Iraqi J Pharm Sci, Vol.26(2) 2017 Ketoprofen nanosuspension 41 Preparation and Evaluation of Ketoprofen Nanosuspension Using Solvent Evaporation Technique Fatimah M. Hussein Wais *, Ahmed N. Abood **and Hayder K . Abbas***,1 *Faculty of Pharmacy , University of Kufa , Najaf ,Iraq. **College of Pharmacy , University of Basrah, Basrah, Iraq. ***Faculty of Pharmacy, Alkafeel University College, Najaf, Iraq Abstract The effective surface area of drug particle is increased by a reduction in the particle size. Since dissolution takes place at the surface of the solute, the larger the surface area, the further rapid is the rate of drug dissolution. Ketoprofen is class II type drug according to (Biopharmaceutics Classification System BCS) with low solubility and high permeability. The aim of this investigation was to increase the solubility and hence the dissolution rate by the preparation of ketoprofen nanosuspension using solvent evaporation method. Materials like PVP K30, poloxamer 188, HPMC E5, HPMC E15, HPMC E50, Tween 80 were used as stabilizers in perpetration of different formulas of Ketoprofen nanosuspensions. These formulas were evaluated for particle size, entrapment efficiency of drug (EE), effect of stabilizer type, effect of stabilizer concentration and in-vitro dissolution studies. All of the prepared Ketoprofen nanosuspensions formulas showed a particle size result within Nano range. The average particle size of Ketoprofen nanosuspensions formulas was observed from 9.4 nm to 997 nm. Entrapment efficiency was ranged from 79.23% to 95.41 %. The in vitro dissolution studies showed a significant (p<0.01) enhancement in dissolution rate of nanosuspension formulas compared to pure drug (drug alone) and physical mixture (drug and stabilizer). The results indicate the suitability of solvent evaporation method for Ketoprofen with improved in vitro dissolution rate and thus perhaps enhance fast onset of action for drug. Keywords: Ketoprofen , Nanosuspension, Particle size, Dissolution rate. انوي للكيتوبروفين باستخدام تقنية تبخير المذيبنتحضير وتقييم معلق 1,***حيدر كاظم عباس و **، احمد نجم عبود *فاطمة محمد حسين ويس كلية الصيدلة ، جامعة الكوفة ، النجف ، العراق . * ** كلية الصيدلة ، جامعة البصرة ، بصرة ، العراق . *** قسم الصيدلة ، كلية الكفيل الجامعة ،النجف، العراق . الخالصة حجم الُجَسْيم وذلك الن الذوبان يحدث على سطح المذاب. المساحة السطحية المساحة السطحية لُجَسْيم الدواء تزداد بواسطة تقليل ك األكبر يرافقها المزيد والسريع في معدل الذوبان. الكيتوبروفين هو ضمن الصنف الثاني )نظام تصنيف الصيدالنيات البيولوجية( والذي يمل بة ومعدل الذوبان للكيتوبرفين من خالل تحضير معلق النانو بطريقة تبخير ذوبانية قليلة مع نفاذية عالية. الغرض من البحث هو زيادة االذا و الهايدروكسي بروبل مثيل سليلوز والتوين تم استخدامها كمثبتات في تحضير 811بيروليدون والبلوكسامير المذيبب. مواد مثل بولي فنيل حجم الُجَسْيم وتأثير نوع المثبت وتركيز المثبت ودراسات من خاللصيغ مختلفة من معلق النانو للكيتوبروفين. هذه الصيغ تم تقييمها ْيم الذوبانية بالمختبر. كل الصيغ المحضرة للُجَسْيمات النانوية للكيتوبروفين أظهرت حجم الُجَسْيم ضمن مدى النانو و ان معدل حجم الُجسَ . %44.98 الى %94.97 . كفاءة التحميل كانت بين نانومتر 449لى نانومتر ا4.9 لصيغ الُجَسْيمات النانوية للكيتوبرفين لوحظت بين في معدل الذوبان لصيغ معلق النانو بالمقارنة للدواء وحده او الخليط الفيزيائي (p<0.01ان دراسات الذوبان بالمختبر أظهرت تحسن مهم ) ين مع تحسين معدل الذوبان بالمختبر وهذا ربما يعزز الفعل األول من الدواء مع المثبت. النتائج تبين مالئمة طريقة تبخير المذيب للكيتوبرف والسريع للدواء. ، معلق نانوي ، حجم الجسيم ، معدل الذوبان .كيتوبرفين الكلمات المفتاحية : Introduction Bioavailability of drug depends on its dissolution and availability as a solution form at the site of absorption. Therefore, the main problems associated with poor soluble drug its low solubility in biological fluids, which results into low bioavailability after administ ration. Poor soluble drug will typically show dissolutionrate limited absorption (1). Drug dissolution in biologic fluids is an important step for systemic absorption. 1Corresponding author E-mail: Hayderkadhim@ymail.com Received: 12/8/2017 Accepted: 29/9/2017 Iraqi J Pharm Sci, Vol.26(2) 2017 Ketoprofen nanosuspension 42 The rate at which drugs with poor aqueous solubility dissolve and release from dosage form in the biologic system frequently controls the rate of systemic absorption of the drug. About 40% of different chemical entities discovered now are poorly water soluble (2). Therefore, the pharmaceutical researches focus on enhancing the solubility and rate of dissolution of poorly water-soluble drugs. According to US food and drug administration (FDA), the drug are classified into four classes based on aqueous solubility and permeation through biological membrane. These classes are follows(3) : class I (highly soluble, highly permeable), class II (poorly soluble , highly permeable), class III (highly soluble, poorly permeable),and class IV ( poorly soluble, poorly permeable) Poor water soluble drug has many problems such as low or variable bioavailability, large dose and delayed onset of action. There are various approaches available for overcoming the solubility of poorly soluble drugs for examples, modification of the crystal habit, self-emulsification, solid dispersion, solubilization by surfactant, salt formation, pH modification, co-crystallization, use of co- solvent, micronization and nanosization. In general, the rate of the drug solubility is related to particle size, as a particle gets smaller one, as (the surface area: volume) ratio increases. The larger surface area allows a better interaction with the solvent, which cause increase in dissolution rate. Since dissolution takes place at the surface of drug particle, the high dissolution rate of drug is associated with large surface area of drug particles. Many drugs are active intravenously but are not effective when taken orally, because of low oral absorption. Reduction of the particle size for drugs with low aqueous solubility to a micronized form has improved the oral absorption of Griseofulvin, nitrofurantoin, and many steroids. In addition smaller particle size enhances water penetration into the particles (4). The Ostwald–Freundlich equation describes the relationship between the saturation solubility of drug and its particle size: 𝑙𝑜𝑔 𝐶𝑠 𝐶∞ = 2 𝜎 𝑉 2.303 𝑅𝑇 𝑝𝑟 ----------1 Where CS is the saturation solubility, C∞ is the solubility of the solid particles with large sizes, σ is the interfacial tension, V is the molar volume, R is the gas constant, T is the absolute temperature, ρ is the solid density, and r is the radius. It is clear that the saturation solubility (CS) of certain drugs will increase by decreasing the particle size (r) (5). Nanosization is a method, where a drug particle is converted into nanoparticles having size less than 1μm. Nanotechnology allowed drug delivery system with improved physical, chemical and biological properties. The main purposes in designing nanoparticles as delivery systems are to control the particle size, surface properties and dissolution of drug, then to reach the action site at a perfect rate and dose level(6). The selection of suitable method for the formulation of nanoparticles depends on the physicochemical characteristics of used polymer and the drug to be loaded. However, the nanosuspension form has an advantage in their ability to increase dissolution rate and to enhance the bioavailability of poor soluble drug. Ketoprofen is an example of class II drug. It is a white crystalline powder and practically insoluble in water (7).It is a non- steroidal anti-inflammatory drug with analgesic and antipyretic properties. The objective of study was to increase its solubility and then the dissolution rate by preparation of nanosuspension using antisolvent precipitation method. Materials and Methods Materials Ketoprofen was purchased from Lishui Nanming Chemical CO.,Ltd (China). Polyvinylpyrrolidone (PVP K30), Poloxamer 188, and Hydroxypropyl methylcellulose (HPMC) E5, E15 and E50 were purchased from Shanghai Send Pharmaceutical Technology co.Ltd (China). Methanol was obtained from Gailand Chemical Company (UK). Disodium hydrogen phosphate and Potassium Dihydrogen Phosphate were supplied by BDH Laboratory Supplies (England) and SPINE- CHEM. Limited, respectively. Methods Determination of Ketoprofen saturation solubility Saturated solubility measurements of Ketoprofen in various solutions were determined by shake flask method. An excess amount of Ketopofen were separately introduced into stoppered conical flask containing 10 ml of Iraqi J Pharm Sci, Vol.26(2) 2017 Ketoprofen nanosuspension 43 0.1 N HCl (pH 1.2), DW and buffers of phosphate (pH 6.8 and 7.4). The sealed flasks were shaken for 24 hours at 37°C .Visual inspection was made to check the precipitation of drug particles in the sample. An aliquot of solution was passed through 0.45μm filter paper and the filtrate was suitably diluted and analyzed on a UV/visible spectrophotometer at 260 nm wavelength (8) .Three determinations were carried out to calculate the solubility of Ketoprofen . Preparation of Ketoprofen nanosuspensions Nanosuspensions of Ketoprofen were prepared by the solvent evaporation technique, which is also termed as antisolvent precipitation method. Ketoprofen powder was dissolved in methanol (2.5 ml) at room temperature preparing drug concentrations (20 and 40 mg/ml). the resultant organic solution of drug (organic phase) was added drop by drop by means of a plastic syringe positioned with the needle directly into aqueous solution of stabilizer(9, 10). The mixture of drug solution and stabilizer was kept at 50°C and subsequently agitated at stirring speed of 500 revolution per minute (rpm) on a magnetic stirrer for about one hour to permit methanol to evaporate(11). Ketoprofen being insoluble in water, therefore, it will precipitate with stabilizer. The ratios (weight: weight) of drug to stabilizer used to prepare the nanosuspension were 1:1, 1:2 and 1:3. Tween 80 was also used at different volumes as explained in table (1). Table (1) :Compositions of ketoprofen nanosuspensions using different stabilizers at different drug: stabilizer ratios with constant volume of injection of organic solution (2.5 ml) Formula NO. Drug (mg) PVP k30 (mg) Poloxamer 188 (mg) HPMC E5 (mg) HPMC E15 (mg) HPMC E50 (mg) Tween 80 (ml) 1 50 50 2 50 100 3 50 150 4 50 50 5 50 100 6 50 150 7 50 50 8 50 100 9 50 150 10 50 50 11 50 100 12 50 150 13 50 50 14 50 100 15 50 150 16 50 0.1 17 50 0.2 18 50 0.3 Iraqi J Pharm Sci, Vol.26(2) 2017 Ketoprofen nanosuspension 44 Continued table (1) Formula NO. Drug (mg) PVP k30 (mg) Poloxamer 188 (mg) HPMC E5 (mg) HPMC E15 (mg) HPMC E50 (mg) Tween 80 (ml) 19 50 50 50 20 50 50 50 21 50 50 50 22 50 50 50 23 50 50 0.1 24 50 50 50 25 50 50 50 26 50 50 50 27 50 50 0.1 28 50 50 0.1 29 50 50 0.1 30 50 50 0.1 31 100 100 32 100 200 33 100 300 34 100 100 35 100 200 36 100 300 37 100 100 38 100 200 39 100 300 40 100 100 41 100 200 42 100 300 43 100 100 44 100 200 45 100 300 Iraqi J Pharm Sci, Vol.26(2) 2017 Ketoprofen nanosuspension 45 Effect of type and concentration of stabilizer on the size of Ketoprofen nanosuspensioins To reach the best formula different types of stabilizers at various concentrations were used in preparation of Ketoprofen nanosuspensions. The formulas (F1-F15) prepared by using these different stabilizers and were subjected to particle size analysis. The combinations of two stabilizers (F19-F30) were also studied to determine their effect on particle size. Characterization of the prepared nanosuspension Particle size and surface area Determination of particle size was done using ABT-9000 nano laser particle size analyzer (Angstrom Advance Inc. USA), which is apparatus of a dynamic light scattering, acts by measuring the light intensity that is scattered by the molecules sample as a time function, at scattering angle (90°) and constant temperature (25°C) without dilution of samples. The particle size can be determined by placing samples of formulas in the analyzer. The average diameters and polydispersity index of samples were measured for each formula. Determination of entrapment efficiency of drug (EE) in Nanosuspension The freshly prepared nanosuspensions of different drug: stabilizer ratios were centrifuged at about 20,000 rpm for 20 minutes at 4°C using cooling ultracentrifuge. The concentration of free drug was detected by measuring the absorbance an appropriately diluted sample of supernatant at 260 nm using UV- spectrophotometer (11-13). EE was determined by subtracting the weight of free drug in the supernatant layer of solution from the initial weight of drug used. For each formula, the experiment was repeated in triplicate and the average was calculated. Percentage of entrapment efficiency (EE) could be calculated by the following equation: 𝑬𝒏𝒕𝒓𝒂𝒑𝒎𝒆𝒏𝒕 𝒆𝒇𝒇𝒊𝒄𝒊𝒆𝒏𝒄𝒚% = 𝒘𝒆𝒊𝒈𝒉𝒕𝒊𝒏𝒊𝒕𝒊𝒂𝒍 𝒅𝒓𝒖𝒈 − 𝒘𝒆𝒊𝒈𝒉𝒕𝒇𝒓𝒆𝒆 𝒅𝒓𝒖𝒈 𝒘𝒆𝒊𝒈𝒉𝒕𝒊𝒏𝒊𝒕𝒊𝒂𝒍 𝒅𝒓𝒖𝒈 × 𝟏𝟎𝟎 (𝟐) In vitro dissolution of Ketoprofen nanosuspensions In vitro dissolution study was performed by USP dissolution apparatus-type II using 900 ml of 0.1N HCl (pH 1.2) as a dissolution medium maintained at 37 ± 0.5°C and stirring speed (50 rpm). The freshly prepared nanosuspensions (equivalent to 50 mg of Ketoprofen ) of drug: stabilizer ratios were added to the dissolution medium, five-milliliter samples were withdrawn at specific intervals of time ( 5,10,15,20 ,30,40.50,60,70,80 and 90 minutes ), then filtered through a 0.45 µm filter paper and analyzed for their drug concentrations by measuring at 260 nm wavelength. The same test was done for pure drug (free drug in dissolution media) and physical mixture of drug powder and stabilizer material (PM) (14). In vitro dissolution / model independent approach The percent of dissolution efficiency (%DE) was detected for comprising the relative performance of Ketoprofen nanosuspension at drug: stabilizer ratios, pure Ketoprofen powder and physical mixture of drug and polymer. The %DE at 60 minutes (%DE60 min) for each formulation was computed (using Microsoft Excel program) as the percent ratio of area under the curve of dissolution up to the time t to that of the rectangle area described by complete dissolution (100%) at the same time (15, 16). DE = {∫ 𝒚. 𝒅𝒕 𝒕𝟐 𝒕𝟏 x /y100 x (t2-t1)} x 100 (3 ) The difference factor (f1) evaluating the percent error between two curves over all time points is given by: f1={ [∑t=1n │Rt – Tt │] / [ ∑t=1n Rt] } x 100 (4) Where t is the number of dissolution sample, n is the dissolution times number, and Rt is the amount of the reference drug and Tt is amount of test drug dissolved at each time point t. The percent error is zero when the test drug and reference outlines are matching and increase correspondingly with the dissimilarity between the two dissolution profiles. The similarity factor (f2) is a logarithmic transformation of the sum-squared error of differences between the test Tt and reference Rt over all time points, and is given by: f2= 50x log { [ 1 + 𝟏 𝒏 ∑ │𝑹𝒕 − 𝑻𝒕𝒏𝒕=𝟏 │ 2 ]-0.5 x 100 (5) The standards for similarity factor are 50- 100, while for dissimilarity factor are 0-15. The dissolution parameters were detected by fitting the data of dissolution using a software Iraqi J Pharm Sci, Vol.26(2) 2017 Ketoprofen nanosuspension 46 program, called DDSolver, which is a menu- driven add-in program for Microsoft Excel written in visual basic for applications. Results and Discussion Determination of Ketoprofen saturation solubility The saturation solubility values of Ketoprofen were found to be about 0.11mg/ml, 0.205mg/ml, 9.82mg/ml and 10.75mg/ml, in D.W, pH 1.2, pH 6.8 and pH 7.4, respectively. The results of saturation solubility of Ketoprofen were illustrated in table (2). Determination of saturated solubility of pure Ketoprofen powder in phosphate buffers (pH 6.8 and 7.4) and 0.1N HCl (pH 1.2) was necessary to maintain the sink condition that is the volume of medium at least three times greater than the necessary volume to form a solution saturated with drug substance (17). According to biopharmaceutical classification system, Ketoprofen is an example of Class ΙΙ drugs having low aqueous solubility and well absorption through gastrointestinal tract (18) due to high permeability and lipophilicity. Ketoprofen is a weak acid drug and will be ionized at higher pH; it is practically in soluble in water (7).Therefore, its solubility increase with pH increase towards alkaline medium (19) as shown in figure (1). Table (2): Saturation solubility values of Ketoprofen in different media Media Solubility (mg/ml) Distilled water D.W 0.11 0.1N HCl (pH1.2) 0.205 Phosphate buffer (pH 6.8) 9.82 Phosphate buffer (pH 7.4) 10.75 Figure (1): Solubility-pH profile of Ketoprofen powder Particle size and surface area The average particle size of all the prepared formulas using ABT-9000 Nano laser particle size analyzer. All of the prepared Ketoprofen nanoparticles formulas showed a particle size result within Nano range. The average particle size of Ketoprofen nanoparticles formulas was observed from 9.4 nm to 997 nm, as shown in tables (3) and (4).The smallest size 9.4 nm for F18 and the largest size 997nm for F26 formula. The specific surface area (SSA) of the particles is the summation of the areas of the exposed surfaces of the particles per unit mass. Where the particle size is inversely related with the surface area(20). The SSA values for the prepared formulas were at range ( 2.07-247.47) m2/g, the largest surface area is recorded in F18 formula and smallest surface area 2.07m2/g in F26 formula as appeared in table (3 and 4). Polydispersity index analysis Polydispersity index is a parameter used to define the particle size distribution obtained from the particle size analyzer. Polydispersity index gives degree of particle size distribution at range from 0.00466 to 0.019 depending on formulation variables. The formula F28 showed lowest PDI (0.00466), as seen in table (3), that indicate good uniformity of nanoparticle size. Uniformity of particle size is determined by polydispersity index values in which the low value means the best uniformity. The range of PDI values (0-0.05) means (monodisperse system), 0.05-0.08 (nearly monodisperse), 0.08- 0.7 (mid-range polydispersity), and >0.7 (very polydisperse) (21). Effect of stabilizer type (optimization of polymer used) The effect of using one stabilizer The average particle size for formulas ( F1-F18) at drug: stabilizer was ranged from 9.4 nm-676.5 nm as seen in table (3). PVP K-30, poloxamer188, HPMC (E5, E15, and E50) and Tween 80 were used as stabilizers in these formulas (F1-F18). PVP K-30 and poloxamer188 are polymeric nonionic stabilizers for nanosuspensions, they form physical barrier on the surface and interrupt the contact of the close particles (22). The non-ionic stabilizers with amphiphilic moieties are usually employed to give steric stabilization, which is dominated by wetting effect. Iraqi J Pharm Sci, Vol.26(2) 2017 Ketoprofen nanosuspension 47 When non-ionic stabilizers are incorporated into nanosuspensions, they are adsorbed onto the surface of the drug particlesth rough an anchor part that is strongly interacted with the suspended particles (23), while the other part that is well-solvated tail will extend into the dispersion medium. HPMC was frequently used as a stabilizer due to its alkyl substituent, which has higher affinity towards the hydrophobic surface of drug particle (24).Figure (2), shows the effect of polymer type on particle size. Generally HPMC at 1:1 of drug: stabilizer ratio shows smaller submicron size (9.4nm) when compared with PVP K30 and poloxamer188 because the hydrophobic moiety of HPMC has a good affinity toward the drug particles and thereby it is able to provide active steric barrier against particles growth. On the other hand, the large size of particle (426.5nm) was obtained in formula F4 that contains poloxamer 188 as a stabilizer at drug: stabilizer 1:1. These results are in agreement with that obtained by El-Badry M et.al. in preparation of Albendazol nanosuspension(25). The size of other formulas lies between the average sizes ranges, since the differences in particle size are due to different in affinity of the polymer molecules toward drug particles. PDI for formulas (50mg of drug and 50mg of stabilizer) F1, F4, F7, F10, F13 and F16 (50mg of drug and 0.1 ml of Tween 80) ranged from 0.008 to 0.016, therefore all these formulas have monodisperse standard. SSA of the formulas F1, F4, F7, F10, F13 and F16 was ranged from 21.02m2/g to 5.34 m2/g. larger surface area was found to be 21.02m2/g in formula F16 which contain Tween 80 as a primary stabilizer, because it had smaller particle size. In contrast to this result, a smaller surface area 5.34 m2/g was obtained in formula F4 that contain poloxamer188 as a stabilizer, since it had lager particle size. Table (3) :Particle size, polydispersity index and specific surface area of Ketoprofen formulas using different types and amounts of stabilizers Formula No. Average Particle size (nm) PD SSA (m2/g) F1 317 0.012 6.75 F2 313 0.009 7.1 F3 282.5 0.009 7.65 F4 426.5 0.01 5.34 F5 317 0.011 6.88 F6 282.5 0.01 7.88 F7 270 0.008 8.23 F8 282.5 0.011 7.65 F9 317 0.01 6.84 F10 282.5 0.01 7.91 F11 426.5 0.024 5.58 F12 479 0.009 4.63 F13 269 0.008 8.23 F14 426.5 0.007 5.24 F15 676.5 0.011 4.17 F16 95.1 0.016 21.02 F17 88 0.012 24.97 F18 9.4 0.017 247.47 Iraqi J Pharm Sci, Vol.26(2) 2017 Ketoprofen nanosuspension 48 Figure (2): Effect of using different types of polymers on particle size at ratio 1:1 (F1, F4, F7, F10 and F13). The effect of Co- stabilizers Combination of stabilizers was preferred for long-term stabilize. Based on the Stokes’principle, drug nanocrystals tend to precipitate in the production media like water or other vehicles (23) .The appearance of agglomeration was largely due to the small particle size, which leads to increment surface- volume ratio that can produce a large amount of Gibbs free energy. Slowly, the particle will collective automatically to decrease the extra surface energy. When it comes to crystal growth, the theoretical background is Ostwald ripening. For that reason, a stabilizing agent is used to solve this problem. Stabilizers can efficiently decrease the surface activity energy to inhibit aggregation that decrease dissolution rate (23). The critical parameter of stabilizer is the stabilization ability by enhancement of the physical stability of nanoparticulate system and maintaining the smallest size of particles. (26). The average particle size of F19-F30 was ranged from 49 nm to 997 nm as shown in table (4). Table (4): Particle size, polydispersity index and specific surface area for ketoprofen formulas using combined stabilizer system Formula Type of mix polymers Average Particle size (nm) PDI SSA (m2/g) F19 PVP+polo188 49 0.008 45.40 F20 PVP+ E5 399 0.012 6.13 F21 PVP +E15 503 0.071 4.39 F22 PVP+E50 564 0.018 3.91 F23 PVP+tween80 99.9 0.019 24.73 F24 Polo188+E5 399 0.007 5.75 F25 polo188+E15 797 0.008 2.73 F26 Polo188+E50 997 0.005 2.07 F27 Polo188+tween80 84.85 0.011 31.45 F28 E5+tween 80 111 0.00466 21.62 F29 E15+tween80 119 0.01 17.87 F30 E50+tween80 141 0.01 15.09 Iraqi J Pharm Sci, Vol.26(2) 2017 Ketoprofen nanosuspension 49 According to the above results, a smaller particle size (49 nm) was achieved in a combination between PVP K30 and poloxamer188. Since this combination show significant (p<0.05) reduction in particle size as when compared with other combinations, and it may be because of the highest affinity to the drug molecules, the same data was reported by XueMing Li et.al. in Fenofibrate nanosuspension(27). A combination of PVP K30 and HPMC (E5, E15, and E50) gave a large particle size as when compared with each polymer separately, this result was in agreement with Shahzeb Khan et.al in preparation of Ibuprofen nanocrystal(28). Furthermore combination of poloxamer188 and HPMC (E5, E15, E50) in formulations F24, F25 and F26 shows larger average particle size with significant differences(p<0.05), these results due to that the combination lead to increase viscosity of the disperse media, so it is ineffective combination and cannot stabilize the nanoparticulate system. On the other hand, a mixture of Tween 80 with each polymer separately gave good reduction in particle size as seen in figure (3).From above results formula F19 was select as a best formula for further evaluation tests. Figure (3): Effect of use of mixed polymers on particle size of Ketoprofen A combination of Tween 80 and PVP K30 or poloxamer188 yields nanoparticles with average size not different significantly (p>0.05) from Tween 80 as single stabilizer. This is due to PVP K30 and poloxamer188 are polymeric molecules, have ability to adsorb on the particle surface and act as a steric barrier, preventing close contact of the particles. Furthermore, it may be because both of them are non-ionic stabilizers so no ionic repulsion occurs. Similar results were gained by P. Kocbek et.al in preparation of Ibuprofen nanosuspension (29). The size of particles in other combination of Tween 80 with HPMC polymer types was larger than that obtained from combination of Tween with poloxamer or PVP K 30. PDI of formulas (F19-F30) was varied from 0.00466 to 0.019, therefore, all these formulas have monodisperse standard. Depending on formulation variables, the formulation F28 (HPMC E5 +Tween 80) showed lowest PDI (0.00466) that indicates good uniformity in average particle size distribution. A narrow size distribution is essential to prevent particle growth, due to Ostwald ripening phenomenon(30). Formula F21 (PVP K30+HPMC E15) exhibited a highest PDI (0.071) so that is in the range of nearly monodisperse. SSA of formulas (F19-F30) was ranged from 2.07m2/g to 45.40 m2/g. F19 formula (PVP K30+poloxamer188) showed large surface area about 24.73 m2/g, since it had showed small particle size. F26 formula (poloxamer188+ HPMC E50) exhibited small surface area, due to large particle size of this formula, as particle size decrease, the surface area per unite mass increase and vice- versa. Effect of stabilizer concentration The concentration of stabilizer may give negative effect (decrease particle size) or positive effect of on particle size (increase particle size). It can also influence on the adsorption affinity of non-ionic stabilizers to particle surface. In general, as the concentration of stabilizer increases the particle size decreases at fixed drug concentration, which indicated that the drug particle surface was sufficiently enveloped by the stabilizer molecules (31). Concentration of stabilizer was played a role in maintaining stability of nanosuspension, if used too low concentration lead to aggregation of particle, and if used high concentration lead to enhance Ostwald ripening (25). As shown in table (3) the size range of particles is decrease in the sequence of F1 (317nm) > F2 (313 nm) > F3 (282.5nm) that correspond to 1:1, 1:2 and 1:3 of drug: stabilizer (PVP K30) ratio, respectively, these results indicated that mean size of particles showed a regular decrease with increasing the concentration of pvpk30. The same result was observed with poloxamer188. The size range of particles is also decreased in the sequence F4 (426.5nm)>F5 (317nm)>F6 (282.5nm) that correspond to 1:1, 1:2 and 1:3 of drug: stabilizer (poloxamer188) ratio, respectively. Figures 4 and 5 explain the effect of PVP K30 and Iraqi J Pharm Sci, Vol.26(2) 2017 Ketoprofen nanosuspension 50 poloxamer188 concentration on average particle size correspondingly. These effects may be due to a process of a primary covering of the newer surfaces competing with the aggregation of the uncovered surfaces. Hence, an elevation in ratio of surfactant in the primary dispersion results in rapid enclosing of the newly formed particle surfaces. There was an optimum concentration of surfactant, above which the increase in concentration did not result in a decrease in particle size due to saturation point; these results are in agreement with that obtained by Chander Parkash Dora et.al in preparation of Glibenclimid nanoparticle when poloxamer188 was used as stabilizer at different ratios (32). Poloxamer188 (pluronic F68)® is a block co- polymer, can act as a surfactant, responsible for the hydrophobic association with the molecules of drug. The inhibition of the crystal growth is mainly related to the hydrophobic part (polypropylene oxide group PPO) in the pluronic polymer, while the second chain which is (the hydrophilic polyethylene oxide) (PEO) can provide steric hindrance against particles aggregation26. On the other hand, as the concentration of HPMC increases the particles size increases in different ratios of grades. For grade E15 the size range is increase in sequence of F10 (282.5nm)