Preparation and Characterization of Domperidone Nanoparticles for Dissolution Improvement

39 Preparation and Characterization of Domperidone Nanoparticles for Dissolution Improvement Malath H. Oudah, Firas A. Rahi and Mohammed S. Al-lami College of Pharmacy, University of Kufa , AlNajaf, Iraq. ** Faculty of Pharmacy, Ibn Hayyan University College, Karbala, Iraq. *** College of Pharmacy, University of Basra, Al Basrah, Iraq Abstract This study was carried out to prepare and characterize domperidone nanoparticles to enhance solubility and the release rate. Domperidone is practically insoluble in water and has low erratic bioavailability range from 13%-17%. The domperidone nanoparticles were prepared by solvent/antisolvent precipitation method at different polymer:drug ratios of 1:1 and 2:1 using different polymers and grades of poly vinyl pyrolidone, hydroxy propyl methyl cellulose and sodium carboxymethyl cellulose as stabilizers. The effect of polymer type, ratio of polymer:drug, solvent:antisolvent ratio, stirring rate and stirring time on the particle size, were investigated and found to have a significant (p≤ 0.05) effect on particle size. The best formula was obtained with lowest average particle size of 84.05nm, which composed from 2:1 of PVPK15:drug and solvent/antisolvent volume ratio of 1:10. This formula was freeze dried and studied for compatibility by FTIR and DSC, surface morphology by Field Emission Scanning Electron Microscope (FESEM) and crystalline state by XRPD. Then domperidone nanoparticles were formulated into a simple capsule dosage form in order to study of the in vitro release of drug from nanoparticles in comparison pure drug and mixture of polymer:drug ratios of 2:1. The release of domperidone from best formula was highly improved with a significant (p≤ 0.05) increase. it can conclude that nanoparticles showed better in vitro dissolution profiles in comparison with pure drug


Introduction
The solubility, dissolution rate and bioavailability of drugs are important factors for achieving in vivo effectiveness. The bioavailability of orally administered medications depends on their capability to be absorbed via gastrointestinal tract. It appears that enhancement 1 of the solubility of poorly water soluble APIs can translate to an increase in their bioavailability. Beside the coming of new technologies in drug detection, combinatorial chemistry, and computer helped drug design, there was growing in the progress of new chemical entities with perfect therapeutic potential. However due to of the complicated chemistry, nearly 40% of the drug applicants in the development pipeline and about 60% of new APIs produced by chemical synthesis are introduced with poor aqueous solubility causing in low and variable bioavailability (1,2) . At the present time nanotechnology offers various methods in the area of dissolution enhancement of low aqueous soluble drugs. nanoparticles formulation technology has achieved a considerable attention by the formulation scientists. Pharmaceutical nanoparticles are defined as solid, submicron-sized drug carrier that may or may not be biodegradable. The advantages of nanoparticles include lower drug toxicity, reduce the dose needed, improved bioavailability, increase drug targeting ability, decrease drug resistance ,increase patient compliance and reduced cost of treatment (3,4) .The aim of this research to formulate domperidone as nanoparticles in capsule dosage form using precipitation method of solvent/antisolvent to improve dissolution rate. Domperidone is antiemetic drug has chemical structure is 5chloro-1-[1-[3-(2,3-dihydro-2-oxo-1Hbenzimidazole-1-yl) propyl] -4piperidinyl1] -1,3 dihydro -2H -benzimidazole -2-one) (5) , domperidone is practically insoluble in water and slightly soluble in ethanol and methanol (6) , and have low oral bioavailability (13-17%) is thought to be due to hepatic first-pass and intestinal metabolism and poor aqueous solubility (7) .

Preparation of domperidone nanoparticles
The domperidone nanoparticles were prepared using solvent/antisolvent of precipitation technique (8) . A certain amount of pure domperidone was completely dissolved in dimethyl formamide (DMF). The drug solution with specific concentration was injected at 1mL/min using ordinary syringe into water solution containing specific concentration of stabilizer of each (PVP-K15, PVP-K30, HPMC-E15, HPMC-E50, andCMC-30) with continuous stirring. Precipitation of nanoparticles in form of colloidal solution occurred gradually upon mixing. The nanoparticles were then lyophilized to obtain the nanoparticles powder. The composition and variable conditions of preparation of different formulas are listed in table (1).

Particle size and poly dispersity index measurement (PDI)
The ABT-9000 dynamic light scattering nano laser (Angstrom-USA particle size analyzer was used to measure the average particle size and poly dispersity index (PDI), as measures for the width of the size distribution, and the specific surface area (SSA) for all prepared domperidone nanoparticles formulas.

Study of variables affecting on size of domperidone nanoparticles Effect of type and concentration of stabilizer
Different stabilizer at two ratios of polymer to drug concentration of 1:1 and 2:1 were used in the preparation of domperidone nanoparticles. Formulas F1-F10, were prepared and used to illustrate the effect of polymer type and concentration on the size of domperidone nanoparticles.

Effect of time of stirring
The effect of time of stirring of 5 and 30min from finished addition of drug solution on size of formed nanoprticles was studied using polymers of PVP-K15, HPMC-E50, and CMC-30 at two ratios polymer to drug concentration of 1:1and 2:1.

Effect of rate of stirring
The effect of stirring rate was studied using 500, 700 and 1100rpm; this effect was examined in all formulas.

Effect of solvent /anti solvent ratio
The effect of the ratio of volume of solvent:antisolvent on the size of the formed nanoparticles was studied in three ratios of 0.5:10, 1:10 and 2:10 in all polymers at polymer:drug ratio of 2:1.

Characterization of lyophilized domperidone nanoparticles Determination of drug content and loading efficiency.
Assay was carried out by taking 6mg powder of lyophilized nanoparticles and dissolved in 20mL DMF in dry volumetric flask and sonicated for 20min and then volume was completed to 60mL with same solvent and filtered on 0.45µm filter. The absorbance of filtrate was then determined using UV-visible spectrophotometer and the drug content was calculated accordingly. The loading efficiency of nanoparticles was determined from the theoretical and actual drug contents (9) . This experiment was done in triplicate.

Determination of saturated solubility
Solubility of pure domperidone and domperidone nanoparticles was determined in each medium of 0.1N HCl of pH 1.2, phosphate buffer solution of pH 6.8, and distilled water using the shacking-flask method (10) . Plus, to ensure increase in saturated solubility of domperidone nanoparticles is due to reduction particle size, physical mixture of polymer of PVP-K15 and pure domperidone in ratio of 2:1 was used and studied. An excess amount of each powder was added in test tube containing 10mL medium, the tubes were sealed well and covered with aluminum foil then incubated in a shacking water bath at 37 o C for 72h. Sample of solution was drawn, filtered and the concentration of domperidone was determined spectrophometrically at the measured λmax using corresponding calibration curve equation in each media. The experiment was performed in triplicate and the average value was calculated.

Field emission scanning electron microscope
Field emission scanning electron microscope (FESEM) (Zeiss-Germany) of pure domperidone powder, PVP-K15 powder were confirmed by direct dusting of powder on carbon tape, while FESEM for liquid formula (F8), sample was done by the droplet evaporation technique. A droplet of liquid was settled on carbon tape and dried at room temperature. Images were taken by secondary electrons using 1kV and different magnification powers.

X-ray powder diffraction (XRPD)
X-ray powder diffraction was used to study crystalline structure of drug, polymer and the prepared nanoparticles. The X-ray diffraction have the operating voltage and current were 60 (kV) and 80 (mA) respectively.

Fourier Transform Infrared Spectroscopy(FTIR)
FTIR spectra were obtained using FTIR spectroscope. Samples that studied were pure domperidone, PVP-K15, physical mixture of PVP-K15 and pure domperidone (at ratio of 2:1), and selected nanoparticles formula. Sample was milled, mixed with potassium bromide and pressed in a form of disc of 13mm in diameter. The disc was analyzed by FTIR spectroscopy at 4000-400cm -1 .

Differential scanning calorimetry (DSC)
DSC (-60, Shimadzu-Japan) was used to determine the crystalline state of drug particularly when converted to nanoparticles and thermal characteristics of samples were determined by an automatic thermal analyzer system. Exactly weighed samples of 5mg were placed in nonhermetically aluminum pan and heated at the rate of 20º C/min against an empty aluminum pan as a reference covering a temperature range of 50 to 300º C (11) .

In vitro dissolution study of domperidone nanoparticles from capsule dosage form
Capsules prepared by simple manual filling of hard gelatin capsule with fixed quantity domperidone and lactose as filler. Then dissolution study for capsule containing domperidone nanoparticles were performed in a paddle type dissolution apparatus according to BP 2009 monograph (12) . Three types of capsule were prepared, containing lyophilized domperidone nanoparticles equivalent to 10mg of domperidone, 10mg of pure domperidone and physical mixture of polymer:pure domperidone using PVP-K15 in ratio of 2:1 equivalent to 10mg of domperidone. Each capsule was dispersed in 900 ml of 0.1NHCl of pH1.2 with aid of special sinker of butter fly to prevent floating at 37±0.5° C and rotated paddle 50rpm. A 5mL sample was draw at specific time intervals from 5-120min for analysis and replaced with same volume of fresh media to maintain sink condition at 37±0.5° C. Then sample was analyzed using UVspectrophotometer at wave length 284nm. Afterward the accumulative percentage of release was calculated and draw against time. The experiment was performed in triplicate and the average value were calculated.

Statistical analysis
The results of the experiments are given as a mean samples ± standard deviation (SD) and were analyzed for differences using one-way analysis of variance (ANOVA) at p≤0.05 and the dissolution profiles data were fitted to f1 and f2 difference and similarity factor to determine the effect of nanoparticles formulation on the dissolution patterns of domperidone from the prepared dosage form (13) .

Evaluations of prepared domperidone nanoparticle Particle size and polydispersity index analysis
The particle size of all the prepared formulas were characterized and found within a range of nanometer sizes as shown in table (2). The most essential parameters for the produced suspended nanoparticles were the mean particle size and poly dispersity index that in turn determine and control the physicochemical properties like saturated solubility and dissolution profile (14) . ABT-9000 nano laser particle size analyzer is a particle size analyzer working on basis of dynamic light scattering theory (DLS), was used to measure the size of domperidone nanoparticles and PDI "The common range of PDI values are 0-0.05 (monodisperse standard), 0.05-0.08 (nearly monodisperse), 0.08-0.7 (mid-range polydispersity), and >0.7 (very polydisperse)'' (15) . All formulas were showed monodisperse PDI except, F10 which showed a nearly monodisperse PDI while F7 and F9, showed medium range of PDI. The specific surface area (SSA) of the particles is the summation of the areas of the exposed surfaces of the particles per unit mass. Result of this study showed a reduction in particle size and consequently high surface area of domperidone nanoparticles when compare with pure drug (16) . Particle size of formulas F1-F5 of different polymer used at same polymer:drug ratio of 1:1 gave different particle size range of 334-667.5nm, this indicates that, polymers have different affinity to domperidone particle, even at same ratio. The lowest particle size was achieved with PVP-K15 polymer in F3, the measured particle size of formula F6-F10 at polymer:drug ratio of 2:1 was significantly (p≤0.05) decreased that are in range from 84.05-265.5nm, the smallest particle size was attained with PVP-K15 polymer (F8) at this ratio. The smallest size obtained in F8 as shown in figure (1) this might be due to high affinity of PVP-K15 to domperidone and low viscosity grade than other polymers of HPMC and CMC. Also that, it is found that the increase in the polymer concentration lead to a decrease in the prepared particle size of domperidone nanoparticles, as result of complete wrapping or covering and stabilize of drug particle in small size. Therefore, F8 was selected and subjected for further studies. The polymers used in this study were anionic and cationic, which might be play an important role in stabilizing of the system by steric effect, this is could be achieved by adsorbing of polymer onto the surface of particle through an anchor part that is strongly interacts with the dispersed particles, while the other solvated tail part extends into the bulk medium (17) . The effect of stirring time on domperidone nanoparticles as shown in figure (2), it is found that the increase in the time of stirring lead to increase size of domperidone nanoparticles. This finding agrees with that obtained by Chopra (18) . The higher stirring rate induces rapid nucleation toward smaller drug particles (19) . The effect of stirring rate on size of the prepared domperidone nanoparticles and was found that, the increase in stirring rate led to a significant (p≤0.05) decrease in the size of the prepared domperidone nanoparticles as shown in figure (3). The effect of ratio of volume of solution containing drug (solvent) to the solution containing polymer (antisolvent) on the size of the prepared domperidone nanoparticles was studied and results are concise in figure (4), and it was found that the solvent: antisolvent volume ratio of 1:10 gave the significant (p≤0.05) lowest mean of particle size in comparison to other ratios, that is might be result of optimum molecular distribution of drug and polymer for stabilization. The same result has been observed by Dong and coworkers in the preparation of spironolactone nanoparticles using 1:10 ratio (20) .

Evaluation of selected formulas of domperidone nanoparticles Drug content and loading efficiency
The measured drug content result from formula F8 was 1.94±0.01mg. The loading efficiency of F8 was 97.3±0.5%, so that the solvent:antisolvent method was effective in preparing domperidone nanoparticles.

Saturated solubility of pure domperidone and nanoparticles.
General statement of solubility increases when particle size decreases, is due to the increase of the surface area was considered. So on, the saturated solubility of domperidone nanoparticles of F8 was increased significantly (p≤0.05) in all solvent media of 0.1N HCl of pH 1.2, phosphate buffer of pH 6.8 and water as shown in table (3), and to ensure increase in saturated solubility of domperidone nanoparticles is due to reduction particle size, physical mixture of PVP-K15 and pure domperidone (at ratio of 2:1) was used and studied. FESEM imaging of pure domperidone powder, PVP-K15 powder and dried liquids of F8 showed a reduction in particle size of domperidone particles to nano range. Pure domperidone particles were presented with irregular shape, rough surface and large particles as shown in figure (5). Polyvinyl pyrrolidone PVP-K15 showed spherical shape as in figure 6  (A and B). FESEM of F8 as in figure 7, explained uniform and small particles size and this result might be caused by adsorption or capping effect of the stabilizer on drug surface and reduce size of domperidone particle to nanometer size.

X -ray powder diffraction analysis
The obtained spectrum of X-ray diffraction test of pure domperidone showed several strong characteristic peaks at 2θ = 9.28º, 14.94º, 15.58º, 19.80º, 24.80º, and 32º as shown in figure (8), which indicates the crystalline state of pure drug. XRPD of PVP-K15 showed low intense peaks due to the amorphous nature as shown in figure (9). Some domperidone peaks were still appeared with the physical mixture as in figure (10). Domperidone nanoparticles of F8 as shown in figure (11) showed less number and low intense of diffraction peaks in comparison to that of pure domperidone, which indicates that the crystalline structure of domperidone was reduced and part of it converted into amorphous state, similar result was also found in candisartan nanoparticles that prepared by Filipcsei's research group (21) .

Fourier Transform Infrared Spectroscopy (FTIR)
FTIR was carried out for domperidone and nanoparticles of F8. Domperidone exhibited a strong characteristic absorbance band at 1714 cm -1 due to C = O stretching vibrations of amide functional group CONHR, and N ̶ H bending characteristic band at 1693 cm -1 , N ̶ H stretching band of secondary amine appeared at 3126 cm -1 as a single band, symmetric and asymmetric C ̶ H stretching bands appeared at 2810 and 2941 cm -1 respectively. As well as aromatic symmetric and asymmetric C ̶ H stretching bands appeared at 3024 and 3074 cm -1 respectively, and the aromatic C = C stretching band appeared at 1624 cm -1 (22) as shown in figure (12). The resulted FTIR of PVP-K15, the physical mixtures of PVP-K15:pure domperidone (2:1) and F8 as in figures (13, 14 and 15) showed the presence of main peaks of domperidone which indicates there is no interaction or complexation between drug and polymer during preparation of nanoparticles.

Differential scanning calorimetry (DSC)
DSC thermogram of domperidone as in figure (16) showed a sharp endothermic peak at 249ºC, this melting point of pure domperidone as referenced (23) and revealed that the drug has crystalline nature with high purity. DSC of PVP-K15 as in figure (17) showed broad peak of water evaporation at 107º C this indicate amorphous nature of this polymer. Physical mixture of PVP-K15:pure domperidone (2:1) showed broad and low intensity peak of domperidone which is nearly at same position within the range of melting point in figure (18). This indicates no chemical reaction or complexation between drug and polymer, DSC of F8 in figure (19) showed remarkable reduction in peak intensity in comparison with pure domperidone, that indicates a reduction of crystalline state of domperidone and conversion of part of it to amorphous state, this result with that obtained X-ray diffraction analysis of formula (F8) agrees with Younis finding (24) .

In-vitro dissolution study of domperidone nanoparticles form capsule dosage form
The percentage of drug release at15min and the time required for released 100% of drug were considered for the comparison of the dissolution results between pure and nanoparticles of domperidone. The release of domperidone from F8 was faster in comparison with pure domperidone, and reached to100% of accumulative % of drug release at 15min., whereas the pure domperidone reached to 58% and 90% at 15min at the end of the study, respectively, as shown in figure (20). In addition, physical mixture of PVP-K15:pure domperidone (2:1) reached 60% and 90.9% at15 and 75min, respectively. These results indicate that, the increase in percentage of release of domperidone from nanoparticles was due to the increase in the surface area of these particles (25) and to the reduction in crystalline state of domperidone. These findings support that, the use of precipitation method was efficient and effective in preparing nanoparticles. The comparison between the dissolution profiles was done using difference and similarity test of f1 and f2 respectively. The data of accumulative percentages of release of drug from selected formula was fitted using a Microsoft Excel program to calculate f1 and f2 and the obtained results as illustrated in table (4). It was noticed that the dissolution profiles of domperidone from nanoparticles was not similar in comparison with the pure drug as a reference, F8were not similar as f1 values was higher than 15, whereas their f2 values were lower than 50 (26) . From these results, it can conclude that nanoparticles showed better in vitro dissolution profiles in comparison with pure drug.

Conclusions
Depending on obtained results, one can concludes that, PVP-K15 gave best formula (F8), the variables of type, concentration of stabilizer, volume ratio of solvent: anti solvent, time and rate of stirring showed considerable effect on decreasing of the size of domperidone nanoparticles. on increase of polymer: drug ratio, the size of produced domperidone nanoparticles was decreased, and 1:10 volume ratio of solvent: antisolvent was best than the other ratios. Analysis by DSC, FESEM and XRPD of nanoparticles of F8 indicated a reduction in crystalline state of domperidone nanoparticles. The dissolution of domperidone was highly improved against pure drug, which give 100% accumulative release at 15min.