FORMULATION,CHARACTERIZATION AND IN-VITRO RELEASE OF ORAL FELODIPINE SELF-NANOEMULSIFYING DRUG DELIVERY SYSTEMS BY

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Introduction
Self-nanoemulsifying drug delivery system (SNEDDS) is isotropic mixtures of oil, surfactant, co-surfactant and drug that form fine oil in water nanoemulsion when introduced into aqueous phase under gentle agitation. SNEDDSs spread readily in the GIT and the digestive motility of the stomach and the intestine provide the agitation necessary for self-emulsification   (1) .
A self-emulsifying / microemulsifying and nanoemulsifying drug delivery system (SEDDS/SMEDDS/SNEDDS), respectively, is a fairly similar liquid lipid dosage form designed for oral delivery which composed of oils, surfactants and possibly co-solvents. These systems have the ability to form fine oil in water (o/w) emulsions, microemulsions or nanoemulsion upon mild agitation following dilution with an aqueous media. This property makes them good candidates for oral delivery of poorlywater soluble drugs (Pouton, 1997; (2,3) .

Solid lipid-based formulations
Solid SEDDS can be used for several dosage forms (dry emulsions, self emulsifying capsules, implants, sustained/controlled release tablets or pellets, beads, microspheres, nanoparticles, suppositories) and can present flexible solution for oral and parenteral administration (Kumar et al., 2010) (4) .

Hypertension
Hypertension known as high blood pressure (HBP), is a long-term medical condition in which the blood pressure in the arteries is persistently elevated (Naish, 2014) (5) . High blood pressure usually does not cause symptoms (High Blood Pressure Fact Sheet, 2016) (6) . Long-term high blood pressure, however, is a major risk factor for coronary artery disease, stroke, heart failure, peripheral vascular disease, vision loss, and chronic kidney disease (Lackland and Weber, 2015;Mendis et al., 2011) (7,8) .

Calcium channel blockers
Calcium channel blockers, or calcium antagonists, treat a variety of conditions, such as high blood pressure, chest pain and Raynaud's disease.Calcium channel blockers prevent calcium from entering cells of the heart and blood vessel walls, resulting in lower blood pressure. Calcium channel blockers, also called calcium antagonists, relax and widen blood vessels by affecting the muscle cells in the arterial walls.Some calcium channel blockers have the added benefit of slowing heart rate, which can further reduce blood pressure, relieve chest pain (angina) and control an irregular heartbeat.

Examples of calcium channel blockers Felodipine
Felodipine is a long-acting 1,4-Dihydropyridine calcium channel blocker (CCB) b. It acts primarily on vascular smooth muscle cells by stabilizing voltage-gated L-type calcium channels in their inactive conformation. Felodipine is used to treat mild to moderate essential hypertension.

Pharmacology
Indication: For the treatment of mild to moderate essential hypertension. Associated Conditions: High Blood Pressure (Hypertension)

Metabolism and Interaction:
Felodipine is metabolized by cytochrome P450 3A4, so substances that inhibit or activate CYP3A4 can strongly affects how much felodipine is present.

Half life:
The Half life of Felodipine in hypertensive patients was 17.5 -31.5 hours; 19.1-35.9 hours in elderly hypertensive patients; 8.5-19.7 in healthy volunteers.Clearance: 0.8 L/min [Young healthy subjects]

Toxicity:
Symptoms of overdose include excessive peripheral vasodilatation with marked hypotension and possibly bradycardia. Oral rat LD 50 is 1050 mg/kg.

Solubility:
Felodipine is class II drug, i.e., low solubility and high permeability. Felodipine has poor water solubility and hence poor dissolution and bioavailability after oral administration. Felodipine undergoes extensive first-pass metabolism with a bioavailability of about 15% (Blychert et al., 1997) (9) .
The major drawback in the therapeutic application and efficacy of Felodipine as oral dosage form is its low aqueous solubility, which is expressed to be approximately 19.17 mg/L at 25°C. Hence, improvement of its water solubility and dissolution is of therapeutic importance (Budavari S. et al., 1996;Moffat et al., 2002) (10,11) .

Determination of Felodipine peak and retention time
Felodipine solution of concentration 1 mg/ml in methanol was prepared and injected in the HPLC (Agilent HPLC 1100 PDA, USA) using the following chromatographic conditions: Hypersil ODS 150 mm x 4.6 mm, 5 m particle size. The mobile phase consisted of 30:70 (v/v) mixture of Sodium acetate: acetonitrile. The column was equilibrated for 30 minutes with the analytical mobile phase before injection; 10 l of the drug solution was injected after filtration with a 0.2 m filter syringe.The mobile phase was pumped isocratically at a flow rate of 2.3 ml/min. The effluent was monitored at 237 nm. Retention time was recorded and sample was analyzed in triplicate.

Solubility study
The maximum solubility of Felodipine was determined in different oils, surfactants and co-surfactants by adding an excess amount of the drug (Felodipine) in 2 ml of each vehicle in 10 ml screw-capped glass vials, and the mixture was vortexed to facilitate solubilization using a vortex mixer (Vortex mixer, Stuart, UK). Mixtures were equilibrated at 25 2˚C for 3 days in an isothermal shaker (BS-11, shaking water bath) and then centrifuged at 7000 rpm for 30 minutes (Beckman model TJ-6 Centrifuge, Korea) to separate the undissolved drug (Nielsen et al., 2007) (12) .
The supernatant was diluted with methanol to make suitable dilution and analyzed for Felodipine content by the previously mentioned HPLC method. The solubility of Felodipine was determined in all of these: Triacetin, Cremophor EL, Span 80, Transcutol HP and ethanol.

Construction of ternary phase diagrams
Series of mixtures were prepared with varying ratios of lipids, surfactants and co-surfactants. The components were weighed into 10 mL glass vials and mixed at 50˚C with a stirring at 300 rpm using isothermal shaker, until the components were completely dissolved. The mixtures were cooled to 37 ˚C and 1 g was transferred to a beaker where 250 mL of distilled water at 37 ˚C was added under gentle stirring of 25 rpm.
The dispersions were visually inspected. A mixture was defined to be a suitable SNEDDS and judged good if spontaneous emulsification was obtained after dispersion in 37 ˚C, followed by the formation of a clear transparent nanoemulsion (El laithy, 2008; Craig et al., 1995; (13)(14)(15) . Ternary phase diagram of surfactant, co-surfactant and oil was plotted; each of them, representing an apex of the triangle (Sunheer, 2012) (16) using sigma plot software.

Determination of drug loading capacity of the formulated SNEDDS
An excess of Felodipine powder was placed in 5 ml of the selected SNEDDSs in 10 ml screw-capped glass vials, and the mixtures were vortexed to facilitate solubilization using a vortex mixer, then sonicated for 10 minutes using bath sonicator (Crest ultrasonic Corp., New York, USA). Mixtures were equilibrated at 25 ˚C for 3 days in an isothermal shaker and then centrifuged at 7000 rpm for 30 minutes to separate the undissolved drug. The supernatant was diluted with methanol for the quantification of Felodipine and analyzed by HPLC.

Preparation of Felodipine SNEDDS
Formulae that could solubilize the highest amounts of Felodipine were chosen to be loaded with the drug (in a concentration below its saturation solubility). In 10 ml screw-capped glass vials, weighed amounts of Felodipine were-added to each system (composed of oil, surfactant and co-surfactant)

Visual inspection
The prepared Felodipine SNEDDSs were inspected for optical transparency and homogeneity by visual observation against strong light. The formulations were also checked for the presence of undissolved drug particles.

Assessment of thermodynamic stability
Felodipine SNEDDSs were subjected to centrifugation for 30 minutes at 3500 rpm. The stable formulations that did not show precipitation of drug were subjected to heating cooling cycles which include six cycles between 45 ˚C and refrigerator temperature 4˚C with storage at each temperature of not less than 48 hours were studied. Furthermore the passed formulae were subjected to freeze thaw cycles (-21 ˚C and +25 ˚C) with storage at each temperature of not less than 48 hours (Shafiq et al., 2007) (17) .

Conductivity measurements
Conductivity measurements were carried out using digital conductometer (Digital Conductometer, Jennway, UK). The measurements were made at a constant frequency of 1 Hz at constant temperature of 35± 0.1˚C. The cell constant was ascertained by using a standard potassium chloride solution. The different SNEDDSs were titrated with water and their electro conductive behavior was determined.Before each measurement, the cell was prewashed twice with the sample in order to avoid the adherence effect of the surfactant and the cosurfactant upon the cell inner wall and the electrodes.

Assessment of the rheological properties
The rheological properties of the prepared Felodipine SNEDDSs were determined by means of Brookfield rotary viscometer (Brookfield digital Rheometer DVIII, USA). fitted with CP-40 cone and plate spindle. Each formula (0.5 ml) was put in suitable container. The rpm was increased gradually in a suitable range to give torque values between 10-100 units, at 37 2 ˚C, with 15 seconds between each two successive speeds.The rheological behavior of each system was investigated by plotting the shear stress versus the shear rate and by plotting the viscosity versus the shear rate. The obtained data were determined using an excelcomputer program. The shear rate in sec -1 and the viscosity in cp were determined from the instrument readings and fitted to the power law constitutive equation (Tung, 1994) (18) .
The two dimensionless quantities: the consistency index (m) and the flow index (n) characteristic for each formulation were obtained. If n = 1 this indicates Newtonian behavior while if n is less than 1, this corresponds to shear thinning flow. The lower value of n the more shear thinning the formulation (Copetti et al, 1997;Owen et al, 2000;Chang et al, 2002) (19)(20)(21) .

Robustness to dilution and phase separation study
In order to mimic physiological dilution process after oral administration of the prepared Felodipine SNEDDSs, the formulae were diluted 50, 100, 1000 times with different aqueous media including distilled water, 0.1 N HCl and phosphate buffer pH 7.4. The diluted formulae were mixed by vortex mixer. The formed mixtures were set aside for 2 hours, and then they were examined by visual inspection for clarity of the formed emulsions and presence of any precipitate of the drug. Formulae that didn't show significant phase separation or drug precipitation at the end of the 2 hours period were used in the subsequent study (Kim and (22)(23)(24) .

Determination of emulsification time, dispersibility and percentage transmittance
The rate of emulsification is an important index for the assessment of the efficacy of emulsification. Evaluation of the self emulsifying properties of SNEDDS formulations was performed by visual assessment. The USP type II dissolution apparatus (Hanson research, USA) was used to evaluate the efficiency of selfemulsification of the selected formulae. One gram of each formula was added drop wise into 500 ml of distilled water maintained at 37˚C with gentle agitation condition provided by rotating paddle at 50 rpm. The time taken for the emulsification (until a clear homogenous system was obtained) formation was assessed visually in triplicates (Bachynsky et al., 1997;Khoo et al., 1998) (25,26) .Transparency of the formed emulsion was determined by measuring % transmittance at 650 nm with purified water as blank through UV Spectrophotometer (Agilent, USA) (Date and Nagarsenker, 2008a) (27) .

Droplet size analysis
Each formula of the prepared Felodipine SNEDDSs was diluted 100 times using double distilled water (Wang et al., 2010) (28) . The mean droplet size and polydispersity index (PDI) of the formed nanoemulsion was determined by using instrument (Nanotrac wave II, Microrack, USA), using the 632 nm line of aHeNe laser as the incident light with angel 90 o .

Zeta potential
For measurement, a dilute suspension of the nanoparticles is subjected to a weak electric field, and the mobility of the particles is commonly determined by NICOMP 380 ZLS. This technique is based on the evaluation of a frequency (Doppler) shift that is observed for the light scattered from the particles, motion in the electric field. As a result, the electrophoretic mobility µ (velocity of the particles/electric field strength) of the nanoparticles is obtained.
Investigation of the effect of different surface-active agents on the zeta potential can provide information on the interaction of the particles with surface-active agents. The effect of variations in preparation procedure as well as the potential influence of drug loading or further processing, such as freeze drying or sterilization, on the zeta potential of SNEDDS has also been studied (Lim et al., 2002) (29) .

Analytical test method of Felodipine by using HPLC technique HPLC Identification
The retention time of the major peak in the chromatogram of the assay preparation corresponds to that in the chromatogram of the standard preparation, as obtained in the Assay.

Method of assay for Felodipine
Chromatographic conditions used for assaying Felodipine in the selected Felodipine SNEDDSs was illustrated in table (1). Buffer solution: Dissolve 6.9 g of monobasic sodium phosphate in about 800 mL of water in a 1000-mL volumetric flask. Adjust with 1 M phosphoric acid to a pH of 3.0 ± 0.05, dilute with water to volume, and mix.

Flow rate 1.0 ml/ min
Standard solution:Transfer 20 mg felodipine reference standard to 100 ml volumetric flask.
Assay solution:Transfer amount of formula equivalent to 10 mg Felodipine to 100 ml volumetric flask, then add 70 ml mobile phase, sonicate for 15 min, dilute to volume by mobile phase (soln. 2). Transfer 10 ml from soln. 2 to 50 ml volumetric flask, dilute to volume and mix by mobile phase.
System suitability: After conditioning the column with mobile phase, inject five replicate injections of the standard solution. These injections should be with relative standard deviation of these replicate injections should not exceed 2.0%.

Identification:
The retention time of Felodipine peak in assay chromatogram should comply with that of working standard.

In-vitro dissolution of Felodipine SNEDDS
Drug dissolution studies of Felodipine SNEDDSs were carried out according to the official method in USP 36-2013. Aliquots of Felodipine SNEDDS each containing 10 mg of Felodipine was installed to the dissolution medium. Also, Plendil 10 mg tablet was tested under the same conditions. Five milliliters of dissolution media was retrieved at timed intervals (10, 30 and 60 minutes) and replaced with fresh dissolution media. The amount of Felodipine was quantified using HPLC method.

Apparatus: USP App II (Paddle)
Medium: pH 6.5 phosphate buffer with 1% sodium lauryl sulfate (Medium is prepared as follows: Transfer 206 ml of 1 M monobasic sodium phosphate monohydrate, 196 ml of 0.5 M dibasic sodium phosphate anhydrous, and 50.0 g of sodium lauryl sulfate to a 5000 ml volumetric flask. Add approximately 4000 ml of water, and mix well. If necessary, adjust with 1 N sodium hydroxide to a pH of 6.5. Dilute with water to volume, and mix well.) Volume: 500 ml in each vessel (60 min) Speed: 50 RPM.

Preparation of Felodipine standard solution
Transfer 20 mg felodipine reference standard to 100 ml volumetric flask, add 70 ml mobile phase, Sonicate for 15 min, dilute to volume by mobile phase to give solution No. 1. Transfer 10 ml from the above solution to 100 ml volumetric flask, dilute to volume and mix by dissolution medium to give solution No. 2. Take 10 ml form solution No. 2 then filtere this solution using filter paper (Whatman No.5)

Preparation of Felodipine assay solution
Transfer amount of the selected formula (either B18 or C19) equivalent to 10 mg Felodipine to 100 ml volumetric flask, add 70 ml mobile phase, Sonicate for 15 min, dilute to volume by mobile phase to give solution No. 3. Transfer 10ml from solution No. 3 to 50 ml volumetric flask, dilute to volume and mix by mobile phase then filtere this solution using filter paper (Whatman No.5) Calculation : A T X C ST X P X 100 In-vitro release % of Felodipine = ‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــ‬ Limit: NLT 75% is released in 60 min.

Kinetics for the in-vitro dissolution of Felodipine SNEDDS
Kinetic orders were used to determine the kinetic parameters of the in-vitro dissolution of Felodipine SNEDDS. Zero-and first -order kinetic, as well as, controlled diffusion model were tried to choose the most suitable kinetic order or systems for Felodipine release. Table (2) summarizes all the orders studied.

Determination of Felodipine peak and retention time
Although Felodipine can be determined spectrophotometrically at max 237nm (Verma et al., 2017) (30) , but the HPLC method was chosen as the preliminary study revealed that the components of SNEDDS may interfere with Felodipine in the spectrophotometric assay.
The chromatogram of 1 mg/ml Felodipine solution in methanol showed an identified sharp symmetrical peak with good base line and no tailing at a retention time of 4.108 minutes, this is shown in figure (1).

Figure (1): The chromatogram of 1 mg/ml Felodipine solution in methanol
Characterization of self-nanoemulsifying drug delivery systems

Solubility study
Values of the maximum solubility of Felodipine determined in different excipients are recorded in table (3).It was found that the Felodipine solubility values (mg/ml) were in the following descending order in Triacetin<Capryol 90< Lauroglycol 90 < Maisine-35-1 < Labrafil M 2125 <Labrafac PG < Isopropyl myristate. The values obtained were 10.9, 7.9, 3.6, 2.4, 1.18, 0.86 and 0.0514 mg/ml, respectively which are greatly higher than its solubility in water (0.019 mg/ml). Generally, oils can solubilize the lipophilic drug in a specific amount. Their ability to facilitate self-emulsification and increase the fraction of lipophilic drug that is transported via the intestinal lymphatic system, thereby increasing absorption from the GIT, making them the most important excipients (Kommuru et al., 2001)  Among the tested co-surfactants; Transcutol HP (9.26 mg/ml), showed the highest solubility of the drug followed by Ethanol (6.11 mg/ml) and 1-propanol (2.03 mg/ml), propylene glycol (1.72 mg/ml) and PEG 400 (1.39 mg/ml).
Transcutol HP was used in formulation of SEDDSs due to its solving power, good water solubility as well as its absorption and permeability enhancement (Gao et al., 1998;Lanlan et al., 2005) (35,36) .

Construction of ternary phase diagram
The existence of self nanoemulsified formulations fields that could self nanoemulsify under dilution and gentle agitation was identified from ternary phase diagram of a system containing oil, surfactant and co-surfactant. Six ternary phase diagrams were constructed with the compositions as shown in table (4) and illustrated in figures (2 and 3). All the components were converted to percent weight per weight before constructing the phase diagram.  The marked points represent all formulations that could self-emulsify in seconds and could be infinitely diluted by purified water indicating that the nanoemulsions formed will be capable of keeping Felodipine solubilized. Within this area, the formulations form fine oil in water nanoemulsion with only gentle agitation (Figure 4). This can be attributed to the fact that the surfactant strongly localized to the surface of the nanoemulsion droplets thus decreasing interfacial free energy and provide a mechanical barrier to coalescence forming a thermos mechanically spontaneous dispersion (Reiss, 1975) (37) . The formed nanoemulsions are clear, isotropic, transparent, and of low viscosity determined by visual inspection.

Determination of drug loading capacity of Felodipine in the formulated self nanoemulsifying drug delivery systems
Since the presence of high surfactant concentrations in the formulation is considered one of the drawbacks of self nanoemulsifying drug delivery system (Grove and Mullertz, 2007) (39) . From each phase diagram, the formulae that contained higher amounts of oil and still able to form clear nanoemulsion when infinitely diluted with water, were selected for drug loading as illustrated in table (5). 30, respectively showed the highest loading capacity of Felodipine (9.44 and 9.78 mg/ml, respectively). The type of oil used did not affect the loading capacity of the formulae. SNEDDSs that contain Capryol are able to dissolve amount of Felodipine comparable to those containing Triacetin which has the higher loading capacity of Felodipine.

Preparation of Felodipine self-nanoemulsifying drug delivery systems
Based on loading capacity of the formulae, the best three formulae from each system were chosen to be loaded with Felodipine. Formulae were prepared and loaded with drug and kept at 25 ˚C for further study, the prepared formulae were listed in table (6).

Visual inspection
Visual inspection of Felodipine SNEDDSs prepared using either Triacetin or Capryol 90 as oily phase showed that all formulae were clear, transparent and homogenous with no recorded signs of phase separation.

Assessment of thermodynamic stability
The physical stability of a lipid-based formulation is important to its performance, which can affect precipitation of the drug in the excipients matrix. In addition, the poor physical stability of the formulation can lead to phase separation of the excipients, which affects not only formulation performance, but also visual appearance of the formulation. The obtained results are tabulated in tables (7-9).   The stability of Felodipine in SNEDDSs was an important issue to be evaluated. Formulae that passed centrifugation test were shown in table (7). Only five formulations showed no precipitation and phase separation. These formulae are the following: A26, B13, B18, C19 and C26. The above selected formulae were subjected to the heating cooling cycles. After heating cooling cycles, only 3 formulae are selected which contain the following formulae B13, B18 and C19 and were able to remain stable as were listed in table (8). Finally the three selected formulae were subjected to freeze thaw cycles and illustrated in table (9). From the twelve starting tested formulae only B18 and C19 were thermodynamically stable without any precipitation of Felodipine or phase separation of the components.

Conductivity measurements
Conductometry is a useful tool assesses nanoemulsion structure, as there is a consistent correlation between structure type and nanoemulsion electro-conductivity behavior (Bumajdad and Eastoe, 2004) (40) .
The electrical conductivity measurements can determine the nature of the continuous phase of nanoemulsions, as O/W nanoemulsions are highly conducting because their external phase is water, while W/O are not, since water is the internal or dispersed phase (Hasse and Keipert, 1997) (41) .
The conductivity of B18 and C19 was checked, as these two formulae passed the thermodynamic stability tests. The observed conductivity curves as a function of water content indicate the use of electroconductimetry to study structural changes in nanoemulsions. Conductivity (O) increased with the increase of the dispersed water volume fraction ( w). The conductivity behavior of system C19 showed a gradual linear increase in conductivity values with the increase of water percent. In contrast, B18 showed an abrupt increase with the increase of water percent.
Results also revealed that the conductivity values of B18 was higher than C19; this may be attributed to the fact that the topological polar surface area of Transcutol that was used in B18 is larger than ethanol that was used in C19, with values of 78.9 and 77.8 A, respectively (Pubchem. ncbi. nlm. nih. gov, 2015) (42) . The results are illustrated in Figures (5 and 6).
By plotting the data of the conductivity values for the two selected Felodipine SNEDDSs (B18 and C19), the values obtained were 0.996 and 0.997 for the correlation coefficients, 2.753 and 0.454 for the intercepts and 0.372 and 0.075 for the slopes, respectively.

Assessment of the rheological properties
The viscosity study of the selected formulae is an important issue. Systems with very high viscosity may create problems in pourability in containers and syringability (Ram and Ajit, 2011;Attwood D, Florence, 2012) (43,44) .
Assessment of the rheological behavior of Felodipine SNEDDSs prepared using either Transcutol HP (B18) or Ethanol (C19) revealed that the formulae exhibited Pseudoplastic flow and Newtonian flow, respectively. It was noticed that B18 showed higher viscosity values than C19; Viscosity values are listed in table (10).
Formula B18 which show pseudoplastic flow (non-Newtonian flow) indicated by its Farrow's index (N = 2.166), as illustrated in table (11). The N value if greater than 1, it indicates pseudoplastic flow while if N value is less than 1, it indicates dilatant flow.
By applying Tung equation, the values of the consistency index and the flow index for formula B 18 showed the following values, 552.41 and 0.4608, respectively. If n = 1 this indicates Newtonian behavior while if n is less than 1, this corresponds to shear thinning flow. The lower value of n the more shear thinning the formulation. Table (10) showed that n value for formula C19 is 1.0750 which is approximately near 1 and conformed that this formula exhibited Newtonian flow.

Robustness to dilution and phase separation study
It is important to ensure that uniform emulsions are formed from self emulsification of SNEDDSs at different dilution. The prepared Felodipine SNEDDSs formulae were exposed to different folds of dilution in different media in an attempt to mimic the in-vivo conditions to predict whether phase separation or precipitation is likely to occur in the GIT where the formulation would encounter gradual dilution (Elnaggar et al., 2009; Gupta et al., 2011) (45,46) .
With all tested media, distilled water, 0.1N HCl and phosphate buffer (pH 7.4), visual examination of the formed solutions at 50 and 100 times dilutions showed nanoemulsions with slight bluish appearance and with no precipitation for B18, but those formed of C19 showed insignificant precipitation in the following order 50 fold dilution < 100 fold dilution; However samples of 1000 times dilution showed clear nanoemulsion with no precipitation or phase separation for both B18 and C19.
So, it was clear that formula B18 was robust to all dilutions with different media and did not show any phase separation even after 24 hours of storage. The insignificant precipitation that occurred with C19 may be attributed to the lower solubility of Felodipine in Ethanol than Transcutol that was found in B18. The results are shown in tables (12-14).

Determination of emulsification time, dispersibility and percentage transmittance
The rate of emulsification is an important index for assessment of the efficiency of emulsification. It was observed that B18 showed less dispersion time when compared with C19, as it showed emulsification time of 30 ± 1.2 seconds while B18 showed 50 ± 1.1 seconds; The results may be attributed to the higher viscosity value of B18 than that of C 19.
Both formulae B18 and C19 showed clear transparent appearance as a result of forming nanoemulsion of grade A rapidly.
Percentage transmittance was carried out to prove that the emulsions formed from dispersion of SNEDDS are clear and transparent systems. % transmittance of C19 and B18 were 97.5% ± 0.5 and 97.4% ± 0.65, respectively, which is closer to 100%, indicating that clear nanoemulsion was formed when diluted with water. The results are shown in table (15).

Droplet size analysis
Droplet size is a crucial factor in self-emulsification performance, because it determines the rate and extent of drug dissolution as well as drug absorption. It has been reported that small size of emulsion droplets may lead to more rapid absorption, thereby improving the bioavailability (Cui et al., 2009a) (47) . The smaller the globule size, the larger the surface area provided for drug absorption (Grshanik and Benita, 2000) (48) . Furthermore, a decrease in the droplet size reflects the formation of a better packed film of the surfactant at the oil-water interface, thereby stabilizing the oil droplets (Cui et al., 2009b) (49) .
Depending upon the size of globules, self-microemulsified drug delivery system (SMEDDS) indicated the formulations forming transparent microemulsions with the oil droplet size rang between 100 and 250 nm. Self-nanoemulsified drug delivery system (SNEDDS) is relatively a recent term indicating the globule size less than 100 nm (Pouton and Porter, 2008) (50) .
The results revealed that the droplet size of Felodipine formula B18 was 64.2 nm (Population % 64.4) and 168.6 nm (Population % 35.6), which is bigger than that of Felodipine formula C19 which was 48.3 nm (Population % 74.2) and 121.4 nm (Population % 25.8). Based on these results both studied Felodipine formulae B 18 and C19 was classified as self nanoemulsified drug delivery system (SNEDDS) as shown in table (16) and illustrated in figures (7 and 8).
The Poly dispersity Index (PDI) is a dimensionless measure of the width of size distribution calculated from the cumulated analysis ranging from 0 to 1. A small value of PDI indicates a broader distribution of droplet size (Tang et al., 2012) (51) . PDI is the ratio of standard deviation to mean droplet size, which signifies uniformity of droplet size within the formulation. The higher the value of PDI the lower is the uniformity of droplet size (Baboota et al., 2007) (52) . PDI of Felodipine formulae B18 and C19 were found to be 0.314 ± 0.665 and 0.139 ± 0.018, respectively.  Zeta potential is a measure for the stability of any formulations based in the nano range. If zeta potential falls in the range of ± 30 mV, this means that the formulations had better stability. The results for the two studied Felodipine formulae B 18 and C 19 showed that zeta potential was 29.133 ± 0.342 mV and 24.057 ± 0.574 mV, respectively. The above values indicated that Felodipine formulae exhibit excellent stability, see table (17).        The obtained areas for Felodipine SNEDDS were converted to in-vitro release percent as shown in table (20). The in-vitro release percent of formula C 19 (system 1) was found to reach 42.16, 53.44 and 81.32% after 10, 30 and 60 minutes of dissolution. Figures (12-14) showed the three chromatograms for formula C 19 corresponding to the three release percent of Felodipine. The above figures represented the chromatographs for Felodipine formula C 19 after in-vitro release for 10, 30 and 60 minutes.    The obtained areas for Felodipine market product were converted to in-vitro release percent as shown in table (21). The in-vitro release percent of the market product was found to reach 37.86, 465.40 and 75.23% after 10, 30 and 60 minutes of dissolution. Figures (15-17) showed the three chromatograms for the market product corresponding to the three release percent of Felodipine. The above figures represented the chromatographs for Felodipine market product after in-vitro release for 10, 30 and 60 minutes.

Kinetic study of Felodipine in-vitro release
According to the results obtained from the in-vitro release, kinetic behaviors of all Felodipine formulae were studied. Zero order, First order and Higuchi diffusion model were tried in this study to investigate the kinetics of the in-vitro release of Felodipine formulations (formula B 18, formula C 19 and Market product). From the obtained data, it is found that in-vitro release of Felodipine formulae follows: Felodipine formulae (B 18 and C 19) obey Higuchi diffusion model while Felodipine market product obey zero-order kinetic.
The previous kinetic data showed that the in-vitro release of Felodipine formulae follows different kinetic orders and no single kinetic order can be used to express the drug release from specific type of these formulations.  Formulae B18 (composed of 30% Triacetin: 40% Span 80: 30% Transcutol HP) and C19 (composed of 20% Triacetin: 50% Span 80: 30% Ethanol) were thermodynamically stable with no precipitation of Felodipine.
Thus, Formulae B18 and C19 were selected for further studies in the following chapters.
Conductivity results showed that, its values increases with increasing the ratio of water added to the prepared formulae. Thus the obtained data indicated the formation of O/W nanoemulsion when water was added to Felodipine SNEDDSs. Felodipine SNEDDSs prepared using Transcutol HP (B18) exhibited pseudoplastic flow while Felodipine SNEDDSs prepared using ethanol (C19) exhibited Newtonian flow. Robustness to dilution and phase separation study showed that B18 was robust to all dilution with the three used media (water, 0.1 N HCl and Phosphate buffer pH 7.4), while C19 Showed slightly precipitation in case of 50 and 100 folds dilution and was robust to 1000 fold dilution with three tested media studied. The passed formulae B18 and C19 were able to form grade A emulsions with rapid emulsification time (30 and 50 seconds) and high % transmittance (97.4 % and 97.5 %), respectively. Formula prepared with Transcutol HP (B18) has larger droplet size and PDI than that prepared with Ethanol (C19). But both formulae, B18 and C 19 were classified as SNEDDS.
Both SNEDDS Felodipine formulae showed negative zeta potential values which indicated the stable nature of nanoparticles owing to electrostatic repulsion.
The results obtained for the in-vitro release of different Felodipine formulae indicated that the studied formulae can be arranged in descending order, concerning to there in-vitro release as follows: Felodipine formula B 18 > Felodipine formula C 19 > Market product, also it was showed that the presence of transcutol in B18 affect positively on the dissolution rather than ethanol in C19.
It was considered that the in-vitro release of C19 was 81% which is higher than the market felodipine 75%.
It was found that in-vitro release of Felodipine formulae follows: Felodipine formulae (B 18 and C 19) obey Higuchi diffusion model while Felodipine market product obey zero-order kinetic.
The previous kinetic data showed that the in-vitro release of Felodipine formulae follows different kinetic orders and no single kinetic order can be used to express the drug release from specific type of these formulations.