mPEG (Mw 5000) and palmitoyl chloride was purchased from Fluka (Bangalore, India) and Aldrich (Bangalore, India) respectively. Curcumin was from Himedia laboratories (Mumbai, India). Porcine pancreatic lipase and pyrene was from Sigma (Bangalore, India). All the solvents used in the study were of analytical grade and obtained from Merck (Mumbai, India).
Human cervical cancer cell line (HeLa) was obtained from National Centre for Cell Science (Pune, India). Cells were maintained in EMEM 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 and 1% antibiotic antimycotic solution (1000 U/ml penicillin G, 10 mg/ml streptomycin sulfate, 5mg/ml gentamycin and 25 µg/ml amphotericin B). Cells were cultured at 37°C in a humidified atmosphere supplied with 5% CO2.
2.2.2 Synthesis of mPEG-PA conjugate
The synthesis of mPEG-PA conjugate was carried out by reacting mPEG with palmitoyl chloride. mPEG (1mM) was dissolved in toluene and mixed with triethylamine (1mM). A
solution of palmitoyl chloride (1.1 mM) in toluene was added drop wise and stirred continuously for 3 h at 60ºC. The solution was then filtered through filter paper (Whatman, grade 1) to remove the precipitated triethylamine hydrochloride salt and clear filtrate was collected in a conical flask. The mPEG-PA conjugate was then precipitated from filtrate solution by adding cold diethyl ether. The precipitate was collected by filtration and dried under vacuum.
2.2.3 Characterization of mPEG-PA conjugate
The purified conjugate was characterized by nuclear magnetic resonance (NMR) spectroscopy and Fourier transformed infrared (FT-IR) spectroscopy. For NMR analysis, the dried conjugate was dissolved in CDCl3 and the spectrum was recorded in 1H NMR (Mercury Plus 400MHz, Varian). The FT-IR spectrum of the conjugate was recorded with a Spectrum One spectrophotometer (Perkin Elmer, USA). A 2% (w/w) mixture of dried conjugate and potassium bromide (KBr) was ground into a fine powder using an agate mortar, compressed into a disc, scanned at a resolution of 1 cmí1 over a frequency region of 450 to 4,000 cmí1 and the characteristic peaks of IR transmission spectra were recorded.
2.2.4 Measurement of critical micelle concentration (CMC) of mPEG-PA conjugate The critical micelle concentration (CMC) of mPEG-PA was determined by using pyrene as a hydrophobic fluorescence probe. The CMC was determined based on the intensity of pyrene excitation spectra and shift of the spectra with increasing mPEG-PA concentrations. The pyrene solutions (6X10-6 M) in acetone were added into the test tubes and evaporated to remove the solvent. Then, solutions of mPEG-PA micelles in PBS (0.01M, pH 7.4) were added to the above test tubes in concentrations ranging from 0.001 to 1 mg/ml, bringing the final concentration of pyrene to 6.0X10-7 M. The solutions were vortexed and kept overnight
at 37ºC to equilibrate pyrene with the micelles. Steady-state fluorescence excitation spectra (Fluoro Max-3, Jobin Yvon, Horiba, USA) of pyrene were recorded from 300 nm to 360 nm keeping emission wavelength fixed at 390 nm with slit width of 2.5 and 5.0 nm for excitation and emission respectively.
2.2.5 Encapsulation of curcumin in micellar nanoparticle
The encapsulation efficiency of curcumin was studied in different concentrations of mPEG- PA. Curcumin solution in methanol was added to the solution of mPEG-PA in chloroform to obtain different drug:polymer ratio ranging from 1:20 to 1:100. Methanol was evaporated under vacuum to produce a film consisting of mPEG-PA/curcumin mixture. Micelles were formed by extensive vortexing of the film in PBS. Nonencapsulated curcumin was separated by centrifugation of the micelle suspension at 5000 rpm (Sigma 4K-15 refrigerated centrifuge, India) for 10 min and quantified spectrophotometrically (Cary 100 BIO UV-VIS spectrophotometer, Varian, USA) at 425 nm. The entrapment efficiency was calculated by following equation:
2.2.6 Physical characterization of drug encapsulated micelle nanostructure 2.2.6.1 Dynamic light scattering (DLS)
Size distribution of empty and curcumin encapsulated mPEG-PA micelles (2.5 mg/ml in 0.01M PBS, pH 7.4) were analyzed by dynamic light scattering (Zetasizer NanoZS, Malvern Instruments, UK) using an argon laser beam at 633 nm and 90° scattering angle.
(Total amount of curcumin - Free curcumin)
Encapsulation efficiency (%) = 100
Total amount of curcumin u amount of curcumin in nanocarrier
Drug Loading (%) = X 100
amount of curcumin loaded nanocarrier Curcumin
= X 100 Curcumin + mPEG-PA
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2.2.6.2 Atomic force microscopy (AFM)
The shape of the free micelle nanoparticles and drug loaded micelle nanoparticles was characterized by atomic force microscopy (Picoscan, Molecular Imaging, USA). A drop of micellar solution (2.5 mg/ml) was placed on freshly cleaved mica. After 1 min incubation the surface was gently rinsed with deionized water to remove unbound micelles. The dried nanoparticle sample was mounted on sample holder using double-sided adhesive tapes and scanned by the AFM maintained in a constant temperature and vibration free environment in non-contact mode.
2.2.6.3 UV-Visible and Fluorescence spectroscopy
The micelle-loaded curcumin was characterized by absorption and fluorescence spectra.
UV-Visible spectra of aqueous solution of curcumin encapsulated in mPEG-PA 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.
2.2.7In vitro stability of drug loaded micelle
100 mg of curcumin-loaded mPEG-PA was dispersed in 10 ml of physiological buffer (0.01M PBS, pH 7.4), simulated gastric fluid (0.2 g sodium chloride and 0.7 ml concentrated HCl in 100 ml water, pH 1.2) and simulated intestinal fluid (0.68 mg KH2PO4 and 7.7 ml of 0.2 M NaOH in 100 ml water, pH 6.8) separately and incubated at 37ºC under gentle agitation. Drug retention was studied at different time intervals ranging from 6 to 48 hours.
The percentage of drug retained was determined with the following equation:
2.2.8 Enzyme catalyzed degradation of micelle and in vitro drug release
To examine the degradation of micelle and site of action of enzyme, 50 mg of mPEG-PA was dissolved in 10 ml of 0.1 M PBS (pH 7.4) containing bovine pancreatic lipase (0.5 mg/ml) and kept at 37ºC. After 48 hr the precipitate was collected and washed several times with water and dried. Dried precipitate was recrystallized from chloroform and analyzed by
1H NMR to confirm the cleavage site.
To estimate the drug release by enzyme catalyzed degradation of micelle, the lipase solution in PBS (0.01 M, pH 7.4) was added to the drug loaded micelle solution at a final concentration of 0.5 mg/ml and incubated at 37°C. Samples were collected at different time intervals ranging from 2 to 48 hours and release curcumin was estimated as described in section 2.2.5. The release was quantified as follows:
To study the drug release by cellular enzymes, cell lysate was obtained from HeLa cells by mechanical lysis followed by centrifugation at 10000g for 10 min at 4°C. The supernatant containing intracellular enzymes was added to the drug loaded micelle solution and drug release was quantified as in section 2.2.5.
2.2.9 Cellular uptake and cytotoxicity studies
To visualize the cellular uptake of drug loaded micelle HeLa cells were grown in 35 mm culture plate up to 80% confluency. Cells were treated with 15 µM of free and micelle encapsulated curcumin. As curcumin is insoluble in aqueous solution the free curcumin was
Released curcumin
Release (%) = 100
Total curcumin u
(Total curcumin encapsulated - Released curcumin)
Drug Retention (%) = 100
Total curcumin encapsulated u
dissolved with the aid of dimethyl sulfoxide (DMSO). The final concentration of DMSO in the culture medium was always < 1%. After 2 hour incubation at 37°C, cells were examined under confocal laser scanning microscope (LSM 510 Meta, Zeiss, Germany) for the intracellular curcumin fluorescence.
The cytotoxicity of empty mPEG-PA nanocariers, free curcumin and nanocarrier encapsulated curcumin was determined by methylthiazoletetrazolium dye (MTT) assay as described by Mosmann (1983). HeLa cells were seeded in 96-well cell culture plate at a density of 1 × 104 cells/well and grown for 24 hours before the assay. The cells were then exposed to a series of different concentrations of free and encapsulated curcumin (1 to 30 µM) for 48 hours. After incubation 100 µl of DMSO was added to each well and absorbance was measured at 570 nm using a microplate reader (Biorad Microplate Reader, Model 680, USA). The cell viability was expressed as a percentage compared to that of the control by following equation:
Where, Nt was the absorbance of the cells treated with free curcumin or curcumin loaded micelles and Nc was the absorbance of the untreated cells. Similarly, different concentrations of empty micelles were used to test the cytotoxicity of the nanocarrier.
Viability (%) = Nt 100 Ncu