Enhanced oral bioavailability of nisoldipine-piperine-loaded poly-lactic-co-glycolic acid nanoparticles

2.2.1 Experimental design and analysis

The Taguchi design method was used to optimize various parameters for preparing nisoldipine and piperine-loaded nanoparticles. The various variables involved in preparing nisoldipine and piperine-loaded nanoparticles were categorized as dependent and independent variables. In this study, three factors namely polymer concentration (coded as A), piperine concentration (coded as B), and ratio of solvent (coded as C) were considered as variable factors affecting the process performance in terms of encapsulation efficiency (Y1) and nanoparticle size (Y2). The three levels of each of the dependent variables are given in Table 1. Nine experiments were performed to find out the factors and their optimized level ranges having a prominent effect on the proficiency of formulation based on Taguchi’s L₉ orthogonal array (Table 2). The run involved the corresponding combination of levels to which the factors in the experiment was set. All nine experiments were performed in triplicate to minimize experimental errors.

The responses of signal-to-noise (S/N) ratio effect plots and analysis of variance (ANOVA) of each response, individually as well as simultaneously, were determined to assess significant optimized levels of factors (Table 3). The optimum conditions were determined to yield an improved performance with the minimum possible influence of the noise factor. The first step was to select the factor/level combination to maximize the response. The second step was to find the condition for attaining optimal desirability. All calculations and statistical analysis of the results were carried out by ANOVA to determine the factors having a statistically significant effect on the response parameters (Table 4).

2.2.2 Fabrication of nisoldipine and piperine-loaded nanoparticles

PLGA-based nanoparticles were prepared using a modified precipitation process [21]. Briefly, 100 mg PLGA (50:50, Mw=40,000–75,000) polymer, 10 mg nisoldipine, and 20 mg piperine were dissolved in 25 ml acetone at room temperature to make a clear solution. Then, this solution was added dropwise with continuous magnetic stirring in 25 ml PVA (1%; Mw=30,000–70,000) for 30 min at room temperature. The organic solvent was evaporated at 26°C first by stirring (2029 g using rotary evaporation; Rotavapor R-124, Buchi, Switzerland) at atmospheric pressure for 6 h and then at reduced pressure for 2 h. Thereafter, nanoparticle suspension was centrifuged (REMI, Mumbai, India) for 25 min at 10,956 g at 4°C to remove the polymer aggregates and washed twice with deionized water to remove acetone and PVA residues, and were freeze dried at −40°C (Eclipse 400; RS Biotech, UK) and stored in a vacuum desiccator at 4°C for further use. The percentage yield was >78%.

2.2.3 Preparation of nisoldipine suspension (control)

The nisoldipine suspension was prepared according to the method of Sandhya et al. [22] with modifications, by dissolving 10 mg nisoldipine in 3.0 ml of ethanol. Mixture 1 containing 30 mg PVP-K30, 0.3 ml Tween 80, and 1.0 ml of 0.01% SLS in 30 ml water was prepared and kept on a magnetic stirrer for uniform mixing. The drug solution was slowly added dropwise to mixture 1 and magnetic stirring was continued for 30 min. Then, the solution was kept in a sonicator for 30 min. The suspension was formed and preserved for further use.

2.2.4 Entrapment efficiency and loading capacity

The entrapment efficiency of the prepared nanoparticles was determined by an indirect method [23]. The concentration of nisoldipine in the supernatant after isolation of nanoparticles was determined spectrophotometrically at 238 nm. The percent entrapment efficiency (EE%) was calculated by the following equation:

EE (%)=[Ntotal−Nsupernatant/Ntotal]×100,

where N_total is the total amount of nisoldipine and N_supernatant is the free nisoldipine in the supernatant.

For calculating drug loading, the nanoparticles were dissolved in a mixture of acetone and ethanol (10:1) and centrifuged at 1677 g for 15 min. The precipitate was then extracted with acetone. The acetone was evaporated and percent drug loading [24] was calculated by the following equation:

Drug loading (%) =  [amount of nisoldipine extracted/ weight of nanoparticles]×100.

2.2.5 Characterization of nanoparticles

The characterization of nanoparticles was achieved using transmission electron microscopy (TEM). Photon correlation spectroscopy (NanoZS90; Malvern Instruments, Worcestershire, UK) was used to measure the particle size, polydispersity index, and zeta potential (ZP) of the prepared formulation. All samples were diluted with ethanol to achieve sufficient concentration before measurement. All parameters were analyzed in triplicate and data are represented as mean±standard deviation (SD). Thermal [differential scanning calorimetry (DSC)] and spectroscopic [Fourier transform infrared (FTIR)] analyses were used to assess the chemical interaction of the drug with excipients.

2.2.6 Dissolution and release studies

In vitro release of nisoldipine from nanoparticles was studied in 0.1 n HCl (pH 1.2) and in phosphate buffer (pH 6.8) using a type II paddle apparatus (DS 8000; Lab India Ltd., Mumbai, India) at 37±0.5°C and 60 rpm for comparing the release pattern at the two different pHs. The nisoldipine release profile is influenced by pH; therefore, the two different pHs were selected to comparatively evaluate the effect on the release profile of nanoparticles, as some researchers claimed it to be independent of pH. Aliquots of 5 ml were withdrawn from the apparatus at specific intervals. Nisoldipine concentration was measured in the withdrawn aliquots. The dissolution medium volume was kept constant by adding the same amount of fresh medium. Drug dissolution and cumulative drug release was determined for nisoldipine-piperine nanoparticles and conventional nisoldipine formulation at 2, 4, 6, 8, 12, 18, 24, 36, 48, and 60 h.

2.2.7 Antihypertensive and pharmacokinetic studies

The study was carried out in Wistar rats of either sex weighing 150–200 g. The animals were maintained under standard laboratory conditions of temperature (22±2°C) and 12 h light/dark cycle. All animals were kept in quarantine for 1 week prior to experiments. Rats were fed with standard laboratory chow and water ad libitum. All experimental procedures were reviewed and approved by the Institutional Animal Ethical Committee of Chandigarh College of Pharmacy, Landran (IAEC/FEB16/018). The animals were fasted overnight before the experiment. Before experimentation, the animals were trained to stay in the restrainer. This ensured that the rats were calm and unaggressive during blood pressure (BP) measurements.

Hypertension was induced by subcutaneous injection of methyl prednisolone acetate (20 mg/kg/week) for 2 weeks [25]. The rats were then randomly divided into three groups with six animals in each group. The groups were methyl prednisolone+saline (methyl prednisolone group), methyl prednisolone+nisoldipine (nisoldipine group), and methyl prednisolone+nisoldipine-piperine nanoparticles (nanoparticle group). In addition, a normal control group treated with saline alone was also included. Drug administration was started 30 min after the last methylprednisolone injection. Nisoldipine was administered orally once in a dose of 0.9 mg/kg and nanoparticles were also administered orally once in a dose of 0.45 mg/kg in solution form by using a cannula attached to a syringe into the mouth of animals. The dose of nisoldipine used was selected based upon the approved clinical dose in humans [26]. This dose was then converted to the equivalent rat dose on the basis of body surface area formula. A non-invasive tail cuff method was used to measure systolic BP [27]. BP in all groups was measured up to 120 h after drug administration starting from 0 h. At each time point, three readings were taken for each animal and the mean was calculated.

The pharmacokinetic profiles of nisoldipine nanoparticles and conventional nisoldipine formulation were also evaluated after oral administration of drugs. Parameters such as peak serum concentration (C_max), time for peak serum concentration (t_max), area under the curve (AUC, 0–∞), half-life (t_1/2), and mean residence time (MRT) were calculated. The blood samples were withdrawn from the tail vein at 0, 1, 2, 3, 4, 6, 8, 10, 12 and 24 h post-dose. About 0.1 ml of blood sample was withdrawn in Eppendorf tubes and centrifuged at 3000 rpm for 30 min. The serum was transferred to another Eppendorf tube and stored at −20°C until further analyzed by high-performance liquid chromatography (HPLC) using a C-18 analytical column (250 mm×4.6 mm and 5 μm in diameter; Merck, Mumbai, India). The mobile phase consisted of acetonitrile-water (78:22) at a flow rate of 1.1 ml/min and detection at λ max of 237 nm. The injection volume was 10 μl [28]. A stock solution was prepared by dissolving accurately weighed 10 mg of nisoldipine in 10 ml volumetric flask to obtain a 1 mg/ml solution using HPLC-grade methanol. Kinetica software (version 5.0; Innaphase Corporation, Philadelphia, PA, USA) was used to calculate the pharmacokinetic parameters. The values are expressed as mean±SD. The data from BP experiments and pharmacokinetic data are presented as mean±SD. The data were analyzed by unpaired Student’s t-test. A value of p≤0.05 was considered statistically significant.

2.2.8 Accelerated stability studies

Nanoformulation stability analysis was done according to International Council on Harmonization [29] guidelines at temperature of 25±2°C and relative humidity of 5%. The nanoparticle formulation samples were collected at prearranged intervals (30, 60, and 90 days) and tested for particle size, shape, appearance, and DSC analysis. Drug content stability and drug entrapment of nanoparticles were also determined by storing the nanoparticles at 4.0±1°C in a refrigerator and 40±2°C in a stability testing chamber for 3 months.