Construction of pseudo-ternary phase diagramsHighest solubility of TFM was found in maisine 35-1 (oil), labrasol (surfactant) and transcutol HP (co-surfactant). The maximum ME area was assessed by using Pseudo-ternary phase diagrams, which indicates that highest stability of a formulation. The phase diagram was exactly fixed ratios of S-Cos (SMix). The SMix ratio of surfactant, labrasol and co-surfactant transcutol HP were selected as 3:1, 2:1, 1:1, 1:2 and 1:3. The oil to SMix ratio has changed for every phase diagram and the selected ratios were taken as 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2 and 9:1 (w/w). Accurately weighing the required quantity of ingredients in the glass vials and titrated by using aqueous titration method where the stepwise addition of water in the mixture of oil to SMix. After equilibrium clarity or turbidity were determined by visual observations, while software CHEMIX VERSION 4 were used for the construction of phase diagrams.
1.2. Formulation of TFM loaded ME and MME Aqueous titration method was used for preparation of TFM loaded ME. Pseudo-ternary phase diagram was suggested the appropriate SMix ratio (3:1) for the preparation of TME formulation. The selected quantity of TFM was added into maisine 35-1 (oil) then add it into 3:1, a ratio of Labrasol:Transcutol-HP (SMix) and continuously stir by using magnetic stirrer (Equip-Tronics EQ-771).
The Exact quantity of aqueous phase (distilled water) was poured drop-wise into the following mixture and continuously stirred thereby homogeneous and clear ME has been formed. There was two different formulations of mucoadhesive microemulsions (MME) was prepared by using carbapol 974P (0.4%) and the mixture of mucoadhesive agent including HPMC K4M (0.
3%) with in situ gelling agent poloxamer 407 (17%). The poloxamer 407 are non-ionic and nontoxic in nature (Illum, 2002). TFM mucoadhesive ME by carbapol 974P TMME (C) and TFM mucoadhesive ME by poloxamer 407 and HPMC K4M TMME (P+H) was prepared by using the minimum quantity of water. Each mucoadhesive gelling agent was separately incorporated into water, this mixture was directly add into optimized ME with continuous stirring to the obtained final concentration of MME. 1.3. Experimental designThe optimization of formulation is mandatory nowadays as per the regulatory submission.
A Box–Behnken statistical design was applied to optimized the ME formulations. The statistical data represents an effect of formulation variables such as oil (A), SMix (B) and water (C) on responses. A statistical design was optimized interaction effects as well as the model effects on responses such as globule size (Y1), polydispersity index (PDI) (Y2), % transmittance (% T) (Y3) and % drug content (Y4).
The Box–Behnken experimental design had suggested significant model for the determination of polynomial equation and 3-dimensional plots with Stat-Ease software (Version 11, Design Expert). The Box–Behnken design was most preferred because it provides minimum 3 factors with more than 3 responses compared to the central composite design (CCD) and other designs (Box and Behnken, 1960). The statistical design obtaining of 17 experimental runs and generated by using quadratic model. The software constructed equation of quadratic model is given in equation 1. Y = a0+a1A+a2B+a3C+a12AB+a13AC+a23BC+a11A2+a22B2+a33C2 (1) Where, the measured response associated with each factor level combination is represented by Y; an intercept is b0; b1 to b33 are regression coefficients computed from the observed experimental values of Y; and independent variables are represented with A, B and CThe responses and independent variables selected on the basis of their -1 (low), 0 (medium) and +1 (high) level, those selected from the results of pseudo-ternary phase diagrams.
The different concentrations of A, B and C were used to formulate 17 ME formulations respectively. 1.3.
1. Measurement of globule size and PDI Globule size and PDI measurements shown nanoscale size and uniformly distribution of particles thereby enhance stability as well as drug permeation of the formulation. Before analysis the ME was 10 times with double distilled water and particle size analyzer (Nanophox, Sympatech, Germany) was used for determination of globule size and PDI. The PDI was calculated by using the given formula.
PDI = (X90 – X10) / X50 (2) Where X50 is mean particle size, X90 and X10 are the sizes of nanoparticles below 10% and 90% respectively of the sample population. 1.3.
2. Drug content and entrapment efficiency (% EE) The drug content or percent assay is important parameter of formulation. The percent assay of optimized ME formulation was investigating by 1 ml of ME in 10 mL solvent mixture of methanol: dichoromethane (3:1) and the solutions were analysed for drug content by directly injecting into the HPLC system after appropri-ate dilutions with mobile phase. To determine the % EE, ENLC was centrifuged 25 _C for 45 min at 25,000 rpm. The amount of EFV entrapped in the pellet was determined by dissolving the pellets in methanol:dichoromethane (3:1) mixture and estimated by HPLC method followed by appropriate dilutions with mobile phase.