Iraqi J Pharm Sci, Vol.27(1) 2018 Darifenacin hydrobromide loaded nanostructured lipid carriers DOI: http://dx.doi.org/10.31351/vol27iss1pp53-68 53 Preparation and Evaluation of Darifenacin Hydrobromide Loaded Nanostructured Lipid Carriers for Oral Administration Ali k. Ala Allah*,1 and Ahmed A. Hussein** *Ministry of Health and Environment, Babylon Health Directorate, Babylon, Iraq. ** Department of Pharmaceutics, College of Pharmacy, University of Baghdad, Baghdad, Iraq. Abstract Darifenacin hydrobromide is a selective M3 receptor antimuscarinic drug and it is used in the management of urinary frequency, urgency, and incontinence in detrusor instability. It is slightly soluble in water, undergoes extensive hepatic first-pass metabolism and has short elimination half-life (3–4 hours). Therefore, It has low bioavailability (15.4 % - 18.6 %). Darifenacin hydrobromide loaded nanostructured lipid carriers (NLCs) were formulated by emulsification sonication using different ratios of solid lipid to liquid lipid, different types and concentration of surfactants. Formula sixteen , containing darifenacin hydrobromide 8.9 mg , solid lipid glyceryl monostearate and olic acid in a ratio equal to 77.5:22.5 , tween 80 (0.5%) , and vitamin E that is added as an antioxidant , was considered as an opti- mized formula based on its particle size, polydispersity index (PDI) , zeta potential and entrapment effi- ciency. This formula was subjected to further characterization such as DSC, FTIR, XRD, AFM, and release study. FTIR and DSC studies indicated no interaction between drug and excipients. XRD study showed a halo pattern which is a significant pattern of amorphous form of the drug. Atomic force mi- croscopy (AFM) study showed discrete lipid nanoparticles with no aggregation. Release study exhibited burst release in the first hour followed by sustained and controlled release up to 12 hours. Keywords: Darifenacin hydrobromide, Nanostructured lipid carrier, Bioavailability. الحامالت الدهنية ذات البنية النانوية المحملة بالداريفناسين هايدروبرومايد المعطاة عن طريق الفم **و احمد عباس حسين 1،*علي كاظم على هللا والبيئة ، دائرة صحة بابل، بابل ، العراق.وزارة الصحة * فرع الصيدالنيات ، كلية الصيدلة ، جامعة بغداد ، بغداد ،العراق. ** الخالصة ويستخدم في عالج سلس البول. هذا العقار قليل الذوبان في M3عقار داريفناسين هايدروبرومايد يعتبر مثبط انتقائي لمستلمات النطاق بالكبد ويمتلك زمن طرح من الجسم قصير جداً من ثالثة الى اربع ساعات لذلك التوافر الحيوي الماء، يتعرض الى ايض واسع في البالزما لهذا الدواء قليل من خمسة عشر بالمئة الى ثمانية عشر بالمئة. حامالت الدهون ذات البنية النانوية المحملة بدرافيناسين صهر مع استخدام الموجات فوق الصوتية مستخدمين نسب مختلفة من الدهون الصلبة الى هايدروبرومايد صنعت بطريقة المستحلب المن الدهون السائلة، انواع مختلفة وتراكيز مختلفة من العوامل التي تقلل الشد السطحي. ملغم ( و نسبة دهون صلبة الى دهون سائلة تســـــــــــاوي 8, 9التركيبة السادسة عشر تتكون من دارافيناسين هايدروبرومايد ) التركيبة السادسة عشر تعتبر أفضل تركيبة اعتماداً يضاف كعامل مضاد لالكسدة . E( , و فيتامين %8, 5) 88توين ,22, 5: 77, 5 برت لدواء بداخلها. التركيبة السادسة عشر اختعلى حجم الجزيئات والتوزيع الحجمي للجزيئات والشحنة السطحية والقابلية على احتواء ا ( ، وكذلك دراسة AFMاي اف ام ) ، حيود االشعة السينية، ( FTIR ) باستخدام المسح الكالوري، مطيافية االشعة تحت الحمراء واد اخل بين الدواء والمالتركيبة السادسة عشر اختبرت باستخدام االشعة تحت الحمراء والمسح الكالوري واظهرت عدم وجود تدالتحرر. اظهر وجود جزيئات AFMاالخرى في التركيبة. اختبار حيود االشعة السينية اظهر شكل غير متبلور , واختبار مجهر القوة الذريـــة دمنفصلة واليوجد تجمع للجزيئات.دراسة التحرر للدواء اظهرت تحرر سريع للدواء خالل الساعة االولى بعد ذلك تحرر بطيء الى ح اثنا عشر ساعة.اظهرت الدراسة الكلية اهمية الحامالت الدهنية ذات البنية النانوية كنواقل لزيادة التوافر الحيوي لعقار داريفناسين هايدروبرومايد مقارنة بالحبوب المعطاة عن طريق الفم . ة، التوافر الحيوي.الكلمات المفتاحية: داريفناسين هايدروبرومايد، حامالت الدهون ذات البنية النانوي Introduction Recently, several approaches have been investigated to develop nanosized drug delivery system such as lipid nanoparticales with a solid matrix which are divided into solid lipid nano- particles (SLNs) and nanostructured lipid carri- ers (NLCs). SLNs are prepared from solid lipids only. Therefore, after preparation at smallest a part of the particles crystallize in a higher en- ergy modification (α or β). During storage, these modifications can transform to the low en- ergy, more ordered β modification. Due to high degree of order of this modification, the number of imperfections in the crystal lattice is small and this leads to drug expulsion. 1Corresponding author E-mail: pharss75@gmail.com Received: 31/10/2017 Accepted: 3/3/2018 Iraqi Journal of Pharmaceutical Sciences http://dx.doi.org/10.31351/vol27iss1pp53-68 http://bijps.com/index.php/bijps/index Iraqi J Pharm Sci, Vol.27(1) 2018 Darifenacin hydrobromide loaded nanostructured lipid carriers 54 NLCs have been developed to overcome the drawbacks associated with SLNs. They are considered to be the second generation of lipid nanoparticles. Compared to SLNs, NLCs show a higher loading capacity for active compounds by creating a less ordered solid lipid matrix, i.e. by blending a liquid lipid with the solid lipid, a higher particle drug loading can be achieved. Therefore, the NLCs have an increased drug loading capacity in comparison to SLNs and the possibility of drug expulsion during storage is less . NLCs have also a lower water content of the particle suspension and a less tendency of unpredictable gelation(1). Darifenacin is a selective M3 antimusca- rinic with actions similar to those of atropine. It has a greater selectivity for the muscarinic re- ceptors of the bladder. It is subjected to exten- sive first-pass metabolism and has a short elim- ination half-life after intravenous and immedi- ate release oral dosage forms (3-4 hr)(2). The absolute bioavailability of darifen- acin from 7.5 mg and 15mg prolonged release tablet was estimated to be 15.4 % and 18.6% re- spectively(2). It is metabolized in the liver by cy- tochrome P450 isoenzymes CYP 2D6 and CYP 3A4 (2). Darifenacin is a P-glycoprotein(P-gp) substrate. It is about 98% bound to plasma pro- teins . Most of the dose is excreted as metabo- lites in the urine and feces(2). The objective of this study is to pre- pare differents darifenacin hydrobromide loaded NLCs to improve the bioavailability of darifenacin hydrobromide which undergoes ex- tensive first-pass effect when formulated in conventional dosage form , characterization of the prepared formulas , and the selection of the best darifenacin hydrobromide loaded NLC which subjected to further characterization . Af- ter that, formulation of the best formula as a dosage form well known to the patient (capsule dosage form) was achieved in order to improve patient compliance . Materials and Methods Materials Darifenacin hydrobromide and glyceryl monostearate ( GMS ) ( hangzhou hyperchemi- cal China ) , oleic acid ( central drug house company India ) , tween80 , stearic acid and pal- mitic acid ( BDH chemical England ) , methanol ( romil, United kingdom ) and distilled deion- ized water was used. All other chemicals were reagent grade. Method Screening of components Prior to the production of NLC formula- tion , lipid, oil , and surfactant screening should be performed to determine the most suitable components for the active ingredient to be in- corporated in the NLC . Solubility in solid lipid The solubility of darifenacin hydrobro- mide in different solid lipids was determined by semi-quantitative method. An accurately weighed fixed quantity (8.9 mg) of the drug was taken in a series of test tubes and solid lipids were added in increments until the drug is com- pletely solubilized. The temperature of the test tubes was controlled at 5-10 °C above the melt- ing point of respective lipids (3). The test tubes were intermittently mixed using cyclone mixer and observed for any drug residues. The amount of lipid (mg) required to completely solubilized the drug in the molten state was determined (3). Solubility in liquid lipid An excess amount of darifenacin hydro- bromide was added to 5ml of oil in a test tube and mixed using cyclone mixer. The mixture was agitated on mechanical shaker for 24 hr at room temperature for equilibration. After equi- librium, each sample was centrifuge at 10,000 rpm for 30 min to separate the undissolved drug. Supernatant that obtained was pulled and filtered through 0.45 μm filter. The filtrate was diluted suitably with methanol and saturation solubility of darifenacin hydrobromide (mg/ml) in oil was determined by recording absorbance using UV- Vis spectrophotometer at respective λ max (4). Preparation of nanostructured lipid carriers ( NLCs ) An accurately weighed solid lipid GMS and liquid lipid oleic acid were mixed and then heated at 5 – 10 °C above the melting point of lipid mixture. To this lipid mixture, the drug was added to obtain a clear melting solution. An aqueous phase was prepared by dissolving sur- factant in deionized water and heated to the same temperature as that of the oil phase. Then, this hot aqueous phase was added dropwise to the lipid phase at a constant rate (2 ml / min) under magnetic stirring. After that, this pre- emulsion was sonicated for 20 minutes using probe sonicator. The resulting hot nanoemul- sion was cooled to room temperature to induce crystallization. Twenty-two formulas were pre- pared by this method as shown in table (1). Vit- amin E was added to the selective formula as antioxidant. The selective formula was freeze- dried by using cryoprotectant to convert NLC to dry powder and was filled in a hard gelatin cap- sule of zero size (5). Iraqi J Pharm Sci, Vol.27(1) 2018 Darifenacin hydrobromide loaded nanostructured lipid carriers 55 Table1. Formulations of darifenacin hydrobromide loaded nanostructured lipid carriers ( NLCs) Formulas No. Amount of drug (Darifenacin Hydrobromide) mg Ratio of solid lipid to liquid lipid glyceryl monostearate: oleic Acid Type of surfactant % ( W / V ) Co- surfactant % ( W / V ) Water Tween20 Tween 80 Poloxamer80 Span80 Myverol F1 8.9 92.5 : 7.5 0.5 Q.S F2 8.9 92.5 : 7.5 1 Q.S F3 8.9 92.5 : 7.5 1.5 Q.S F4 8.9 85 : 15 0.5 Q.S F5 8.9 85 : 15 1 Q.S F6 8.9 85 : 15 1.5 Q.S F7 8.9 77.5 : 22.5 0.5 Q.S F8 8.9 77.5 : 22.5 1 Q.S F9 8.9 77.5 : 22.5 1.5 Q.S F10 8.9 92.5 : 7.5 0.5 Q.S F11 8.9 92.5 : 7.5 1 Q.S F12 8.9 92.5 : 7.5 1.5 Q.S F13 8.9 85 : 15 0.5 Q.S F14 8.9 85 : 15 1 Q.S F15 8.9 85 : 15 1.5 Q.S F16 8.9 77.5 : 22.5 0.5 Q.S F17 8.9 77.5 : 22.5 1 Q.S F18 8.9 77.5 : 22.5 1.5 Q.S F19 8.9 85 : 15 0.5 Q.S F20 8.9 85 : 15 1 Q.S F21 8.9 85 : 15 0.5 0.2 Q.S F22 8.9 77.5 : 22.5 0.5 Q.S Iraqi J Pharm Sci, Vol.27(1) 2018 Darifenacin hydrobromide loaded nanostructured lipid carriers 56 Characterization and evaluation of nanostruc- tured lipid carriers ( NLCs ) Particle size and polydispersity index measurement The particle size analysis of formulas was performed using ABT- 9000 Nano Laser Particle Size Analyzer. Before measurements, NLCs dispersion was diluted suitably using de- ionized water. Data was analyzed by software and values of mean particle size, polydispersity index (PDI) and particle size distribution curve were recorded (6). Zeta potential measurement The zeta potential analysis of formulas was performed using Zeta Sizer. Before meas- urements, NLCs dispersion was suitably diluted (7). Entrapment efficiency measurement Entrapment efficiency corresponds to the percentage of drug encapsulated within the lipid matrix. Certain volume of NLCs disper- sion was accurately taken and subjected to cen- trifugation at 25000 rpm for 30 min at 4° C . After centrifugation, 1 ml of supernatant was taken and suitably diluted with methanol and the free drug concentration determined using UV-Vis Spectrophotometer and (%EE) meas- ured using the following equation (8): 𝐸𝐸 ( % ) = 𝑊𝑖𝑛𝑖𝑡𝑖𝑎𝑙 − 𝑊 𝑓𝑟𝑒𝑒 𝑊𝑖𝑛𝑖𝑡𝑖𝑎𝑙 × 100 EE(%) = percentage of entrapment efficiency Winitial = initial drug concentration Wfree = free drug concentration ( unentraped drug ) Differential scanning calorimetry ( DSC ) study The possibility of any interaction be- tween darifenacin hydrobromide and excipients was assessed by carrying out thermal analysis of the formulation using DSC. The analysis was performed on the pure darifenacin hydrobro- mide, GMS and lyophilized darifenacin hydro- bromide NLCs . Each sample was weighed ac- curately and kept in aluminum pans and scanned between 30 ºC – 400 ºC at a heating rate of 10 °C/min and cooling rate of 40 °C/min under nitrogen gas. An empty aluminum pan was used as reference in the study (9). FTIR spectroscopy study FTIR helped to confirm the identity of the drug and to detect the interaction of the drug with carriers.FTIR spectral measurement for pure darifenacin hydrobromide drug, lipid glyc- eryl monostearate, oleic acid, tween80, vitamin E and optimized NLCs formulation were ob- tained on FTIR using KBr disk method. The scanning range was 400- 4000 cm -1 (10). X- Ray diffraction (XRD) study Powder X-ray diffraction (PXRD) was performed to analyze crystalline or amorphous nature of darifenacin hydrobromide loaded NLCs. PXRD studies were performed by pow- der X-ray diffractometer where CUK α radia- tion of 1.5405°A was used as X-ray source. For the measurements , samples were Kept in the glass sample holders followed by scanning from 2° to 60° with scan angular speed (2 θ /min ) of 2°/ min , 40 KV working voltage and 30 mA current. Samples used for study were pure da- rifenacin hydrobromide, glycerl monostearate, and lyophilized dariferacin hydrobromide NLC (11). Atomic Force Microscopy (AFM) study To study morphological changes and also the particle size of NLCs , AFM micro- graphs were imaged by using atomic force mi- croscopy (AFm). The images were obtained by measurement of interaction forces between the tip and sample surface . The experiments were done in air at room temperature (25°C) operat- ing in noncontact mode droplets of dispersion were placed on a small mica disk. The measure- ments were performed in different sample loca- tions. image data were analyzed with software (12). In-vitro drug release and release kinetic stud- ies The in-vitro release of darifenacin hy- drobromide from NLCs was carried out in 500 ml phosphate buffer solution (pH 6.8) by using the dissolution testing apparatus with rotating basket at 100 rpm and temperature 37+̅ 0.5 °C (13). This method involved placing the capsule of the selected formula inside wire basket that is rotated while immersed in the dissolution me- dium. Five milliliters aliquots were withdrawn at 1 , 2 , 4 , 6 , 8 , 10 , and 12hr from dissolution medium and replaced with 5 ml of fresh buffer to maintain sink condition . The aliquots with- drawn were filtered by using 0.45μm filter, suit- ably diluted if necessary, and analyzed by using UV-Vis Spectrophotometer. The cumulative percentage of the released drug was plotted ver- sus time (13). The in-vitro release profile was fitted us- ing several kinetic models such as zero-order (cumulative percentage of drug released versus time), first – order (log cumulative percentage of drug remaining versus time), Higuchi (cumu- lative percentage of drug released versus square root of time), and Korsmeyer–peppas ( log cu- mulative percentage of drug released versus log time ) equations (13). Statistical analysis Statistical analysis of the data was car- ried out using one-way analysis of variance Iraqi J Pharm Sci, Vol.27(1) 2018 Darifenacin hydrobromide loaded nanostructured lipid carriers 57 (ANOVA) test and the level of statistically sig- nificance difference was selected as P < 0.05. Results and Discussions Selection of components A selection of suitable lipids and other excipients was significant to develop NLCs for slightly water-soluble darifenacin hydrobro- mide. To keep the drug in soluble form, it was of prime importance that drug must possess higher solubility in solid lipid and oil. Selection of solid lipid Solid lipid was selected by checking the solubility of the drug in melted solid lipid by means of visible observation with the naked eyes under normal light. Lipids used for this study were stearic acid, palmitic acid and GMS. It was found that GMS showed highest darifen- acin hydrobromide solubilizing capacity. Table (2) shows the comparative solubility of drug in different lipids. Table2. Amount of solid lipid required to sol- ubilize 8.9 mg of darifenacin hydrobromide No . Lipids Amount of lipid 1 Stearic Acid more than 1000 mg 2 Palmitic Acid more than 1000 mg 3 Glyceryl monostearate 400 mg Selection of liquid lipid Liquid lipid was selected based on the maximum solubility of darifenacin hydrobro- mide in different liquid lipids. Lipids used for this study were oleic acid, castor oil, and ethyl oleate. It was found from the result that oleic acid exhibited maxi- mum darifenacin hydrobromide solubilizing ca- pacity than the others as shown an table (3). Therefore, it was selected as liquid lipid to make a matrix with solid lipid GMS for the de- velopment of NLCs. Table3. Solubility of darifenacin Hydrobro- mide in different oils No . Oil Saturation solubility mg / ml 1 Caster oil 11.5 2 Oleic Acid 13.7 3 Ethyl oleate 12.43 Preparation of darifenacin hydrobromide loaded nanostructured lipid carriers Emulsification sonication is a simple and popular method for preparation of NLCs and considered the method of choice for drugs showing high solubility in molten lipids (14). Solid lipid GMS and liquid lipid (oleic Acid) were utilized to provide a core composed of highly lipophilic environment to accommo- date darifenacin hydrobromide, thus becoming a suitable and optimum nanocarrier or reservoir for the drug. The incorporation of solid and liq- uid lipids mixture in the lipid matrix promoted imperfect crystallization, thus lowering the probability of the entrapped drug expulsion dur- ing storage. Also, the presence of liquid lipid in formulations allowed more flexibility for mod- ulation of drug release and better drug-entrap- ment efficiency (15). Characterization and evaluation of nanostruc- tured lipid carriers particle size and polydis- persity index determination Particle size and PDI were important characteristics in the evaluation of stability of darifenacin hydrobromide loaded NLCs (16). Four darifenacin hydrobromide formulas ( F19, F20 , F21 and F22 ) from the prepared formulas were in microsize range , therefore they are not subjected to further characterization . Eighteen darifenacin hydrobromide for- mulas in nano size range, from the prepared formulas, were successfully prepared as shown in table (4). A nanoscale particle exhibited unique physical and biological properties, mak- ing it particularly ideal for drug entrapment, and provided a large surface area for the reaction with its site of action (17). Also, the nanoscale size minimized the probability of drug being phagocytized by macrophage of the mononu- clear phagocytic system, hence decreasing the destruction of darifenacin hydrobromide NLCs in the body (18). Particle size plays a crucial role in the gastrointestinal uptake and their clearance by the reticuloendothelial system. Therefore, the precise determination of the particle size is very important where particle size less than 300 nm is advisable for the intestinal transport(19). Polydispersity index (PDI) is a measure- ment of particle size distribution that varies from 0 to 1. The polydispersity index (PDI) of darifenacin hydrobromide loaded NLCs formu- las was within the acceptable range and it indi- cated that all the prepared NLCs were almost in monodispersity and homogeneous with narrow size distribution as shown in table (4). The closer the value of PDI to zero, the higher the homology between the particles. The PDI of less than 0.5 indicates that there was no aggre- gation of the nanoparticle of darifenacin hydro- Iraqi J Pharm Sci, Vol.27(1) 2018 Darifenacin hydrobromide loaded nanostructured lipid carriers 58 bromide-NLCs. PDI more than 0.5 is an indica- tion of particle aggregation. The aggregates do not interact with the site of action in the way smaller individual particles do. The aggregation or agglomeration impedes the targeting effi- ciency of the nanoscale particle to the target or- gan. Also, the degree of cellular uptake might be decreased due to the presence of unwanted aggregates since , aggregation increases the par- ticle size and lower the surface area (20-22). Effect of concentration of surfactants on particle size It was observed that increasing the concentration of surfactants had statisti- cally significant effect (p>0.05) on parti- cle size.The particle size was found to decrease with an increase in concentration of surfactant tween80 and tween20 for for- mulas (F1-F18) when the ratio of solid lipid GMS to liquid lipid oleic acid con- stant .The higher surfactants (Tween 80 and Tween 20) concentrations reduced the surface tension and facilitated particle partition. The decrease in the particle size is accompanied by a rapid and tremendous increase in the surface area . Thus, an in- crease in the surfactants (Tween 80 and Tween 20) concentration in the primary dispersion resultsed in rapid coverage of the newly formed particle surface (23). Zeta potential determination Zeta potential is essential for evaluating the storage stability of colloidal dispersions(24). The zeta potential of the different formulas of darifenacin hydrobromide NLCs was found within the range of (- 11.78 mv to-32.44 mv) as shown in table (4). Zeta potential referred to the electrostatic charges on the surface of the nano- particle in the dispersion, which was used to predict the long term stability of the nanoparti- cles (24). Since, the zeta potentials above 30 mv or below–30 mv were required for full electro- static stabilization (25). Many experiments demonstrated that it is not only electrostatic re- pulsion had an effect on the stability of any na- noparticles, but also the use of steric stabilizer that favoured the formation of stable nanoparti- cle dispersion (26). The steric hindrance from tween80, that was used in the production of da- rifenacin hydrobromide-NLCs as a stabilizer , had an additional effect in increasing the parti- cle stability (26). Also, surface charge of the NLCs has an effect on tissue permeability and cellular up take where high positive or negative zeta potential lead to superior phagocytosis (27) . Effect of ratio solid lipid to liquid lipid on zeta potential The negative zeta potential value in the darifenacin hydrobromide loaded NLCs formu- las related to deprotonation of carboxyl group of oleic acid. The increase in ratio of liquid lipid to solid lipid had significant effect (p>0.05) on zeta potential. The value of zeta potential in- creased when the ratio of oleic acid to glyceryl monostearte increased (28). Entrapment efficiency determination The entrapment efficiency of the differ- ent formulas of darifenacin hydrobromide loaded NLCs is shown in table (4) . It was con- sistently reported that the increase in entrap- ment efficiency in NLCs related to the presence of solid and liquid lipids that results in great im- perfections in crystal lattice providing higher space for drug accommodation (29 - 31). Also, higher drug solubility in liquid lipid will in- crease the entrapment efficiency (32). Effect of concentration of surfactants on entrapment efficiency It was observed that increasing the concentration of surfactants had statisti- cally significant effect (p>0.05) on the entrapment efficiency of darifenacin hy- drobromide. The entrapment efficiency of darifenacin hydrobromide loaded NLCs was found to decrease with an increase in the concentrations of surfactants (tween 80 and tween 20) for formulas (F1-F18) when the ratio of solid lipid glyceryl monostearate to liquid lipid oleic acid was constant. The high surfactants (tween 80 and tween 20) concentrations reduced the particle size and this decreased the amount of darifenacin hydrobromide en- trapped in the core of darifenacin hydro- bromide NLCs and adsorbed on the sur- face of darifenacin hydrobromide NLCs (33). Iraqi J Pharm Sci, Vol.27(1) 2018 Darifenacin hydrobromide loaded nanostructured lipid carriers 59 Table4. Particle Size, Zeta Potential, PDI and Entrapment Efficiency of Darifenacin Hydrobromide Loaded NLCs. Formula No. Particle size Zeta potential Entrapment effi- ciency PDI F1 98.9nm -14.32 56.27% 0.22 F2 79.1 nm -13.82 56.17% 0.22 F3 78.6 nm -15.09 53.93% 0.37 F4 87.9 nm -16.72 68% 0.15 F5 86.6 nm -14.71 55.11% 0.11 F6 19.7 nm -17.29 53.39% 0.23 F7 989 nm -18.97 83.51% 0.03 F8 436 nm -25.72 58.42% 0.01 F9 99.3 nm -21.58 47.44% 0.53 F10 191 nm -17.9 65.77% 0.1 F11 106 nm -11.78 43.97% 0.22 F12 61.9 nm -13.92 38.46% 0.01 F13 151 nm -19.23 65.78% 0.04 F14 139 nm -17.36 62.79% 0.07 F15 133 nm -17.81 61.06% 0.02 F16 249 nm -32.44 74.44% 0.2 F17 139 nm -18.46 65.38% 0.49 F18 78.7 nm -27.06 23.82% 0.33 Selection of the optimum formula Formula sixteen regarded as the opti- mum formula depending on entrapment effi- ciency measurement which was equal to 74.44% and zeta potential which was equal to– 32.44mv in addition to the particle size which is equal to 249 nm and polydispersity index which was equal to 0.2 . Formula sixteen containing ratio of solid lipid GMS to liquid lipid oleic acid equal to 77.5:22.5 , tween 80 (0.5%), darifen- acin hydrobromide 8.9 mg, and vitamin E that is added as antioxidant. Differential Scanning Calorimetry ( DSC ) study Differential scanning calorimetry was performed to characterize the polymorphism and the degree of crystallinity of darifenacin hy- drobromide loaded NLCs. Figures (1 - 3) and (3) showed the DSC thermograms of darifen- acin hydrobromide, GMS and darifenacin hy- drobromide loaded NLCs respectively. The study showed that the melting point of darifen- acin hydrobromide NLCs (69.76 °C) was lower than that of the bulk material GMS (76.65 °C) also disappearance of melting peak of darifen- acin hydrobromide (235.17 °C) indicated that darifenacin hydrobromide was dissolved in the lipid matrix and encapsulated in the nanostructured lipid carrier. During the prepa- ration , darifenacin hydrobromide was dis- solved in the melted lipid phase. Following the cooling of the dispersion to room temperature, darifenacin hydrobromide melting peak was not detected anymore. The absence of this thermo- dynamic transition could be due to a molecular dispersed state of darifenacin hydrobromide in the mixture (34). The decrease in the melting point of darifenacin hydrobromide NLCs (69.76 °C) which was below that of GMS (76.65 °C) is described as melting point depression (35). The addition of oil (oleic acid) into the matrix pro- voked an additional shift of the melting point to lower temperature. Decrease in melting en- thalpy in darifenacin hydrobromide NLCs as compared to GMS and darifenacin hydrobro- mide was due to less–ordered arrangement of nanoscale particles. Therefore lesser amount of energy was needed to overcome the lattice force in the NLCs than GMC . Also, incorporation of darifenacin hydrobromide inside the lipid ma- trix resulted in a further increase in the number of defects in the lipid crystal lattice (35). Iraqi J Pharm Sci, Vol.27(1) 2018 Darifenacin hydrobromide loaded nanostructured lipid carriers 60 100.00 200.00 Temp [C] -4.00 -2.00 0.00 2.00 mW DSC 235.17x100C Figure 1. DSC thermogram of darifenacin hydrobromide 50.00 100.00 150.00 200.00 250.00 Temp [C] -30.00 -20.00 -10.00 0.00 mW DSC 76.65x100C Figure 2. DSC thermogram of glyceryl monostearate. 100.00 200.00 Temp [C] -5.00 0.00 mW DSC 69.76x100C Figure 3. DSC thermogram of dariferacihy- drobromide loaded NLCs. FTIR Spectroscopy study FTIR spectra of darifenacin hydrobro- mide , GMS , oleic acid, Tween 80 , vitamin E , and darifenacin hydrobromide loaded NLCs ( F16 ) are shown in figures (4 - 9) illustrated that , there was no interaction between drug and excipients since, the characteristic peaks of the drug and the major constituents are still ob- served in IR spectrum of the selected formula . Figure 4 . IR spectrum of darifenacin hydrobromide. Iraqi J Pharm Sci, Vol.27(1) 2018 Darifenacin hydrobromide loaded nanostructured lipid carriers 61 Figure 5. IR spectrum of glyceryl monostearate (GMS). Figure 6 . IR spectrum of oleic acid Iraqi J Pharm Sci, Vol.27(1) 2018 Darifenacin hydrobromide loaded nanostructured lipid carriers 62 Figure 7. IR spectrum of tween80. Figure8 . IR spectrum of Vitamin E. Iraqi J Pharm Sci, Vol.27(1) 2018 Darifenacin hydrobromide loaded nanostructured lipid carriers 63 Figure 9 . IR spectrum of darifenacin hydrobromide loaded NLCs ( F16 ). X–Ray Diffraction study X–ray diffractograms of pure darifen- acin hydrobromide , GMS and freeze dried da- rifenacin hydrobromide NLCs were presented in figures (10-12) . The X – Ray diffractogram of darifenacin hydrobromide exhibited sharp peaks at diffraction angles (2θ) with a typical crystalling patten. All major characteristic crystalline peaks ( 11.47° , 18.20° and 24.55° ) disappeared in the diffractogram of darifenacin hydrobro- mide NLCs. This indicated that darifenacin hy- drobromide converted from crystalline to amorphous form (36). Figure 10. X – Ray diffractograms of darifenacin hydrobromide. Iraqi J Pharm Sci, Vol.27(1) 2018 Darifenacin hydrobromide loaded nanostructured lipid carriers 64 Figure 11. X – Ray diffractograms of glyceryl monostearate (GMS ). Figure 12 . X – Ray diffractograms of darifenacin hydrobromide loaded NLCs Iraqi J Pharm Sci, Vol.27(1) 2018 Darifenacin hydrobromide loaded nanostructured lipid carriers 65 Atomic Force Microscopy ( AFM ) study The morphological analysis performed by AFM showed three-dimensional structure (figure 13) and discrete lipid nanoparticles with no aggregation. The particle size equal to 260nm as shown in the histogram of particle size distri- bution in figure (14) (37). Figure 13. Three –dimensional morphology of darifenacin hydrobromide loaded NLCs ( F16) Figure 14. Granularity cumulation distribu- tion of darifenacin hydrobromide loaded NLCs ( F16) In-vitro drug release and release kinetic stud- ies The in-vitro drug release of darifenacin hydrobromide loaded NLCs showed an interest- ing biphasic release with an initial burst effect followed by controlled and sustained release (38) as shown in figure (15). The initial burst release of darifenacin hydrobromide might be due the presence of unentrapped darifenacin hydrobro- mide in the NLCs (39). Another reason might be due to most of the liquid lipid ( oleic Acid ) be- ing located in the outer shell of NLCs which lead to darifenacin hydrobromide enriched shell that easily released drug by diffusion or matrix erosion (40). The third supportive factor for the burst release that if NLCs prepared with high temperature and optimum concentration of sur- factant, it may produce drug burst release (40 ,41) . At the end of first hour, 30 % of drug is released, after that the drug release follow steady pattern of controlled and sustained re- lease up to 12 hs. The kinetic of release and the mechanism of the release from NLC was eval- uated by fitting the release date into first order , zero order , Higuchi and korsemeyer – peppas as shown in table (5 ). The darifenacin hydro- bromide loaded NLCs was fitted well with Hi- guchi model since R2 value equal to 0.9425 . The n value ( < 0 .5 ) indicated that the release behavior of darifenacin hydrobromide loaded NLCs followed fickian diffusion mechanism (41) . Figure 15. The percentage of drug release from formula sixteen per time at pH 6.8 and 37°C Iraqi J Pharm Sci, Vol.27(1) 2018 Darifenacin hydrobromide loaded nanostructured lipid carriers 66 Table5.The kinetic and the mechanism of the release data of darifenacin hydrobromide from NLC F o r m u la Drug release kinetic Krosmeyer -pepas n value Zero order R2 First order R2 Higuchi R2 16 0.2225 0.9298 0.9425 0.419 Conclusion In this work, darifenacin hydrobromide loaded NLCs with sustained release for about 12 hours with biphasic profile effect was suc- cessfully prepared using solid lipid GMS and liquid lipid oleic acid in a ratio 77.5 : 22.5 in presence of 0.5 % tween80 by using emulsifica- tion sonication method . Future study Stability study for the prepared darifen- acin hydrobromide loaded NLCs capsules , bi- oavailability and clinical study are to be done References 1. Müller RH, Radtke M, Wissing SA. Nanostructured lipid matrices for improved microencapsulation of drugs. 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