2.1 Materials
Felodipine was a kind gift from Cadila Healthcare Limited (Ahmedabad, India). Poly (D, L-lactic-co-glycolic) acid (PLGA 50:50) (Mw = 18,000) was procured from Sigma Aldrich. Lutrol® F-68 (Poloxamer 188) was purchased from SD Fine Chemicals. Distilled- deionized water was prepared with Milli-Q plus System (Elix 10, Millipore corp. India). All other chemicals used were of the highest available grade.
2.2 Preparation of drug loaded PLGA nanoparticles
The felodipine loaded nanoparticles were prepared by single emulsion solvent evaporation in accordance with method reported with slight modification [17]. Briefly, 100 mg of PLGA (copolymer ratio 50:50) polymer and weighed quantity of felodipine (equivalent to 20% w/w dry weight of polymers) was dissolved in acetone at room temperature to make clear solution. The solution was mixed with 25 ml of aqueous phase containing 1% (w/v) of poloxamer-188 with a constant flow rate of 1 ml/min. The mixture was then homogenized using a probe homogenizer (VIRTIS, Cyclone IQ, USA), at constant agitation speeds of 15000 rpm in an ice bath to form oil-in-water (O/W) emulsion. Then the emulsion was kept at room temperature under gentle stirring for 24 h to evaporate the organic solvent. The prepared nanosuspension was then centrifuged at 30,000 rpm, for 15 min (Sorvall Ultracentrifuge, USA). The pellets were collected and washed with double distilled water to remove any unbound poloxamer-188 and free drugs. Finally, the pallets were freeze-dried (Freezone 6lt, Labconco Corp., MO) to get powdered nanoparticles and kept in freeze for further use.
2.3 Physico-chemical characterization of nanoparticles
2.3.1 Particle size and zeta potential
Dynamic laser scattering (DLS) was used to measure the hydrodynamic diameter (d nm) and Laser Doppler Anemometry (LDA) was used to determine zeta potential (mV). The DLS and LDA were analyzed using Zetasizer 3000 (Malvern Instruments, Malvern, UK). The particle size and zeta potential was determined by using a dilute suspension of nanoparticles (100 μg/ml) prepared in double distilled water and sonicated on an ice bath for 30 seconds. For each batch of sample, the mean diameter ± standard deviations of three determinations were calculated under identical conditions. The polydispersity index (PDI) was also measured to determine particle size distribution.
2.3.2 Estimation of drug entrapment efficiency and loading capacity by RP-HPLC method
The entrapment efficiency (EE) and loading capacity (LC) of felodipine loaded nanoparticles were measured by reverse phase High Performance Liquid Chromatography (RP-HPLC) method [18] with slight modification. Briefly, 1 mg/ml drug loaded nanoparticle solution was prepared in methanol and 20 μL of the sample was injected manually to HPLC equipped with Shimadzu LC-20 AD PLC pump and SPD-M20A PDA detector. The output signal was monitored and integrated using Shimadzu CLASS-VP Version 6.12 SP1 software. The chromatographic separation was carried out using analytical column Phenomenex C18 (150 × 4.6 mm, 5 μ). The measurements were made at 240 nm maintaining the flow rate at 1.0 mL/min and ambient condition using thermostat. The amount of the drug present in the sample was determined from the peak area of the chromatogram correlated with the standard curve.
2.3.3 Fourier transforms infrared spectroscopy (FTIR)
Infrared spectroscopy study was carried out using FT-IR spectrophotometer (Perkin Elmer, FT-IR Spectrometer, SPECTRUM RX I, USA) and the spectrum was recorded in the range of 4000–400 cm−1 with resolution of 2 cm−1. Samples were mixed separately with potassium bromide (200–400 mg) and compressed by applying pressure of 200 kg/cm2 for 2 min in hydraulic press to prepare the pellets. The pellets of the native drug, polymer and the nanoparticles were analysed by placing it on the light path and the spectrum was obtained.
2.3.4 Differential scanning calorimetry (DSC)
DSC is a thermo-analytical technique used to observe fusion and crystallization events of the drug in the prepared nanoparticles. Thermogram was obtained by the DSC analysis (DSC-60, Shimadzu, Japan). Approximately, weighed 2 mg of native drug, polymer and nanoparticles were placed separately into sealed standard aluminium pan and scanned between 25°C to 300°C with heating rate of 10°C/minute under nitrogen atmosphere. An empty aluminium pan considered as reference.
2.3.5 Scanning electron microscopy (SEM)
The shape and surface morphology of prepared nanoparticles were examined by scanning electron microscopy (SEM) (ZEISS EVO18, Carl Zeiss SMT GmbH, Germany). Moisture free lyophilised powdered samples were consigned on aluminium stubs using adhesive tapes and coated with gold using sputter coater and photomicrographs were taken at an acceleration voltage of 10–30 kV.
2.3.6 Atomic force microscopy (AFM)
Atomic force microscopy (AFM) studies were carried out to characterize the surface morphology of the drug loaded nanoparticles. The suspension of freeze dried powder was prepared with milli-Q water and dried in air on a clean glass surface for overnight. The observation was performed with AFM (JPK NanoWizard II, JPK instrument, Berlin, Germany) with silicon probes with pyramidal cantilever having force constant of 0.2 N/m. The scan speed of 2 Hz and 312 kHz resonant frequency was used to obtain the images [19].
2.3.7 In vitro drug release study and release kinetics
The in vitro drug release study was carried out by using rotating basket method with some modification [20],[21]. The felodipine loaded nanoparticles (containing 5 mg felodipine) were suspended in glass bottles containing 100 ml of phosphate buffer pH 6.8. Glass bottles were placed in beaker and kept in incubator shaker (50 rpm) throughout the study, with temperature adjusted to 37°C. At specified time interval 10 ml samples were withdrawn and centrifuged at 12,000 rpm for 30 min. The precipitates were resuspended with 10 ml of fresh phosphate buffer and added to the glass bottle and supernatants were collected and analyzed by RP-HPLC. All the measurements were carried out in triplicate.
The drug release mechanism from the nanoparticulate systems were analyzed by various mathematical models. The drug release data were fitted with mathematical models including zero order kinetic [Eq. (1)], first order kinetic [Eq. (2)], Higuchi kinetic [Eq. (3)] and Korsmeyer-Peppas model [Eq. (4)].
(2)
The plots were made: Qt vs. t (zero order kinetic), ln (Q0 - Qt) vs. t (first order kinetic) and Qt vs. t1/2 (Higuchi model), where Qt is the percentage of drug release at time t, Q0 is the initial amount of drug present in the formulation and K0, Kt and Kh are the constants of the equations. The first 60% drug release was fitted in Korsmeyer-Peppas model, where Mt/Mα are the fraction of drug release at time t, Kp is the rate constant and “n” is the release exponent. The value of “n” is calculated from the slop of the plot of log of fraction of drug released (Mt/Mα) vs. log of time to characterize the different release mechanism [20],[21].
2.4 In vivo studies
2.4.1 Animals
Wistar albino mice were procured from Central Animal House, Rajah Muthiah Medical College, Annamalai University, India and housed in the Institutional animal house under standard environmental conditions (22 ± 3°C, 55 ± 5% humidity and 12 h/12 h dark/light cycle) and maintained with free access to standard diet and water ad libitum. All animal experimentations were executed in compliance with the guidelines of the Committee for the Purpose of Control and Supervision of Experimental Animals (CPCSEA), New Delhi, Government of India.
2.4.2 Acute oral toxicity study in mice
The animals were randomly selected and assigned to following four test groups (6 mice in each group) namely Group I (Control groups, treated with normal saline), Group II, III, IV and V (Test groups, treated with felodipine loaded nanoparticles equivalent to 60, 120, 240 and 480 mg/kg BW of felodipine in distilled water). The respective doses of felodipine loaded nanoparticles were freshly prepared and administered by oral gavaged in a single dose. Acute toxicity was measured by mortality and survival time and also by clinical picture of intoxication and behavioural reactions. Animals on study were observed for any adverse reaction, like changes of body weight, condition of eye, nose and motor activity. On completion of the treatment, the animals were bled via the retro orbital plexus and sacrificed by cervical dislocation and necropsied to facilitate gross pathological examination of organs, viz. size and appearance of heart, lungs, liver, spleen and kidney [22].
2.4.3 Histopathological studies
The animals were sacrificed at the end of the 14 days and the organic tissues of heart, liver, spleen and kidneys were collected and fixed in 10% formalin then subjected to histopathological examination. The tissues of organ samples were embedded in paraffin blocks, then sliced and placed onto glass slides. After histological staining the slides were observed and photos were taken using topical microscope and histopathological examination performed [23].
2.4.4 Biochemical assay
The blood samples were collected at 14th day and centrifuged at 4000 rpm for 5 min. The serum was kept at −20°C until analyzed. The levels of serum glutamate oxaloacetic transaminase (SGOT), serum alkaline phosphate (SAP), serum glutamic pyruvic transaminase (SGPT), serum creatinine, serum bilirubin and proteins were analyzed with automatic analytical instrument (Hitachi, Japan) [24],[25].
2.5 Statistical analysis
All values are expressed as mean ± SD (standard deviation). Significant statistical differences were analyzed by one-way analysis of variance (ANOVA). In all comparisons, the difference was considered to be statistically significant at p < 0.05 (*).