INTRODUCTION:

Nifedipine is an inadequately water soluble medication which comes under BCS Class II. This medication experiences hepatic metabolism activity in liver and gut wall. The drug is poor water soluble, bioavailability rate is lesser, also, drug is fluctuating in plasma levels and greater nutrition reliance is the mainly imperative and normal issues with this medication. Significant hard work has been made for the advancement of modified medication bearers to conquer the annoying in vivo results of the medication (Barratt GM et al., 2003) [1]. Consequently, increasing necessity for a unique system that shall tackle detailing associated issues related with the release of hydrophobic medications keeping in mind the target in boost their systematic feasibility and upgrade healing as for pharmacoeconomics.

The rate of dissolution for ineffectively water soluble medications frequently turns into a factor limiting process in their fascination progress from GI tract (Chiba Y et.al., 1991) [2]. Different solubilization approaches been utilized to enhance medication solubility and disintegration parameters, comprise utilization of surfactant, water-soluble bearers, polymeric conjugates, and solid dispersions.

Nanosuspension is a proficient and brainy approach used to delivery of water insoluble medications where the medication is diminished to the submicron extend as the saturation solubility and the surface area available for dissolution enhancement subsequently increasing its dissolution rate and thus its bioavailability (White SR., 2005) [3]. Stabilizer assumes an imperative part in the preparation of nanosuspensions. Without a suitable preservative, the high surface energy of nano sized particles can initiate agglomeration of particles. The principle elements of a preservative are wetting the drug particles completely and preventing Ostwald's ripening (Moschwitzer J., 2004) [4].

Nanoprecipitation strategy displays various points of interest, in that it is a clear method, quick and simple to execute. In this methodology, the drug substance is disperse in organic solvent, for example, acetone, acetonitrile etc. The organic solvent is removed by pressure reducing or by continual stirring. Size of the drug particles was observed to be impacted by the kind of stabilizer, concentrations of stabilizer, and speed of homogenizer (Wongmekiat et.al. 2002) [5]. In the present work, nanosuspension formulation was developed by nanoprecipitation methodology; in which drug substance is disperse in a solvent, which is then mixed to non solvent that causes precipitation of the fine drug molecules and framework is balanced out by polymer and surfactant.

MATERIALS AND METHODS:

Materials:

Nifedipine drug sample was procured from reputed pharmaceutical company. Hydroxy Propyl Methyl Cellulose (HPMC) was procured from Loba chem. Pvt. ltd, Chennai. Poly Vinyl Alcohol (PVA) was purchased from procured from Meru Chem Private Limited., Chennai. Ethyl alcohol, 95% v/v and tween 80 were purchased from Drugs India, Bangalore. Analytical grade chemicals and reagents were used in the study.

METHODS:

Formulation of nifedipine nanosuspensions:

Nanosuspensions formulations were developed and synthesis by the nanoprecipitation method (Itoh K et.al., 2003) [6]. In short, nifedipine (10mg) and stabilizers (PVA, Tween 80, PVP K44 and HPMC K4M) were made soluble in organic solvent (15ml of 95% ethanol) to produce a series of organic solutions at room temperature (25±2°C) consisting of different concentrations of stabilizers. Distilled water holding a surfactant (1% tween 20), which serves as antisolvent system was cooled at lower temperature (below 5°C). Consequently the organic solution was added in to aqueous solution at a slower rate (1ml/min.) with syringe, higher-speed reflex agitation of 6000rpm by means of sonicator for 10 minutes to produce the required nanosuspensions formulation. The temperature less than 15ºC was persistent during the process by means of an ice-water bath to maintain the rate of SLN. The significant coarse predispersion was comminuted using zirconium oxide beads (milling media) on a magnetic stirrer. Zirconium oxide beads were used in the preparation of nanosuspensions due to cost effective and easy availability for lab scale production of nanosuspensions in comparison to silver beads. Different formulation batches were prepared in respect to the formulation design. Later the prepared nanosuspensions were kept under vacuum at 25°C for 3 hour for removal of organic solvents.

Characterization of nanosuspensions Particle size and poly dispersity Index:

The formulated nifedipine nanosuspensions were characterized by means of Photon Correlation Spectroscopy (PCS) with a Zetasizer Nano ZS apparatus. In the process of analyzing, a superannuate of the formulation was shrink before the quantification. All experiments conducted in thrice at 90°C scattering angle at ambient temperature.

Determination of zeta potential:

Prepared nanosuspension formulations were subjected for zeta potential measurement by means of an additional electrode in the particle size analyzer i.e. Zetasizer, Malven. For analysis the concentration of samples were lowered with water and kept in electrophoretic cell as the electrophoretic mobility was transformed to zeta potential by means of Smoluchowski equation (Keck CM and Muller RH., 2006) [7]. Average values were estimated for every individual sample by measured thrice at room temperature.

The percent of the total drug content:

All formulations were conducted for assay test to determine the amount of pure drug present in the formulation. In brief the procedure is as follows; 0.5ml of aliquot sample was first made soluble in 10mL phosphate buffer (pH 7.4 buffer) and filtrated through 0.22µm size whatman filter paper. The drug quantity was estimated after suitable dilution with respective medium and phosphate buffer as a blank on UV Spectrophotometer (Lab India, UV 3000) at a wavelength of 238nm. The drug content and percentage of drug content were estimated by using following equations;

Total drug content = (Total volume/Aliquot volume) X Drug amount in aliquot x 100

% Total drug content = (Total drug content/Total added drug) x 100

Here, Total Volume/ Aliquot Volume is the ratio of total nanosuspension quantity to the volume taken in aliquot and the total quantity of drug taken for the preparation of nanosuspension, total added drug (Chorny M et.al.,2002) [8].

Dissolution studies (In vitro) of prepared formulae:

Nanosuspension formulations of nifedipine were evaluated for dissolution studies against dissolution profile of pure drug. The procedure is as follows; pure drug and nanosuspension formulation (all equivalent to 10mg of nifedipine) were weighed accurately for the study and made soluble in adequate quantity of phosphate buffer pH 7.4. The samples were placed in dialysis bag (Mw cut-off = 12,000Da). After this procedure, the formulations formulae were transferred in a beaker containing phosphate buffered (500ml) and stirred at a steady speed of 200 rotational per minute with a magnetic stirrer by maintaining temperature at 37±0.5°C. At specified time point, sample (5ml) was withdrawn and replaced with a dissolution medium at 5, 10, 15, 20, 30 and 45 minutes followed by filtration and diluted suitably for the measurement of drug concentration using UV at λmax 238nm. Experiments were conducted for three times for each of the selected formulation. Drug release kinetics of the formulations was determined for zero-order, first-order and Higuchi by using Microsoft Excel Add-Ins DD Solver software.

Lyophilization of selected nanosuspensions:

To facilitate the removal of water content from the nanosuspensions, lyophilization technique is most commonly employed (Kumar MP et al., 2008) [9]. In the present work, we have subjected the formulations for the same purpose. The optimized formulations were initially exposed to lowest temperatures to make them to frozen and then lyophilized by means of a freeze dryer (Thermo Fischer Scient, Micromodul YO 230). During process formulation was placed in ampoules and pre-frozen in a deep freezer at -45ºC for a period of 24 hours, followed by ampoules were moved to glass flasks and these flasks were connected with adapter (vacuum) of freeze dryer.

Optimization of nifedipine nanoparticles Process yield determination:

The percentage yield of the experimental procedure was calculated by the formula;

% process yield = [Mass recovered/Mass entered into the experiment] X100

Nifedipine entrapment efficiency in the dry lyophilized powder form was estimated by disperse 1mg of powder in 10ml of alcohol (ethanol). As a result of this a suspension was obtained and this was subjected for sonication in a water bath for about 30 minutes then centrifuged at 2000rpm, in order to remove insoluble solid particles. After this nifedipine content was estimated in supernatant layer by using UV spectrophotometers at a wavelength 238nm. The obtained weight was divided by the mass initially taken in the process and calculated in terms of percentage.

Scanning electron microscopy (SEM):

The optimized nanosuspension formulations were studied for morphological characters by scanning electron microscopy (SEM). Suitable concentration of samples was obtained by diluting with ultra purified water. Further, the samples were kept on a sample holder and dried using vacuum. Sample was later coated with gold (JFC 1200 fine coater, Japan) and monitor by a Scanning Electron Microscopy (SEM).

Fourier transforms infrared spectroscopy (FTIR):

In order to identify the incompatibilities in drug and polymers and other excipients in the formulation, the nanosuspensions were analyzed by IR spectroscopy method and spectra’s of drug sample optimized formulations were recorded by means of FTIR spectrophotometer. The samples scanned in the range of 4000- 400 cm-1.

Differential scanning calorimetry (DSC):

Further incompatibility studies of drug were confirmed by means of Differential Scanning Calorimetric method (TA Instruments, Q20). Instrument for differential scanning calorimetric is performed by DSC Q200 V24.4 Build 116. Calibration of the instrument performed with indium for melting point and heat of infusion. A heating rate at 20°C/minute is employed throughout the analysis in the range of 25–200°C. Standard aluminum sample pans were utilized for all samples; Empty pan is analysed as reference. The thermal behavior was calculated under a nitrogen purge; analysis was carried out on each sample in triplicate to execute the reproducibility.

In vivo drug absorption study:

An in vivo pharmacokinetic study was performed in accordance with the ethical guidelines for investigations in laboratory animals, approved by the Institutional Animal Ethics Committee (IAEC). Six Rabbits weighing 2.30±0.12kg (divided into three groups) were fasted overnight. The animals were allowed seven days to adapt and were given adlibitum access to standard rat chow (0.5% NaCl) and tap water until the start of the study. For dosing, each group of two rabbits was given either a 10 mg/kg subcutaneous dose of nifedipine formulated as regular suspension. At the initiation of study, the animals weighed from 297 to 329g. Blood samples (approximately 0.2mL per sample) were collected from each animal via jugular vein cannulae at the following time points: pre dose; 5, 15, and 30 min. post dose; and 1, 2, 4, 8, and 24 h post dose. All samples were collected into sample tubes containing anticoagulant (potassium EDTA). Blood samples were centrifuged within 30 minutes of the collection, and plasma was collected. Plasma samples were examined for drug concentrations by a HPLC assay method.

The plasma samples were determined using validated HPLC method using verapamil as internal standard for nifedipine. Chromatographic system consisted of model Shimadzu and rheodyne injector with 20µL fixed volume loop and shimadzu PDA detector controlled by lab solution software. Separation was carried out at room temperature (25ºC) purosphere C18 (150mm × 4.6mm) 5 µ. A mixture of pH 3.0 phosphate: acetonitrile (80:20) was used as mobile phase with flow rate 0.6ml/minute and pressure was maintained at 90-150kg/cm2. Column temperature was maintained at 35ºC. Mobile phase was filtered through 0.2µm membrane filter before use. The detector wavelength set at 238nm.

Dose simulation:

A model based on the Wagner-Nelson equation was established in-house and was used to calculate the drug absorbed and the amount of drug absorbed as a function of time (Van Eerdenbrugh et al., 2009) [10]. The utilization of the equation allow us to obtain the entire drug that is absorbed (including excreted) at different time points. This concludes the estimated relationship and the impact on the absorption on the surface area changes of the drug.

Fig.1 Scanning electron microscope image of Nifedipine nano suspension

RESULTS AND DISCUSSION:

Influence of various parameters on particle size and size distribution:

Stirring time effect on particle size was evaluate by maintaining 50:50 ratio of different diameter (0.5mm to 0.8mm and 1.4mm to 1.8mm) of zirconium oxide beads and ratio of the drug: surfactant: milling media volume (1:3.0:50) constant. Mean particle size at lowest 325nm was achieved after stirring at 50:50 ratio of zirconium oxide beads for 24 hrs. Further stirring is increased up to 28hrs may lead to increased particle size due to increased surface free energy.

Optimized formulation demonstrate mean particle size of 296nm with Polydispersity index of 0.310 (before lyophilization), with 3.0% w/v of PVA was used as a preservative and 50% v/v of milling media. After lyophilization a mean particle diameter was found to be 298nm with Polydispersity index 0.321, so in lyophilization process there was no considerable change in particle size and size distribution.

The nature of the stabilizer and its quantity is an important factor in controlling the size and firmness of the nanosuspensions in the process. The results were indicated decreasing the particle size with increasing the stabilizer concentration.

The results showed particle size had been decreased with the increasing of stabilizer concentration as the particle size of a batch which contains 0.1% stabilizer was 618.45±26.44 nm compared with 238.78±12.18nm for a batch contains 0.8% stabilizer. This could be attributed to the increase in the molar substitution ratio of the polymer per drug. The increase of the hydrophilic corona surrounding the polymer to protect the nanoparticles enhances the stability and prevents particles from coalescence and preventing aggregation. On the other hand, the particle size increased with the high concentration of PVP K30 which might be due to the higher viscosity of the resulting solution that might hinder particle attrition at same milling energy. Moreover Ostwald ripening might cause agglomeration, and consequently, higher particle size values resulted (Merisko E et.al., 1996) [11]. On the other side, the poly dispersity index (PI) values were ranged from 0.08-0.517 which indicates acceptable uniformity level for most of the preparations.

Zeta potential analysis:

Zeta potential of the completed formulations is observed range -9.45 to -18.34. Zeta potential of nifedipine nanosuspensions was relatively low due to the shielding effect of the hydrophilic chains of the polymers used. These chains formed what is called hydrophilic corona that is surrounding the particles and prevent the true measure for the zeta potential (Saindane., 2013) [12]. On the other hand, the importance of the colloidal stability of the nanosuspension is reduced because these formulae will be kept in dry state which is reducing the importance of zeta potential as a controlling factor.

The percent of the total drug content:

The drug content for the prepared formulae was calculated from the experimental observations. The drug content for all formulae was calculated as a percent of the initially added drug. The amount of the drug within the formulations was more than 90% in all samples.

In vitro dissolution:

The most significant feature of nanosuspensions is higher the rate of dissolution not only because of increase in surface area but also due to use of hydrophilic surfactant. The in-vitro dissolution of nifedipine was performed for all synthesized nanosuspensions formulations and then compared to that of the pure drug moiety. The cumulative percentage of the drug dissolved was 97.85% at 35 minutes for selected nanosuspension, compare to pure drug was 36.46 at 35 minute. The difference was significance at p<0.05 when t-test for unpaired data was applied, and the release kinetics was found to obey first-order kinetics with R >0.98.

Process Yield:

The process yield of the mass recovered for developed nanosuspensions was estimated after lyophilization process and was considerably high (96±3.25%) it indicated efficient processing with minimum batch variability, therefore it representing a negligible loss of drug during preparation.

Morphology evaluation:

The characteristics of drug morphological were investigated using scanning electron microscopy (SEM). The SEM image of the drug and nanosuspension showed a considerable difference in the morphology of particles. Nanosuspension sample was appeared to be spherical with the mean particle size of 213nm. They are having narrow distribution index, while SEM of the drug revealed coarse, irregular, more elongated, and within a micro range. The results showed the formation of uniform non-aggregated particles that adsorb the hydrophilic corona around them. Two distinct layers are shown; where the hydrophobic part of the polymer is directed inward the particles and the hydrophilic part of the polymers are directed outward.

Fourier transform infrared spectroscopy (FTIR):

IR spectra of nifedipine concealed that characteristic peaks of the NH stretching at 3331 cm-1 and a band with main peak at 1688 cm-1 indicative of the C=O stretch of the esteric group. The above characteristic peaks emerge in the spectra of both physical mixtures and formulations of drug with stabilizers. From these results it was established that there was no interaction between the drug and stabilizers used in the nanosuspension formulations. Further characterization was done by DSC.

Differential scanning calorimetry (DSC):

DSC thermogram of pure nifedipine illustrate a attributed sharp endothermic melting peak at about 176.7 °C with peak onset at 171.91°C and peak end at 175.17 °C and the heat of transition was (171mJ/g). The thermogram of nanosuspension formulation showed endothermic melting peak at 120.5°C which is close to the predictable value for the drug addition, melting enthalpies of endotherm were at a lower-energy state as compared to a crystalline form of the drug. The shift in the drug peak to a lower temperature and decrease in the area of the peak in the nanosuspension compared to pure drug may be due to lesser drug crystals. Additionally, this decrease in enthalpy value indicates low lattice energy, and it was well statement that the particles with lower lattice energy are easier to dissolved (Wei L et.al., 2011) [13].

In vivo pharmacokinetics study:

A pharmacokinetic study conducted in mice proved that the bioavailability was enhanced when nanosus- pension formulation of nifedipine was compared to the market formula. There was a statistically considerable variation in the T max, C max, AUC (0-24) and MRT data between the market formula and the nanosuspension optimized formulation. Cmax value of nifedipine nanosuspension formulation was significant (p<0.05) greater than reference formulation. The AUC (0-24) assessment of nifedipine nanosuspensions after oral administration was almost 2 folds higher than those obtained of the marked formulation.

CONCLUSION:

Nanosuspensions of nifedipine were prepared successfully by nanoprecipitaion method. The prepared nanosuspenions were found stable with appropriate concentrations of stabilizer. It has been concluded that this methodology is a novel and more reproducible for developing novel drug delivery system for nifedipine to overcome the problem of solubility and to enhance its oral bioavailability. The prepared formulations were found significantly enhanced dissolution characteristics in comparison to the available marketed formulation. Hence nifedipine nanosuspensions proved to be most potential new drug formulation for oral drug release with enhanced oral bioavailability.

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Received on 08.05.2020 Modified on 13.07.2020

Asian J. Res. Pharm. Sci. 2021; 11(1):1-6.

DOI: 10.5958/2231-5659.2021.00001.1