Drug encapsulation and loading in micelle nanocarriers was calculated as follows (Park et al., 2005):
3.2.3 Physicochemical characterization of micelles
UV-Visible spectra of aqueous solution of curcumin encapsulated in Pluronic F68 and F127 micelles were recorded with Cary-100Bio spectrophotometer (Varian). The fluorescence emission spectra of curcumin in same micelle solutions were taken using excitation wavelength of 420 nm with FluoroMax-3 spectroflourimeter (Jobin Yvon, Horiba). The excitation and emission slit widths were 2 nm and 5 nm respectively and the scan rate was 1nm/sec.
amount of curcumin in nanocarrier
Drug Loading (%) = X 100
amount of curcumin loaded nanocarrier Curcumin
= X 100
Curcumin + Pluronic
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amount of curcumin encapsulated
Drug Encapsulation (%) = X 100
amount of curcumin used
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Atomic force microscopy (AFM, Picoscan, Molecular Imaging, USA) was used to determine the shape and morphology of the micellar nanocarriers. 10 Pl of the sample solutions were placed on the freshly cleaved mica and dried under stream of nitrogen. The samples were dried under a stream of nitrogen and mounted on the microscope scanner. The micelle shape was observed and imaged at non-contact mode.
3.2.4In vitro release of curcumin from Pluronic micelle
Release of curcumin from Pluronic micelles was studied at physiological condition (pH 7.4, 37º C). Curcumin (1 mg) loaded Pluronic (100 mg) was prepared in 50 ml physiological buffer (0.01M PBS, pH 7.4) by thin film rehydration method. Non-encapsulated curcumin was separated out by centrifugation. Curcumin loaded micelle solution was placed in incubator at 37º C under gentle agitation (120 rpm). Samples were withdrawn periodically and curcumin was quantified spectrophotometrically. The percentage of curcumin released from the micelles at various time points was calculated by following equation:
3.2.5 Stability studies
Stability of the curcumin encapsulated Pluronic micelles was evaluated for both in solution form and lyophilized form respectively. Curcumin loaded Pluronic aqueous dispersions were stored at 25º C and 4º C in the dark and characterized for drug retention at fixed time intervals up to 10 days after preparation. Lyophilized formulation was obtained by freeze- drying of curcumin encapsulated pluronic micelles in a freeze dryer (Christ, Germany).
During freeze drying the chamber pressure was maintained at 0.035 mbar and temperature Released curcumin
Drug Release (%) = X 100
Total encapsulated curcumin
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was –55 ºC. Stability of the lyophilized formulations was checked similarly as above at the temperature 25 ºC.
3.2.6In vitro cell viability and cellular uptake assay
The cytocompatibility of Pluronic copolymers and the activity of curcumin entrapped into Pluronic micelles for the proliferation inhibition of HeLa carcinoma cells was investigated by in vitro MTT assay and compared with that of free curcumin. Cells (1 × 104/well) were seeded in 96-well cell culture plates and after 24 h were treated with different concentrations of empty pluronics, free curcumin and Pluronic encapsulated curcumin. After 48h of treatment media were carefully removed, cells were washed twice with PBS and 100 µl fresh medium containing 0.5 mg/ml MTT was added to each well. The plates were incubated at 37 °C for 4 h. Then the medium was totally removed and 100 µl DMSO was added to each well to solubilize the insoluble formazan crystals. The absorbance, which was proportional to cell viability, was subsequently measured at 570 nm in each well using a 96 well plate-reader (Bio-Rad, USA). The percentage of cell viability was calculated as follows:
Where, Asample was the absorbance of the cells treated with free or nanocarrier encapsulated curcumin, while Acontrol was the absorbance of the cells without curcumin treatment.
To visualize the uptake of drug encapsulated nanocarriers, HeLa cells were grown on 35mm cell culture plates up to ~80% confluency. After that, the growth medium was replaced with medium containing 20 µM curcumin encapsulated in Pluronic F127 and F68 micelles separately. Cells were incubated at 37 ºC for 4h to allow the uptake of nanocarriers and visualized under inverted fluorescence microscope (Nikon TS100, Japan). We also
sa m p le c o n tro l
C e ll v iab ility (% ) = A x 1 0 0 A
visualized the cells after 48h of treatment with nanocarrier loaded curcumin to observe the change in cellular morphology.