Cyclosporine (Purity 99%) was purchased from Poli Industria Chemica S.P.A. (Rozzano, Milano, Italy). Poly(lactide-co-glycolide) (PLGA, lactide:glycolide = 50:50, inherent viscosity: 0.58 dL/g in hexafluoroisopropanol, Mw≈31,000 Da) was purchased from Lactel International Absorbable Polymers (Pelham, AL, USA). Dichloromethane and chloroform (HPLC grade) was obtained from Fisher Scientific Co. (Norcross, GA, USA). Sodium lauryl sulphate and sodium azide was purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals and solvents used were analytical or HPLC grade.
2.1. Design of experiment
Traditional development of pharmaceutical formulation is based on time and energy consuming approach of changing one variable at a time while keeping other variables constant. Use of experimental design (DOE) technique allows testing of large number of variables simultaneously in a few experimental run. Screening design are the most powerful DOE techniques that determine the most critical factors in the pharmaceutical development. Most common screening design is Plackett–Burman (PB) design that screens large number of factors and identify critical one in a minimal number of run with good degree of accuracy. Generally, number of run needed to investigate the main effects are equal to 2n
or multiple of 4 in PB designs instead of 2 as in the case of full factorial design (Plackett and Burman, 1946
). PB screening design with 12 experiments was constructed using software JMP version 7.0.1 (SAS, NC, USA). The linear equation of the model is as follows:
is the response, b0
is the constant and b1
are the coefficient of factor X1
(representing the effect of each factor ordered within −1, +1).
Independent process and formulation variables selected were drug (X1
), polymer (X2
), and surfactant concentration (X3
), stirring rate (X4
), type of solvent (X5
) and organic to aqueous phase ratio (X6
). The parameter level selection was based on preliminary study and on literature. Parameter studied in preliminary investigation was homogenization time and mechanical stirrer speed. Homogenization time did not have a significant impact on particle size and was kept constant for all the experiments. Mechanical stirrer speeds have impact on nanoparticle size and entrapment and included in the design. Solvents are selected based on the report of Italia et al. who reported effect of solvent on particle size (Italia et al., 2007
). Similarly, level of drug, polymer, surfactant level and external volume are selected based on the literature (Shi et al., 2009
).We could not conduct study all the variables. That is why we selected the ones that we thought are critical. The two levels of independent factors for the screening design and experiment domain of each variable were summarized in and . The dependent variables were encapsulation efficiency (Y1
), particle size (Y2
), zeta potential (Y3
), burst release (Y4
) and dissolution efficiency (DE) (Y5
Experimental factors and their level.
2.2. Preparation of CyA-PLGA nanoparticles
CyA-PLGA nanoparticles were prepared according to emulsification-solvent evaporation technique (Kawashima et al., 1999
). Briefly, CyA and PLGA were codissolved in 10 ml of organic solvent (dichloromethane or chloroform). Sodium lauryl sulphate solution (0.05%, w/v or 0.10%, w/v) was prepared in deionized water. Drug and polymer solution was added drop-wise to surfactant solution to make organic to aqueous phase ratio of 1:10 or 1:20 while stirring at 300 rpm and homogenizing by probe type homogenizer PowerGen 125 (Fisher Scientific, PA, USA) and continued homogenization for 10 min at 6000 rpm after complete addition of organic phase into aqueous phase. The nanoparticles formation and subsequent hardening was effected as a result of solvent evaporation by mechanical stirring at 600 rpm or 900 rpm at room temperature. The nanoparticles were retrieved from the aqueous solution by centrifugation at 49,500 × g
(RC-5C, Sorwall Instruments/Thermo Scientific, MA, USA) for 30 min. The obtained nanoparticles were washed twice with 20 ml of deionized water, frozen at −80 °C and freeze dried in Freeze Dry/Shell Freeze System (Labconco Corp., MI, USA) at −10 °C for 48 h. The dried particles were stored in fridge until further study.
2.3. Drug entrapment efficiency
Five milligrams of freeze-dried nanoparticles were dissolved in 5ml of chloroform, sonicated for 5min and vortexed. Hundred microliters of solution was diluted with 900 µl of the mobile phase for CyA quantization using a Hewlett–Packard (HP) HPLC instrument (Agilent technologies, CA, USA) that consist of a quaternary HP 1050 pump, HP 1050 autosampler, and 1050 HP UV detector set at a wavelength of 203 nm and column compartment thermostated at 70 °C. The HPLC stationary phase was composed of a C8, 4.6mm × 250mm (3.5 µm packing) reverse phase chromatography Zorbax SB-C8 column and a C8, 4.6mm × 12.5mm (5 µmpacking) Zorbax SB-C8 reliance guard column (Agilent technologies, CA, USA). The mobile phase consisted of acetonitrile:methanol:water:phosphoric acid (8:4:3:0.05) and was pumped isocratically at a flow rate of 1.25 ml/min. Each experiment was performed in triplicate and entrapment efficiency (EE) was calculated according to the formula:
2.4. Particle size and zeta potential measurements
Dried nanoparticles were suspended in CyA saturated MiliQ water and sonicated for 2–3 min to obtain a uniform suspension before measurements. The particle size distribution was expressed as mean number and determined by photon correlation spectroscopy at 23 °C (Particle Size/Zeta Potential PSS NICOMP 380 ZLS, Particle sizing Systems, Santa Barbra CA, USA). For zeta potential measurements, nanoparticles were suspended in MiliQ and measurements were made at 23 °C, at a diffraction angle of 14°, under an electrical field of 15 V/cm, by Zetasizer (NICOMP 380 ZLS). The measurements were conducted in triplicate.
2.5. In vitro drug release studies
The dried nanoparticles were also evaluated for in vitro drug release studies by horizontal shaker method. The CyA-PLGA nanoparticles equivalent to 5mg of drug were suspended in 200 ml of phosphate buffer (0.20M) pH 7.4 containing 0.1% (w/v) sodium lauryl sulphate and 0.02% (w/v) sodium azide. Sodium lauryl sulphate was used to maintain sink condition and sodium azide was used to prevent the microbial growth in the release medium. Various replicates were placed on biological shaker at 37 ± 0.5 °C and 120 rpm. 0.5 ml samples were withdrawn at specified time intervals (0.08, 0.16, 0.33, 1, 2, 3, 4, 5 and 7 days) and centrifuged at 14000 rpm for 15 min and supernatant were analyzed for percentage of drug released by RP-HPLC. The experiment was performed in duplicate.
2.6. SEM measurements
Surface morphology and shape of freeze-dried nanoparticles were investigated by SEM (JSM-6390 LV, JEOL, Tokyo, Japan) measurements at the working distance of 15 mm and an accelerated voltage of 20 kV. Nanoparticles were gold coated with sputter coater (Desk V, Denton Vacuum, NJ, USA) before SEM observation under high vacuum and high voltage 10 mV to achieve film thickness of 30 nm.
2.7. Differential scanning calorimetric studies
DSC of CyA, PLGA, physical mixture of drug and polymer and nanoparticles were performed with SDT 2960 Simultaneous DSC/TGA (TA Instruments Co., New Castle, DE, USA). The physical mixture prepared with blank nanoparticles of PLGA and CyA by blending in mortar and pastle. Accurately weigh sample (2–4 mg) were sealed in an aluminum pan and empty pan was used as a reference. The samples were scanned from 50 to 300 °C at a scanning rate of 10 °C/min. Nitrogen was used for purging the sample holders at a flow rate of 20 ml/min.
2.8. Powder X-ray diffraction studies
X-ray diffraction (XRD) experiments were performed on X-ray diffractometer (MD-10 mini-diffractometer, MTI Corporation, Richmond, CA, USA) using Cu K2α rays (λ = 1.54056 Å) with a voltage of 25 kV and a current of 30 mA, in flat plate θ/2θ geometry, over the 2θ ranges 25–70°, with a step width 0.05° and a scan time of 2.0 s per step. Diffraction patterns for CyA, PLGA, physical mixture of drug and PLGA and drug loaded nanoparticles were obtained.
2.9. FTIR studies
FTIR spectra of drug, polymer, their physical mixture and drug loaded nanoparticles were performed by ATR–FTIR (Thermo Nicolet Nexus 670 FTIR, GMIInc., Ramsey, Minnesota, USA), and OMNIC ESP software (version 5.1) was used to capture and analyze the spectra.