FORMULATION, OPTIMIZATION, AND IN VITRO EVALUATION OF POLYMERIC NANOSUSPENSION OF FLURBIPROFEN

Objective: At present, more than 40% of drugs are poorly water-soluble that leads to reduced bioavailability. The objective of the present investigation was to overcome the issue of poor aqueous solubility of drug; therefore, stable flurbiprofen (FBF) nanosuspensions were developed by nanoprecipitation method. Materials and Methods: Based on particle size, zeta potential, and entrapment efficiency, the polymeric system of hydroxypropyl methylcellulose E15 and poloxamer 188 was used effectively. The prepared formulations were evaluated for Fourier transform infrared spectroscopy, transmission electron microscopy, differential scanning calorimetry, powder X-ray diffraction, saturation solubility, entrapment efficiency, particle size, zeta potential, dissolution profile, and stability. Results: The resultant FBF nanosuspensions depicted particles in size range of 200–400 nm and were physically stable. After nanonization, the crystallinity of FBF was slightly reduced in the presence of excipients. The aqueous solubility and dissolution rate of all FBF nanosuspensions were significantly increased as compared with FBF powder. Conclusion: This investigation demonstrated that nanoprecipitation is a promising method to develop stable polymeric nanosuspension of FBF with significant increase in its aqueous solubility.


INTRODUCTION
The large number of active pharmaceutical ingredients emerging from the drug discovery process exhibits poor aqueous solubility resulting in a low dissolution rate and oral bioavailability [1,2]. Solubility, dissolution, and permeability of drugs are rate-limiting parameters for its oral absorption [1,2]. Various physicochemical and physiological parameters of drug affect the oral bioavailability of drugs [1,2]. Size reduction of drugs improves oral bioavailability of drug by increasing its effective surface area and thus increasing solubility and dissolution rate of drugs [1,2]. High log p value and molecular weight of the substance are important factors regarding nanosuspension of less aqueous solubility of drugs [2]. Nanosuspension is the novel approach to overcome the problem of low dissolution rate and compromised oral bioavailability and reduce the delivery issues by maintaining the drug in preferred crystalline state [3][4][5][6][7][8]. Nanosuspension signifies sufficient safety and efficacy [4][5][6]. According to Nernst-Brunner diffusion layer model, the peripheral layer of the solid particle gets saturated by small portion of an adjacent solvent. Afterward steady-state mass transfer takes place into the bulk solution [8][9][10][11][12]. The formulation can be achieved by top-down (fracturing larger particles to smaller particles) or bottom-up (generation of smaller particles by precipitation at molecular level) approaches [1,[9][10][11][12][13]. Nanoprecipitation is one of the promising techniques for the development of nanosuspension of low water-soluble drug molecules [14]. However, particle agglomeration and crystal growth due to Van der Waals forces or Ostwald ripening can be prevented by addition of one or more stabilizer (s) [15]. The selection of polymers and stabilizers is very crucial in the development of nanoformulations. Hydroxypropyl methylcellulose E15 (HPMC E15) and poloxamer 188 (Pluronic F68) are steric stabilizers provide stabilized dispersion by steric hindrance [1,13]. Nanosuspension formulations of several drugs such as Rapamune (sirolimus) and Tricor (fenofibrate) are already successfully marketed [16].
Flurbiprofen (FBF) is a phenylalkanoic acid derivative (Fig. 1), nonsteroidal anti-inflammatory and classified as Biopharmaceutics Classification System Class II drug due to its practical insolubility in water. Its oral bioavailability is affected by low aqueous solubility having pKa value ~ 4.03. The high log p value of FBF is an important feature in the development of its nanosuspension [17,18].
This study was focused to develop stable polymeric nanosuspension for enhancement of dissolution and oral bioavailability of FBF. The solidification of formulations was carried out by freeze-drying.

Materials
FBF, HPMC E15, and poloxamer 188 (Pluronic F68) were kindly gifted by Sun Pharma Pvt., Ltd., Ahmednagar. Polyvinylpyrrolidone K30 (PVP K30), polyethylene glycol 6000 (PEG 6000), and sodium dodecyl sulfate (SDS) were procured from BASF Ltd. All used supplementary chemicals and reagents were of analytical grade and utilized without additional purification. Double distilled water was used during the experimental work.

Screening of stabilizer based on settlement volume ratio
To select the optimal stabilizer, the FBF (0.5% w/v) nanosuspensions were prepared using different stabilizers (0.5% w/v) such as PVP K30, PEG 6000, SDS, and poloxamer 188, respectively, by nanoprecipitation technique. The obtained nanoformulations were analyzed by settlement volume ratio (F) for a week, and suitable stabilizer was selected based on the stability of the system [19].

Jadhav and Yadav
Screening of stabilizer based on solubility study The solubility of FBF was determined in the solutions of stabilizers. Briefly, excess amount of FBF was added to the stabilizer solution (5 ml) in sealed glass vials. The vials were shaken at rotary shaker (Remi RS BL) at 75 rpm (rotations per minute) for 72 h at 37°C and centrifuged at 40,000 rpm for 10 min at 4°C (Remi C30 PLUS) to remove non-dissolved drug. The supernatant was suitably diluted with distilled water and was analyzed for FBF by ultraviolet (UV) spectrophotometer (Shimadzu UV Spectrophotometer 1800) at 247 nm. Each experiment was carried out in triplicate.

Experimental design
Initial screening studies were carried out to check the effect of process parameters and formulation parameters on FBF nanosuspension as well as its stability. The stirrer speed of mixing was identified as a critical process parameter and polymer:surfactant ratio as a critical formulation parameter. The design of experiment was used systematically to evaluate and optimize the selected process and formulation parameters at three levels (−1, 0, and +1) using 3 2 factorial design to find out their effects on critical quality attributes of nanosuspension. The batch size 100 ml, drug concentration (0.1% w/v), HPMC E15 as a polymer and poloxamer 188 as a stabilizer, drug:polymer ratio (1:1), and stirring time (1 h) were kept constant in experimental process.

Preparation of a physical mixture of FBF, HPMC E15, and poloxamer 188
A physical mixture of FBF, HPMC E5, and poloxamer 188 was prepared by mixing them in same proportion and used for comparison with optimized formulation. The homogenous mixture was obtained by mixing in mortar, passed through 40# mesh sieve, and stored in desiccator.

Preparation of FBF nanosuspension
Nanoprecipitation technique was used to prepare FBF loaded nanosuspensions. Nanosuspensions were obtained using 500 mg FBF at different HPMC E15:poloxamer 188 ratios and stirring speed.
Accurately weighed 500 mg of FBF and 500 mg of HPMC E15 were dissolved in 10 ml of methanol (cosolvent) by sonication. The prepared organic phase of drug was added in 100 ml distilled water containing poloxamer 188 at different ratios using a syringe (26 G) with constant speed (0.5 ml/min). Stirring was continued for 1 h by mechanical stirrer at different speeds of stirrer. An excess amount of methanol was evaporated by air drying. The resulting nanosuspension was sonicated for 15 min. The optimized formulation FB8 was lyophilized without cryoprotectant.

Lyophilization of optimized batch (FB8) of nanosuspension
An optimized batch of nanosuspension (FB8) was converted into the dry powder without mannitol using laboratory scale lyophilizer (Christ, Alpha, 12 LD PLUS) by freeze-drying for 48 h at predetermined conditions. The freeze-dried product was placed in airtight container for further characterization.

Evaluation of optimized FBF nanosuspension
Particle size distribution and zeta potential The mean particle size analysis and polydispersity index PDI of suspended particles in nanosuspensions were carried out using Nanoparticle Analyzer SZ-100 with a Zetasizer (Horiba Scientific, Japan). Zeta potential is an index of the stability of suspension depending on the surface charge of particles. The zeta potential was determined by a laser Doppler anemometer coupled with Nanoparticle Analyzer SZ-100 (Horiba Scientific, Japan). The samples were properly diluted with de-ionized water before analysis. Each sample was assessed in triplicate [13].

Determination of % drug entrapment efficiency
FBF nanosuspensions (10 ml) were centrifuged at 10,000 rpm and 6°C using a cooling centrifuge (Remi C30 PLUS) for 30 min. The supernatant was separated out, and the absorbance was measured for the free drug content by UV spectrophotometer (Shimadzu UV Spectrophotometer 1800) at 247 nm. Entrapment efficiency was determined by subtracting the amount of free drug from the initial amount of drug. The % drug entrapment efficiency calculated by the following equation [20],

 
Initial drug concentration -Final drug concentration 100 Initial drug c %Entrapment oncentration efficiency

Fourier transform infrared spectroscopy (FTIR)
FTIR spectra were recorded on the infrared spectrophotometer (Alpha T Bruker). Samples about 2-3 mg were mixed with dry potassium bromide and scanned over the range 4000-400 cm −1 .

Differential scanning calorimetry (DSC)
The DSC thermograms of FBF, HPMC E15, poloxamer 188, physical mixture, and lyophilized powder of optimized nanosuspension (FB8) were recorded on DSC (Mettler Toledo, Staresw 920) at a rate of 10°C/min over a temperature range of 25-200°C in an atmosphere of nitrogen having flow rate of 40 ml/min.

Transmission electron microscopy (TEM)
Surface morphology of suspended nanoparticles in FBF nanosuspensions was observed by TEM (A Hitachi H-7500, Japan). The images were viewed at different magnifications.    [21].

Dissolution testing
In vitro dissolution study of crude FBF, physical mixture and lyophilized powder of optimized nanosuspension FB8 were investigated by USP 37 Type II dissolution test apparatus (Electrolab EDT-08 Lx) in three different dissolution media 0.1 N HCl (pH 1.2), phosphate buffer (pH 4.5), and phosphate buffer (pH 7.2), respectively. The samples equivalent to 100 mg of FBF were added in 900 ml of dissolution medium maintained at 37±0.5°C. The paddle speed was 75 rpm. Samples (5 ml) were collected after 5, 10, 20, 30, 45, and 60 min with immediate replacement by fresh dissolution medium to maintain the sink condition. All samples were filtered through 0.10 μm PTFE filter and analyzed by UV spectrophotometer at 247 nm.

Physical stability
The physical stability of optimized nanosuspension FB8 was evaluated at 4°C and 25°C for a period of 3 months. Small aliquots of nanosuspension were withdrawn after 3 months of storage for analysis of particle size, PDI, and zeta potential. Each sample was analyzed in triplicate.

Formulation optimization by stabilizer
The selection of suitable stabilizer is an important aspect of the stability of nanosuspension. In the present investigation, settlement volume ratio (F) and aqueous solubility of FBF in the presence of different stabilizers were used as stability index for the nanosuspension.

Selection of stabilizer by settlement volume ratio (F) method
Settlement volume ratio (F) is the ratio of the volume of sedimentation after and before for a given time. Larger the F value more is the stable suspension. The settlement volume ratios for FBF loaded nanosuspensions formulated with different stabilizers are recorded in Table 1.
The stabilization effect observed was followed by Pluronic F68 ˃SDS ˃PVP K30 ˃PEG 6000. From the results, it could be concluded that reasonably poloxamer 188 should be selected for development of stable nanosuspension.
Selection of stabilizer by solubility study Fig. 2 represents the apparent solubility profiles of FBF in the presence with different stabilizers. FBF represents higher aqueous solubility in the presence of SDS. This could be due to SDS micelle formation. Increased solubility can exert Ostwald ripening, resulting in increased particle size during storage. Comparatively minimum solubility was observed in the presence of poloxamer 188; therefore, it was selected as a steric stabilizer.

Experimental design
Regression and graphical analysis of data acquired from the experimental runs generated following equations in which F ratios were statistically significant (p<0.05) with Adj-R 2 value in the range of 0.8-1 (Table 2) and with a statistically non-significant lack of fit values (p>0.05). These model equations fitted the data well. A positive sign indicates a synergistic effect, while negative sign indicates an antagonistic effect.
Equations (2) and (3) represent that increase in stirring time decreases particle size and increase in the concentration of surfactant to certain level increases entrapment efficiency. An increased concentration of surfactant decreases zeta potential.
Effect of formulation parameter and process parameter on entrapment efficiency, particle size, and zeta potential Fig. 3a and c displays that the ratio 1:1:2 of drug, polymer, and surfactant, respectively, showed greater entrapment efficiency. An increase in the concentration of surfactant above optimum level depicted decreased entrapment efficiency probably due to surface adsorption of drug by it. A small decrease in zeta potential was observed with increase in concentration of surfactant. This could be explained by the reality that micelle formation and increased surface adsorption of drug. As shown in Fig. 3b, the increase in stirring speed gradually decreases the particle  Entrapment efficiency (%)

Mean particle size (nm)
Zeta potential (mV)  size. It could be due to increased attrition and counter diffusion of particles during nanoprecipitation, and additional mechanical energy induces the repulsion between nanocrystals.

Model verification
The desirability function was evaluated by Design-Expert software to obtain the optimized FBF nanosuspension. The model verification results are displayed in Table 3 that compare observed and predicted values of entrapment efficiency, particle size, and zeta potential using model equations.

Evaluation of optimized FBF nanosuspension
Particle size and zeta potential analysis Table 4 depicts mean particle size, % entrapment efficiency, and zeta potential data of all formulations. The observed mean particle size of raw FBF powder was 16.41±2.41 μm while that of optimized nanosuspension (FB8) was 263.3±2.73 nm which represents significant (62-fold) reduction in particle size. The PDI of all batches of nanosuspension was found to be in the range of 0.258±0.01-0.411±0.02 indicates monodispersity of nanosuspensions. The PDI   showed that optimized nanosuspension (FB8) was 0.266±0.02. Zeta potential is the stability index of prepared nanosuspension. Sterically stabilized systems show lower zeta potential values with sufficient stabilization [12]. Zeta potential of all batches of nanosuspension was found to be in the range from −15.91±0.08 mV to−7.91±0.37 mV. The zeta potential (Fig. 4) depicted that optimized nanosuspension (FB8) was −10.2±0.42 mV.

Drug entrapment efficiency (%) analysis
Entrapment efficiency is the percentage of the actual mass of drug entrapped in the polymeric carrier compared to the initial amount of loaded drug. The results (Table 4) suggested that the formulation FB8 having FBF: HPMC E15:poloxamer 188 (1:1:2) ratio showed increased drug entrapment efficiency due to formation of polymeric matrix with optimum viscosity. The drug entrapment efficiency of with FB6 by one-way ANOVA followed by Dunnett's test.

FTIR analysis
FTIR spectra of FBF and its nanosuspension (Fig. 5) depicted a typical broad peak of FBF in the range of 3000-3500 cm −1 due to hydrogen bonding in the compound which was broadened in the spectra of lyophilized formulation. The distinctive sharp peak at 1404 cm −1 represents stretching of C-F and peak at 1687 cm −1 represents the carbonyl stretch (C = O) in the FBF. The O-H stretch of carboxylic acid group represented by peak at 3072 cm −1 and C-H stretch characterized by peaks at 2945 cm −1 and 2981 cm −1 . Slight shifting of peaks in lyophilized nanosuspension FB8 from 2976 cm −1 to 2981 cm −1 and 3072 cm −1 to 3074 cm −1 indicates an enhancement of hydrogen bonding. The spectra of physical mixture and lyophilized nanosuspension showed the same absorbance pattern indicating the compatibility of drugs and polymers. Therefore, no shifting of position of the functional groups and no major interaction between FBF and polymers have been observed. Results likewise indicated that upgrade in the dissolution rate of FBF was not caused because of any chemical interaction among FBF and polymers. Fig. 6 represents the DSC thermograms of crude FBF, HPMC E15, poloxamer 188, physical mixture, and lyophilized nanosuspension. FBF demonstrated a sharp endotherm at 117.88°C ascribed to its crystalline nature. The peak of FBF (117.54°C) was maintained in the physical mixture of FBF with HPMC E15 and poloxamer 188 which noticeably indicates the absence of the physical interaction between drugs and excipients. Lyophilized nanosuspension revealed a sharp endotherm at 117.08°C which may represent the decreased particle size or miscibility of drugs with excipients. The above results signify the fact that there PXRD analysis X-ray diffraction study (Fig. 7

Surface morphology by TEM study
The morphology of FBF nanoparticles suspended in nanosuspension was illustrated by TEM analysis, and obtained TEM micrographs of optimized nanosuspension are shown in Fig. 8. The images indicate spherical shapes with better dispersity of particles and level of particle agglomeration.

Saturation solubility study
Saturation solubility data of FBF, physical mixture and lyophilized powder in 0.1 N HCl (pH 1.2), phosphate buffer (pH 4.5), and phosphate buffer (pH 7.2) is presented in Fig. 9

Dissolution study
FBF loaded nanosuspension is illustrated in Fig. 10 biphasic drug release outline. Rapid release of drugs was observed in the first phase due to the presence of free drugs not entrapped in polymer system. The second phase of slow release of drugs was observed due to slow diffusion of FBF through the polymer matrix. All the batches were optimized on the basis of minimum initial burst release and maximum release profile of drug. The batches with smaller particle sizes and lower entrapment of drugs showed high burst effect.  When dissolution pattern of FB8 in 0.1 N HCl was compared with that of in phosphate buffer pH 7.2; the difference factor (f 1 ) and similarity factor (f 2 ) were found to be 14 and 64, respectively, exhibiting similar pattern of drug dissolution.

Physical stability study
Physical stability is a fundamental problem in the development of nanosuspension due to aggregation of nanoparticles and Ostwald ripening effect. Ostwald ripening is the process of growth of larger particles from smaller particles that contribute to instability of nanosuspension. The physical stability study of optimized FBF nanosuspension was performed at 4°C and 25°C over 3 months. The minor increase in particle size and PDI was seen at both storage conditions after the completion of 3 months (Table 6). Therefore, the increase in particle size was insignificant. The initial and at the end of 3 months absolute zeta potential values of nanosuspension remained in between −15.91±0.08 mV and −7.91±0.37 mV indicating that nanosuspensions were physically stable. It might be due to the presence of HPMC E15 and poloxamer 188, which probably served as crystal agglomeration inhibitor by adsorbing onto the surface of FBF nanoparticles.

CONCLUSIONS
In the present work, FBF nanosuspensions were successfully developed by the nanoprecipitation method. Prepared nanosuspensions exhibited a great degree of aqueous solubility, dissolution rate, and stability. The resultant FBF nanosuspensions were physically stable with mean particle size range of 200-400 nm. There was a slight reduction in crystalline nature of FBF in the nanosuspensions due to the presence