Iraqi J Pharm Sci, Vol.22(1) 2013 Theophylline pulmonary delivery system 65 Response Surface Methodology for Development and Optimization of Theophylline Pulmonary Delivery System Ali A. Yas *,1 , Yehia I. Khalil * and Mohamed S. Mohamed ** * Department of Pharmaceutics, College of Pharmacy, University of Baghdad, Baghdad, Iraq. ** Department of Pharmaceutics, Fakulti Farmasi, Universiti Teknologi Mara, Selangor, Malaysia. Abstract The aim of the present study was to develop theophylline (TP) inhalable sustained delivery system by preparing solid lipid microparticles using glyceryl behenate (GB) and poloxamer 188 (PX) as a lipid carrier and a surfactant respectively. The method involves loading TP nanoparticles into the lipid using high shear homogenization – ultrasonication technique followed by lyophilization. The compositional variations and interactions were evaluated using response surface methodology, a Box – Behnken design of experiment (DOE). The DOE constructed using TP (X1), GB (X2) and PX (X3) levels as independent factors. Responses measured were the entrapment efficiency (% EE) (Y1), mass median aerodynamic diameter (MMAD, daer) (Y2), zeta potential (ZP, ξ) (Y3), fine particles fraction (% FPF) (Y4) and percentage of dissolution efficiency at 420 minutes (% DE420) (Y5). The optimized formula was characterized by differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and X – ray powder diffraction (XRD) demonstrated that prolonged release physically due to the loaded TP exists mostly in its crystalline state . Analysis of dissolution data of the optimized formula indicated that the best fitting is with Higuchi model, whereas the mechanism of drug release pattern follows anomalous or non – Fickian diffusion. Key words: Glyceryl behenate, solid lipid microparticles, theophylline nanoparticles مىهج الرد السطحً لحطىٌر وظام جحرٌر رئىي للثٍىفلٍٍه علً احمد ٌاس *،1 ، ٌحٍى اسماعٍل خلٍل * و محمد سالمة محمد *** * .فرع انصيذالَيبث ، كهيت انصيذنت ، جبيؼت بغذاد ، بغذاد ، انؼراق ** .فر انصيذالَيبث ، كهيت انصيذنت، انجبيؼت انخكُٕنٕجيت يبرا ، صيالَكٕر، يبنيزيب الخالصة انٓذف يٍ انذراصت ْٕ ححضير صيغت دٔائيت بطيئت انخحرر يٍ انثيٕفيهييٍ بشكم جضيًبث دُْيت يجٓريت يؼذة نالصخُشبق ببصخخذاو طريقت انخحضير حخضًٍ ححًيم انجضيًبث انُبَٕيت نهثيٕفهييٍ في انُبقم . صطحيكًثبج 188غهيضيريم بٓيُيج كُبقم دُْي ٔ انبٕالكضبيير حقييى انخغييراث انخركيبيت ٔ انخفبػهيت . ايٕاج فٕق صٕحيت يهيٓب حجفيف ببنخجًيذ – شذةانذُْي ببصخخذاو حقُيت يؼذنت حشًم حجبَش ػبني ال و انخجريبي حى حشيذِ ببػخًبد ػٕايم يضخقهت ٔ ْي يانخصى. انخجريبيبيُكيٍ –حصًيى بٕكش , ببصخخذاو االصخجببت انًُٓجيت انضطحيت يخٕصظ انقطر , (Y1)ردٔد قيبصيت ٔ ْي كفبءة االَحببس ٔ( X3) 188 انبٕالكضبييرٔ ( X2) غهيضيريم بٓيُيج, (X1)انثيٕفيهييٍ بؼذ انًثبنيت نصيغت لثبج (. Y5)انكفبءة انًئٕيت نالَحالل ٔ َضبت( Y4)َضبت انجضيًبث انذقيقت , (Y3)انزيخب االحخًبنيت , (Y2)االيرٔدايُبييكي االنكخرَٔي ٔ حيٕد االشؼت انضيُيت نهًضحٕق يانًجٓرانًضح , رييت انطيفي نالشؼت ححج انحًراءٔححٕيم ف, حرارياجراء انًضح انخفبضهي ال ٔ نكٍ انيت انخحرر حخبغ اٌ انخحرر انطٕيم االيذ صببّ اٌ يؼظى انثيٕفيهييٍ انًحًم يٕجٕد بصيغت بهٕريت ٔ اٌ انخحرر يخبغ ًَٕرج ْيجٕحشي .غير فيكيبٌ .غلٍسٍرٌل بهٍىٍث ، الدهىن الصلبة المجهرٌة ، جسٍمات الثٍىفلٍٍه الىاوىٌة :الكلمات المفحاحٍة Introduction Pulmonary drug delivery in comparison to other routes of administration is most promising for local respiratory diseases (e.g., asthma, chronic obstructive pulmonary disease, tuberculosis, cystic fibrosis and cancer) as well as systemic diseases (e.g., thrombosis and diabetes) (1) . Drug targeting via inhalation result a rapid drug deposition in a higher concentration at the site of action within lung tissues, thereby reducing the dose required and the side effects (2) . Dry powder inhalers (DPIs) are more preferable for delivering dry particulate drug to the respiratory tract, since they do not contain propellants as in the pressurized metered – dose 1 Corresponding author E-mail: alistein76@yahoo.com Received: 13/10/2012 Accepted: 16/3/2013 Iraqi J Pharm Sci, Vol.22(1) 2013 Theophylline pulmonary delivery system 66 inhalers (pMDIs), ensure drug stability and better patient compliance due to breath – actuated (3) . For an efficient conducting airways deposition, the particle size should be in the range of 2.5 – 6 µm, suitable shape, density and surface chemistry (4, 5) . Sustained delivery inhalation therapy prolongs the duration of action and hence reduces dosing frequency (6) . Although liposomes – based systems are biocompatible and tolerable, they are costly to be prepared and unstable during storage and nebulization (7) . Polymeric nano – and micro – particles incorporated drug are prone to pulmonary toxicity, inefficient biodegradation, polymer accumulation and requirement of organic solvents for their production (8) . Solid lipid microparticles which are solid lipid cores stabilized by surfactants; represent a unique sustained release inhalation therapy, prepared from biocompatible and biodegradable natural lipids with good tolerability and less respiratory toxicity (9) . Nocturnal asthma is when asthma symptoms show exacerbation in the early hours of the morning and is associated with increased morbidity and lowered quality of life (10) . Uniphyl ® 400 – 600 mg extended release tablets of TP, produced using CONTIN ® chronopharmaceutical technology and administered once daily at evening for treating nocturnal asthma (11) . The inter – individual variability of TP absorption especially during night and the ubiquitous of cyclic nucleotide phosphodiesterases (PDEs) enzymes that TP non – selectively inhibit results in a wide systemic side effects, necessitate pulmonary targeting preparation (12, 13) . According to the biopharmaceutics classification system (BCS); TP is a Class I drug and in order to enhance loading and prolong its release from lipid microparticles, TP nanoparticles were prepared (14, 15) . This research focuses on the preparation, optimization and characterization of TP inhalable solid lipid microparticles. Materials and Methods Materials Pure theophylline (TP) and stearic acid (SA) were purchased from (Sigma – Aldrich / USA). Glyceryl behenate (GB) (Compritol ® 888 ATO) was a gift from (Gattefosse´ / France). Poloxamer 188 (PX) (Lutrol ® F 68) was purchased from (BASF – Ludwigshafen / Germany). All other chemicals / solvents used were of analytical grade. Methods Design of experiment Box – Behnken Design is a class of second – order designs based on three – level incomplete factorial designs (16) . A design of three parts, each of two fully – leveled factors and a third factor set at zero level. The dots on the surface of a sphere are lying at on the middle of each edge of multidimentional cube and center point replicate (n = 3) was designed using (Design – Expert ® Software Version 8.0.7.1 / USA). This model is described by the following quadratic equation: Y = T0 + T1X1 + T2X2 + T3X3 + T12X1X2 + T13X1X3 + T23X2X3 + T11X1 2 + T22X2 2 + T33X3 2 (Eq. 1) where Y is the measured response associated with each factor level combinations; T0 is the intercept and T1 to T33 are the regression coefficient computed from the observed value of Y; X1, X2 and X3 are coded level of independent variables. The XiXj (i = 1, 2 or 3 and j = 1, 2 or 3) and Xi 2 (i = 1, 2 or 3) are interaction and quadratic terms, respectively. The independent factors selected were TP (X1), GB (X2) and PX (X3). The dependent responses studied were the % EE (Y1), MMAD, daer (Y2), ZP, ξ (Y3), % FPF (Y4) and % DE420 (Y5). Box – Behnken design exhibit important advantages in comparison with other experimental designs: three factors are needed, and only twelve runs plus three replicates at the center point are required, costing less time and energy, each factor is studied and coded at three basic levels and it does not concern factors at extremely high or extremely low levels to avoid experiments in extreme conditions under which undesirable results might occur (17) . The composition of the Box – Behnken experimental design / factors levels are shown in (table 1). Preparation of theophylline nanoparticles Theophylline 250 mg and stearic acid 75 mg were dissolved in a mixture of dimethylformamide: ethanol, 45: 5 ml and then allowed to mix overnight. The solution was added to a beaker contained distilled water 50 ml via a microsyringe under sonication using a probe sonicator (Vibra – Cell TM / USA) at 35% amplitude – 20 second cycles for 6 minutes. The resulting nanosuspension was stored at – 80 ° C freezer (GFL ® / Germany) and then lyophilized using a freeze dryer (Labconco ® / USA). Directly after nanosuspension preparation, sample of 2 ml was added to the quartz cell of the photon correlation spectroscopy ( Malvern Iraqi J Pharm Sci, Vol.22(1) 2013 Theophylline pulmonary delivery system 67 Zetasizer 1600 / UK) for zeta potential, mean particle size diameter and polydispersity index measurements. After lyophilization, the recovery was calculated using the following equation: Recovery = Wp Wi X 100 (Eq. 2) were Wp – produced solid powder weight after lyophilization, Wi – initial powder weight added of both TP and SA (18) . Preparation of theophylline solid lipid microparticles The process parameters were optimized for the preparation of microparticles. Theophylline nanoparticles (TP – NPs) weight equivalent to TP (0.025, 0.050 or 0.100 gm) was added to a beaker containing molten GB (1.25, 2.50 or 5.00 gm) on a hot plate (Favorit / Malaysia) at 10 ° C above GB melting point. The mixture was then subjected to high shear homogenization using (X 120 CAT / Germany) at 15000 rpm for 3 minutes to allow dissolution / dispersion of TP. The still molten mass was then rapidly cooled at – 20 ° C freezer (Hitachi / Japan) for 2 days. The solidified mass was thoroughly grounded in a mortar and the obtained microparticles were dispersed in a 4 ° C – 25 ml aqueous phase of (1, 2 or 3 % w / v) PX, homogenized at 15000 rpm for another 3 minutes and finally sonicated at 70 % amplitude – 20 second cycles for 12 minutes for cavitation forces to aid in further size reduction. The obtained suspension was stored at – 80 ° C freezer and then lyophilized to obtain water free microparticles (19) . Entrapment / Loading efficiency (% EE – Y1) Solid lipid microparticles weight approximately equivalent to TP 2 mg from each formula was washed on a filter with distilled water to remove the uncoated drug particles. Then the washed microparticles were added to 100 ml of distilled water heated up to 10 º C above excipients melting point on a hotplate magnetic stirrer using (Daihan Hotplate Stirrer / Korea) and then stir at 1500 rpm for 5 minutes to extract TP. After being cooled to room temperature, the extract is filtered through 0.45 µm syringe filter and the content was determined spectrophotometrically at 274 nm against filtered extract from TP free solid lipid microparticles as a blank using spectrophotometer (Hitachi / Japan). The entrapment efficiency (EE) and the loading efficiency (LE) were calculated by the following equations: Table 1: Box–behnken experimental design/factorslevels Formulation TP (gm, X1) GB (gm, X2) PX (% w / v, X3) F1 +1 0 +1 F2 -1 0 +1 F3 +1 0 -1 F4 0 +1 -1 F5 -1 +1 0 F6 +1 +1 0 F7 0 0 0 F8 0 +1 +1 F9 0 -1 -1 F10 +1 -1 0 F11 -1 0 -1 F12 0 0 0 F13 0 0 0 F14 -1 -1 0 F15 0 -1 +1 Factor Level (-) Level (0) Level (+) X1 0.025 0.050 0.100 X2 1.25 2.50 5.00 X3 1 2 3 EE = Wf Wi X 100 (Eq. 3) where Wf – TP weight in the finished microparticles and Wi – TP weight initially added in the formulation. LE = WTP WM X 100 (Eq. 4) where WTP – TP weight in the microparticles and WM – microparticles weight (20) . Mass Median Aerodynamic Diameter (MMAD, daer – Y2) The solid lipid microparticles mass median aerodynamic diameter (MMAD, daer) for Iraqi J Pharm Sci, Vol.22(1) 2013 Theophylline pulmonary delivery system 68 each prepared formula was calculated by the following equation: daer = d X 𝜌 𝜌1 (Eq. 5) where daer – aerodynamic diameter, d – mass mean geometric diameter, ρ – particles density and ρ1 – reference density (1 gm / cm 3 ) (21) . The mean mass geometric diameter (d50) for each formula was measured by diluting a certain amount of solid lipid microparticles with distilled water, sonicated for 1 minute and then added to the sample dispersion unit of the laser diffractometer (Malvern Mastersizer 2000 / UK) (22) . The particles density (ρ) measurement was done by placing a certain amount of microparticles in a graduated cylinder, heated up to 10 ° C above excipients melting point and after being cooled to room temperature the final volume measured, according to the following equation: ρ = m v (Eq. 6) where ρ – density of the solid lipid microparticles, m – weight of the solid lipid microparticles and v – volume of the molten lipid microparticles at room temperature (23) . Zeta potential (ZP, ζ – Y3) After samples being diluted with distilled water and sonicated for 1 minute, the zeta (ξ) – potential is determined automatically by the photon correlation spectroscopy, by placing the solid lipid microparticles suspension in an electric field and measuring their mobility which is then related to the ξ at the interface, using the Smoluchowski equation: ξ = UE η ε (Eq. 7) where ξ – zeta potential (mV), UE – electrophoretic mobility (cm / sec), η – medium viscosity (centipoise) and ε – dielectric constant (24) . Fine particles fraction (% FPF – Y4) In order to evaluate the in vitro aerosolization efficiency of the solid lipid microparticles; Andersen Cascade Impactor (ACI) (Graseby / USA) was used. The cascade impactor consists of a throat, a preseprator and eight stages of impaction plates (0 – 7) with cutoff aerodynamic diameters of 9, 5.8, 4.7, 3.3, 2.1, 1.1, 0.65 and 0.43 µm respectively. The impaction plates were precoated with a 2 % w / v of hydroxypropylmethylcellulose (4000 cps) gel in water to prevent particles bouncy and re – entrainment. A size ―2‖ hard gelatin capsules were filled with 20 mg powder from each prepared sample and aerosolized using Rotahaler ® (Cipla / India). The inhalation test was performed at an inhalation rate of 28.3 L / minute for 10 seconds. The fine particles fraction, which is total percentage deposited at stage 2 – 7 of the cascade impactor was used to evaluate aerosol performance using the following equation: FPF = FPD TD X 100 (Eq. 8) where FPF – fine particles fraction, FPD – fine particles dose (i.e., total weight of the solid lipid microparticles with size ≤ 5 µm) and TD – total dose weight of the solid lipid microparticles delivered from the mouthpiece of the inhaler into the apparatus (25) . Dissolution efficiency (% DE420 – Y5) The in vitro release was performed using Franz diffusion cell system (PermeGear / USA). The receiver compartment was filled with 21 ml phosphate buffer pH 7.4, maintained at 37 ± 0.5 º C and stirred magnetically at 100 rpm. A cellulose acetate membrane (0.45 µm pore size and 2.5 cm 2 surface area) was inserted between the donor and the receptor compartments. A suitable aliquot of the solid lipid microparticles equivalent to TP 0.4 mg from each prepared sample was evenly spread on the cellulose acetate membrane that is pre – moistened with phosphate buffer pH 7.4 containing 0.1 % w / v Tween 80 as a wetting agent and was occluded with a paraffin film. At time intervals of (5, 10, 15, 30, 60, 120, 180, 240, 300, 360 and 420 minutes), 3 ml aliquots of the receptor fluid were withdrawn, replaced by an equal volume of fresh medium and assayed spectrophotometrically against phosphate buffer pH 7.4 as a blank (26) . Relying on correction for sampling, TP cumulated amount released Q was calculated using the following equation: Q = Vs X cn − 1 n n =1 + Vm X Cn (Eq. 9) where Vs – volume of sample withdrawn, Cn – 1 – drug concentration of the sample and Vm – volume of the receptor medium (27) . The magnitude percentage of dissolution efficiency at 420 minutes (% DE420) for each prepared sample was calculated from the area under the curve at time t and measured using the trapezoidal rule and expressed as a percentage of the area of the rectangle described by 100 % dissolution in the same time using the following equation (28) : % DE420 = ( y X dt t 0 y 100 X t ) X 100 (Eq. 10) The release data of TP formulas was fitted into various release kinetic models (29) (Table 2). Iraqi J Pharm Sci, Vol.22(1) 2013 Theophylline pulmonary delivery system 69 Table 2: Mathematical models and dissolution mechanisms describing release kinetics. Where % r – cumulative % TP released at time t, K – rate constant of the model and n – release exponent Checkpoint analysis The checkpoint analysis was performed to confirm the reliability of the equations and plots in responses prediction. Independent variables values were taken at three points / levels (0, - 0.5, 0.5), (0.5, 0, - 0.5) and (- 0.5, 0.5, 0). Solid lipid microparticles formulations at these three checkpoints were prepared experimentally as shown in (Table 3) and evaluated for the responses by analyses in triplicate to ensure reproducibility (30) . Table 3: Checkpoint formulas Formulas X1 X2 X3 F16 0 -0.5 0.5 F17 0.5 0 -0.5 F18 -0.5 0.5 0 Optimization of the Solid Lipid Microparticles Preparations In order to get the optimized formula (FO), the desirability function was run using Design – Expert ® Version 8.0.7.1 Software. The optimum formula was based on list criteria of maximum entrapment / loading efficiency, mass median aerodynamic diameter (MMAD ≤ 5 µm), zeta potential (≥ ± 30 mV), maximum fine particles fraction and maximum cumulative percentage release in 420 minutes. Differential scanning calorimetry Thermal analyses were performed using differential scanning calorimeter (DSC – Perkin Elmer / USA). Under nitrogen flow of 20 ml / minute, approximately 2 mg of pure TP, GB, SA, TP – NPs, TP – GB physical mixture (1: 3) and FO was placed in a crimp – sealed aluminum pan and heated from 10 – 300 º C at a scanning rate of 10 º C / minute. An empty aluminum pan was used as a reference. Fourier transform infrared spectroscopy Fourier transform infrared spectroscopy was performed using (FTIR – Perkin Elmer / USA) in order to characterize the possible interactions between the drug and the carrier in the solid state. Samples of pure TP, GB, SA, TP – NPs, TP – GB physical mixture and FO were prepared according to the KBr disc method. The scanning range was 4000 – 450 cm – 1 and the resolution was 4 cm – 1 . Scanning electron microscopy The scanning electron microscopy analyses were performed using (FESEM – FEI / Netherland). Samples of pure TP, TP – NPs and FO were mounted onto aluminum stubs and coated with a thin platinum layer. The scanning electron microscope was operated at an acceleration voltage of 5 KV and working distance of 7 – 10 mm. X – ray powder diffraction The powder X – ray diffraction patterns were recorded using (Rigaku XRD / Japan), under the following conditions: target CuKα monochromatized radiation, voltage 40 KV and current of 20 mA at ambient temperature. The data of pure TP, GB, SA, TP – NPs, TP – GB physical mixture and FO were collected in the continuous scan mode from 2 º – 40 º (2θ) at an angular increment of 0.02 º / second and count time of 1second / step. Results and Discussions Theophylline nanoparticles characterization Stearic acid was chosen as a stabilizer because it is naturally found in a small amount in the surfactant layer lying the lung epithelium, solid at room temperature and an amphiphilic surfactant that will act as an interface between TP – NPs and water phase (31) . The TP – NPs characterizations are shown in (Table 4) and (Figure 1). Table 4: Theophylline nanoparticles racterizations Model Mechanism Equation Zero – Order Concentration Independent % r = K0 t (Eq. 11) First – Order Concentration Dependent % r = 100 (1 – e – K 1t) (Eq. 12) Higuchi Diffusion % r = KH t 0.5 (Eq. 13) Hixson – Crowell Erosion % r = 100 (1 – (1 – KHC t 4.6416 )) (Eq. 14) Korsmeyer – Peppas Diffusion % r = KKP t n (Eq. 15) ζ (mV) d50 (nm) PI %Recovery - 41.205 ±1.453 722.040 ±6.677 0.558 ±0.031 75.361 ±16 Iraqi J Pharm Sci, Vol.22(1) 2013 Theophylline pulmonary delivery system 70 Figure 1: TP – NPs Size distribution – size statistics report by intensity Theophylline solid lipid microparticles preparation The rapid cooling leads to formation of solid solution (homogenous distribution) of drug in lipid matrix. Drug release takes place by diffusion from the matrix and upon lipid degradation in vivo (Figure 2) (32) . Figure 2: Three incorporation models of drugs in the solid lipid matrix Table 5: Observed responses Entrapment / Loading efficiency (% EE – Y1) The entrapment efficiency (EE) varies from 34.762 % (F14) to 95.989 % (F3) for various factors level combinations (Table 5). The independent factors affecting the EE were TP (X1) and PX (X3) levels (P < 0.05, Tables 6 – 7) and (Figure 3). TP has a significant – positive effect on the EE. Although TP hydrophilicity favors the external aqueous phase, its EEs in some formulations were high, because TP localized at the interface (i.e., solid lipid microparticles surfaces) will induce outer surface saturation at higher concentrations and besides the internal phase viscosity (i.e., GB solidity due to its high melting point) will reduce TP leakage by reducing the external aqueous phase entrance into the lipid matrix (33, 34) . Figure 3: Response surface contour plot (Up) and 3D plot (down) for TP and PX effect on EE. Formulation % EE (Y1) MMAD daer µm (Y2) ZP ξ mV (Y3) % FPF ≤ 5 µm (Y4) % DE420 (Y5) F1 75.964±5.463 4.849±0.456 -34.366±1.187 36.792±1.055 73.782±5.533 F2 47.596±6.458 4.902±0.227 -26.850±1.282 35.816±1.773 44.156±3.109 F3 95.989±4.146 6.168±0.083 -32.066±0.375 24.725±0.800 75.189±6.496 F4 67.017±7.147 6.514±0.149 -23.566±0.301 23.742±1.606 50.547±4.601 F5 51.578±4.542 6.104±0.229 -22.450±1.837 34.598±0.888 47.786±2.512 F6 85.263±3.917 6.225±0.196 -31.316±0.957 33.808±0.862 76.210±4.414 F7 56.428±5.931 6.232±0.209 -27.300±0.312 32.466±0.781 52.135±3.791 F8 41.578±3.605 5.278±0.478 -25.433±0.301 38.736±0.885 38.629±1.254 F9 78.247±5.174 6.456±0.106 -28.216±0.028 24.674±0.596 70.089±4.460 F10 83.789±6.955 6.099±0.064 -30.683±0.161 35.878±0.962 77.708±6.095 F11 58.754±5.777 6.354±0.205 -27.733±0.189 22.677±0.808 46.812±3.213 F12 55.357±5.896 6.181±0.208 -28.166±0.602 32.343±0.503 51.971±4.218 F13 55.714±5.786 6.283±0.211 -27.983±0.425 32.905±0.639 51.553±3.989 F14 34.762±4.301 6.256±0.213 -20.200±0.100 34.615±1.356 42.878±2.214 F15 39.368±3.568 5.115±0.224 -25.916±1.537 37.703±1.114 47.799±2.121 Iraqi J Pharm Sci, Vol.22(1) 2013 Theophylline pulmonary delivery system 71 Table 6: Statistical regression analysis Table 7: Analysis of variance (ANOVA) *Probability > F is the significance level and a value < 0.05 considered significant PX has a significant – negative effect on the EE. TP becomes partly negatively charged at pH < 9 (i.e., neutral pH) and this result in the absence of attractive electrostatic interactions with PX polyethylene oxide chains. In addition TP characteristic of pH independent solubility will precludes its hydrophobic interactions with the PX hydrophobic cores and the PX swelling upon hydration, all result in TP within the PX layer release increment (35) . The effects on Y1 can be explained by the following quadratic equation: Y1 = 62.22 + 18.24X1 + 0.86X2 – 11.75X3 – 1.79X1X2 – 0.7X1X3 + 1.84X2X3 + 4.88X1 2 – 3.25X2 2 + 3.12X3 2 (Eq. 16) There is an inverse relationship between the aqueous surfactant concentration and the sonication time but to a certain limit, because less energy will be required to achieve maximum dispersion of the produced solid lipid microparticles. Any increase in PX concentration will not aid in coverage, in contrary will lead to an increment in the micelles formation which has more negative effect on TP – solid lipid microparticles EE (36) . Mass median aerodynamic diameter (MMAD, daer – Y2) The aerodynamic diameter is the diameter of a sphere of unit density, which reaches the same velocity in the air stream as a non – spherical particle of arbitrary density and expresses the mechanism of particle deposition in the Iraqi J Pharm Sci, Vol.22(1) 2013 Theophylline pulmonary delivery system 72 respiratory system (37) . The mass median aerodynamic diameter [MMAD, daer] ranged from 4.849 µm (F1) to 6.514 µm (F4) (Table 5) with the selected levels of variables. The most factors affecting the MMAD are PX (X3 and X3 2 ) and TP (X1 2 ) (P < 0.05, Tables 6 – 7) and (Figures 4 - 5). PX has a significant – negative effect on the MMAD. Figure 4: Response surface contour plot (Up) and 3D plot (down) for TP and PX effect on MMAD Generally, independent on the PX molecular weight, the preserving of the MMAD values is due to the PX efficient coverage of the surfaces and sterically stabilizing microparticles by its long hydrophilic polyethylene oxide chains (poloxamer 188 average number of ethylene oxide ≈ 153 units) extend into solution and shield the particles surfaces preventing their agglomeration during the homogenization and sonication processes. Also, these hydrophilic arms will potentially influence the shape of the resulting particles (38) . TP has a significant – negative effect on the MMAD. This is attributed to the concentration effect of the non – incorporated TP molecules during lyophilization results in the formation of highly concentrated solutions of TP that its ionization will affect the ZP and particles agglomeration. (39) . Y2 can be described by the following quadratic equation: Y2 = 6.24 – 0.024X1 + 0.035X2 – 0.66X3 + 0.061X1X2 + 0.026X1X3 + 0.034X2X3 – 0.17X1 2 + 0.11X2 2 – 0.49X3 2 (Eq. 17) A part of the PX covering layer of the solid lipid microparticles surfaces removed, a reduction in the repel force impart by PX steric hindrance, enhance particles agglomeration through Van der Waals forces of attraction between particles and hence there will be an increase in the mean solid lipid microparticles size after freeze drying process (40) . Figure 5: Particle size distribution of the FO Zeta potential (ZP, ζ – Y3) The zeta (ξ) – potential (ZP) is the electrostatic potential at the boundary dividing the compact layer (charged solid surface – immobile conuterions) and the diffuse layer (liquid counterions – mobile counterions) (41) . The ZP (> 30 or < - 30 mV), indicates electrostatic repulsion among particles and good stability (42) . The ZP values varied from – 34.366 mV (F1) to – 20.200 mV (F14) (Table 5). TP (X1) and GB (X2 2 ) have statistical influential effects on the ZP (P < 0.05, Tables 6 – 7) and (Figures 6 – 7). TP has a significant – negative effect on the ZP. This is attributed to the free not incorporated TP that will be partly negatively charged at neutral pH and the increase in the ZP will be in the negative direction (43) . Figure 6: Zeta potential distribution of the FO Iraqi J Pharm Sci, Vol.22(1) 2013 Theophylline pulmonary delivery system 73 Figure 7: Response surface contour plot (Up) and 3D plot (down) for TP and GB effect on ZP GB has a quadratic significant – positive effect on the ZP. As GB content increased, there will be a reduction in the solid lipid microparticles pH together with ZP increment in the positive direction due to the solid lipid microparticles surfaces TP protonation at low pH (pH < 4) (44) . Both factors effects on Y3 are shown in the quadratic equation below: Y3 = - 29.38 – 3.94X1 + 0.24X2 – 0.43X3 – 0.22X1X2 – 0.88X1X3 – 0.96X2X3 – 0.12X1 2 + 3.34X2 2 – 1.04X3 2 (Eq. 18) The negative charge of the solid lipid microparticles core matrixes are involved in electrostatic interaction with the weakly basic TP molecules. Thus, the solid lipid microparticles surfaces negative charge is the main contributor to the negative ZP of the solid lipid microparticles (45) . The changes in the interfacial properties will have an influential effect on the ZP and hence solid lipid microparticles size. This is due to the PX layer covering the solid lipid microparticles surfaces will reduce the ZP but will provide steric stability instead if solid lipid microparticles being efficiently covered with PX (46) . Fine particles fraction (% FPF – Y4) Andersen Cascade Impactor (ACI) is a primary technique used for both development and testing of the inhaler products. Size determination is based on the inertial impaction of aerosolized particles passing through decreasing nozzle apertures onto subsequent deposition stages each with a defined aerodynamic cut – off diameter (47) . The fine particles fraction (FPF) is lowest at 22.677 % (F11) and highest at 38.736 % (F8) (Table 5). PX (X3 and X3 2 ) and GB (X2 2 ) are the most factors affecting FPF (P < 0.05, Tables 6 – 7) and (Figure 8). Simply by decreasing the cohesive forces between particles a lower aggregation tendency might gain and consequently lower surface energy was necessary to increase the flowability and finally the powder FPF. Also; by reducing the particle density and tolerating an increase of the average particle size will enhance the aerosol efficiency. PX has a significant – positive effect on the FPF. PX percentage used has a special role concerning particles morphology, dispersibility and flowability. As PX concentration increased, the FPF increased up to a certain limit that beyond highly charged particles produced (48) . Figure 8: Response surface contour plot (Up) and 3D plot (down) for GB and PX effect on FPF Although GB has a quadratic significant – positive effect on the FPF, but it is a slight increment and only at higher GB concentration and that is shown from the surface truncated shape of the 3D plot for the response Y4 range. Effects of factors on response Y4 explained by the following quadratic equation: Iraqi J Pharm Sci, Vol.22(1) 2013 Theophylline pulmonary delivery system 74 Y4 = 32.37 + 0.33X1 – 0.35X2 + 6.69X3 – 0.64X1X2 – 0.34X1X3 + 0.56X2X3 + 0.37X1 2 + 1.99X2 2 – 3.08X3 2 (Eq. 19) GB enables the production of low density aerodynamically inhalable particles and thus improving their respirable fraction by avoiding the natural clearance mechanism of the lungs (e.g., alveolar macrophage upake) due to the higher geometric diameter of the particles (49) . Dissolution efficiency (%DE420 – Y5) The cumulative percentage of TP release at 420 minutes (% DE420) varied from 38.629 % (F8) to 77.708 % (F10) (Table 5).TP (X1) is the only factor affecting the % DE420. TP has a significant – positive effect on the % DE420 (P < 0.05, Tables 6 – 7) and (Figure 9). Concerning TP effect only; TP release is biphasic, the first phase was a slight burst release due to the free non – incorporated TP on the solid lipid microparticles surfaces followed by prolong release due to the incorporated stable crystalline anhydrous TP (50) . Figure 9: Response surface contour plot (Up) and 3D plot (down) for TP and GB effect on %DE420 Concerning GB non – significant – negative effect on the % DE420 is due to the cold homogenization technique employed in the solid lipid microparticles production which results in a solid solution drug incorporation model and GB low crystallization degree. Therefore, TP release rate will be prolonged for some formulations because the drug which is molecularly dispersed in the colloidal particles has a limited motion (51) . Percentage TP released in 420 minutes can be described by the following quadratic equation: Y5 = 55.23 + 15.15X1 – 3.17X2 – 4.3X3 – 0.04X1X2 + 1.56X1X3 + 1.35X2X3 + 4.15X1 2 + 1.77X2 2 – 0.64X3 2 (Eq. 20) The reasonable explanation for the prolonged release is the three steps govern TP release from solid lipid microparticles; entrance of the dissolution medium into the solid lipid microparticles matrixes, dissolution of the dispersed TP particles / crystals and diffusion of the dissolved TP through the inert solid lipid microparticles matrixes (52) . Checkpoint analysis Three checkpoint formulations were prepared and evaluated for dependent responses (Table 8). Comparing the predicted and experimental values using Student t – test, the differences were found to be insignificant (p > 0.05) indicate that the obtained mathematical equation is valid for predicting the dependent responses values. (Eq. 21) was used in the percentage relative error calculation between the experimental and predicted values of each response: % Relative Error = 𝑷𝒓𝒆𝒅𝒊𝒄𝒕𝒆𝒅 𝑽𝒂𝒍𝒖𝒆−𝑬𝒙𝒑𝒆𝒓𝒊𝒎𝒆𝒏𝒕𝒂𝒍 𝑽𝒂𝒍𝒖𝒆 𝑷𝒓𝒆𝒅𝒊𝒄𝒕𝒆𝒅 𝑽𝒂𝒍𝒖𝒆 X 100 (Eq. 21) Optimization of the solid lpid microparticles preparations Comprehensive search through desirability function revealed that the FO with 0.889 desirability has the composition of 0.1 gm TP, 1.27 gm GB and 3 % w / v PX. By preparing the FO, the experimental responses are in good agreement with the predicted values (Table 9). Iraqi J Pharm Sci, Vol.22(1) 2013 Theophylline pulmonary delivery system 75 Table 8: Checkpoint formulations comparing experiemntal / predicted Values (n = 3) No. Ex. Pr. % RE F16 % EE Y1 60.187±3.074 56.740 0.060 MMAD daer µm Y2 6.025±0.435 6.634 0.091 ZP ζ mV Y3 -25.480±0.314 -23.399 0.088 % FPF Y4 28.356±0.367 32.225 0.120 %DE420 Y5 67.344±3.773 61.142 0.101 F17 Ex. Pr. % RE % EE Y1 87.434±4.933 81.055 0.078 MMAD daer µm Y2 7.234±0.763 6.139 0.178 ZP ζ mV Y3 -35.132±1.145 -30.434 0.154 % FPF Y4 17.315±0.310 15.727 0.100 %DE420 Y5 53.640±2.206 58.342 0.080 F18 Ex. Pr. % RE % EE Y1 54.345±2.629 45.132 0.204 MMAD daer µm Y2 6.431±0.173 5.985 0.074 ZP ζ mV Y3 -27.340±1.415 -24.156 0.131 % FPF Y4 29.346±0.381 32.637 0.100 %DE420 Y5 38.343±1.146 46.635 0.177 Table 9: Optimized formula experiemntal / predicted values (n = 3) F O Ex. Pr. % RE % EE Y1 76.246±3.582 71.923 0.060 MMAD daer µm Y2 6.035±0.463 4.890 0.234 ZP ζ mV Y3 -29.734±2.509 -31.580 0.058 % FPF Y4 35.819±1.343 38.731 0.075 %DE420 Y5 71.069±2.136 74.723 0.048 Mechanism of dissolution The regression parameters obtained after fitting various release kinetic models to the in vitro dissolution data are listed in (Table 10). The fit for various models investigated for drug release of the FO can be arranged in the following descending order: Higuchi > Korsemeyer – Peppas > Zero – Order > Hixson – Crowell > First – Order. The exact mechanism of release is non Fickian or anomalous from the slope value of Korsemeyer – Peppas which is lie in the range of (0.450 < n < 0.890). The in vitro release profiles of TP, TP - NPs and FO are shown in (Figure 10). The low release percentage of crude TP powder is due to the larger particle size in comparison with the TP – NPs and FO. Table 10: Release kinetic parameters of the FO Figure 10: in vitro release profiles of TP, TP – NPs and FO Differential scanning calorimetry (Figure 11) shows the DSC of pure TP, GB, SA, TP – NPs, TP – GB physical mixture and FO endothermic peak at 274 ° C, 77.41 ° C, 69.30 ° C, 64.83 – 272.20 ° C, 75.11 – 270.38 ° C and 76.18 ° C respectively. The endothermic peak corresponding to the melting point of TP is reduced to 272.20 ° C in the DSC thermogram of TP – NPs and 270.38 ° C in the DSC thermogram of TP – GB physical mixture respectively, and this indicated that in TP – NPs there is a lack of significant changes in TP crystalline state, whereas in the TP – GB physical mixture indicates TP saturation in the carrier. The disappearance of TP melting peak in the FO was noticed due to its percentage of (≈ 5 % w / w) so it is lowered, broadened and becomes DSC undetectable (53) . Model Slope Intercept R 2 Zero – Order 0.179 12.510 0.916 First – Order 0.003 0.933 0.578 Higuchi 3.982 -0.952 0.990 Hixson – Crowell 0.006 2.013 0.633 Korsmeyer – Peppas 0.687 0.187 0.966 Iraqi J Pharm Sci, Vol.22(1) 2013 Theophylline pulmonary delivery system 76 Figure 11: Differential Scanning Calorimetry Thermograms of TP (a), GB (b), SA (c), TP – NPs (d), TP – GB Physical Mixture (e) and FO (f) Fourier Transform Infrared Spectroscopy As shown in (Figure 12), the FTIR spectrum of pure TP, GB, SA, TP – NPs, TP – GB physical mixture and FO. The spectrum of TP shows characteristic peaks at 1710 cm -1 and 1665 cm -1 attributable to the (C = O stretching vibrations). The band at 1563 cm -1 is attributable to the (pyrimidine ring stretching vibration) (54) . The GB shows distinctive bands at 2916 cm -1 and 2849 cm -1 attributable to asymmetric and symmetric (aliphatic CH2 stretching vibrations). The band at 1737 cm -1 is attributable to the (ester C = O stretching vibration) (55) . The spectrum of SA resembles that of GB with little shift, i.e. the asymmetric and symmetric (aliphatic CH2 stretching vibrations) will be at 2920 cm -1 and 2851 cm -1 respectively, whereas the (ester C = O stretching vibration) will be at 1703 cm -1 . The spectrum of TP – NPs still showed the SA 2920 cm -1 and 2851 cm -1 bands, whereas 1703 cm -1 disappear due to the TP 1710 cm -1 and 1665 cm -1 bands in the same region. The spectrum of TP – GB physical mixture still showed the GB bands at 2916 cm -1 , 2849 cm -1 and 1737 cm -1 , whereas TP 1710 cm -1 , 1665 cm -1 and 1563 cm -1 still shown with different intensity as a consequence of TP existence on the microparticles surfaces. The spectrum of FO shows GB bands at 2916 cm -1 , 2849 cm -1 and 1737 cm -1 , whereas TP band at 1563 cm -1 is the only shown due to the TP bands at 1710 cm -1 and 1665 cm -1 close proximity with respect to the GB 1737 cm -1 band. Scanning electron microscopy The scanning electron microscope images for pure TP, TP – NPs and FO are shown in (Figure 13). Both pure TP and TP – NPs show a prismatic TP crystal habit, whereas the FO image shows irregular shape particles (56) . This irregularity is due to sonication done at 4 ° C by using an ice bath to prevent lipid melting by heat generated and so drug loading and homogeneity maintained. X – ray powder diffraction The XRD patterns of pure TP, GB, SA, TP – NPs, TP – GB physical mixture and FO are shown in (Figures 14). Pure TP diffractogram has an important distinctive peak of high intensity at 12.63 ° (2θ), another two small and isolated peaks are present at 7.11 and 14.33 ° (2θ), whereas a group of small peaks covers the range 20 – 30 ° (2θ). GB has a high intensity peak at 21.22 ° (2θ) and a smaller one at 23.43 ° (2θ). SA has a high intensity peak at 2.22 ° (2θ) and two successive peaks at 21.5 ° (2θ) and 23.8 ° (2θ). TP – NPs, TP – GB physical mixture and FO diffractograms still showed TP, SA and GB peaks but at reduced intensity. Although TP melting peak disappearance in the FO corresponding DSC curve, TP isolated peaks in the above diffractograms confirms its crystalline state. Iraqi J Pharm Sci, Vol.22(1) 2013 Theophylline pulmonary delivery system 77 Figure 12: Fourier transform infrared images of TP (a), GB (b), SA (c), TP – NPs (d), TP – GB physical mixture (e) and FO (f) Figure 13: Scanning electron microscopy images of TP ( a), TP – NPs (b) and FO (c) Iraqi J Pharm Sci, Vol.22(1) 2013 Theophylline pulmonary delivery system 78 Figure 14: Powder X – ray diffraction Spectra of TP (a), GB (b), SA (c), TP – NPs (d), TP – GB physical mixture (e) and FO (f) Conclusions The selected variables main and interaction effects on the critical quality attributes of the inhalable solid lipid microparticles were determined by a Box – Behnken design of experiment. 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