International Journal of Pharmaceutics 485 (2015) 235–243
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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical nanotechnology
Design and optimization of topical methotrexate loaded niosomes for enhanced management of psoriasis: Application of Box–Behnken design, in-vitro evaluation and in-vivo skin deposition study Aly A. Abdelbary * , Mohamed H.H. AbouGhaly Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Cairo University, Cairo, Egypt
A R T I C L E I N F O
A B S T R A C T
Article history: Received 5 February 2015 Received in revised form 6 March 2015 Accepted 9 March 2015 Available online 12 March 2015
Psoriasis, a skin disorder characterized by impaired epidermal differentiation, is regularly treated by systemic methotrexate (MTX), an effective cytotoxic drug but with numerous side effects. The aim of this work was to design topical MTX loaded niosomes for management of psoriasis to avoid systemic toxicity. To achieve this goal, MTX niosomes were prepared by thin film hydration technique. A Box-Behnken (BB) design, using Design-Expert1 software, was employed to statistically optimize formulation variables. Three independent variables were evaluated: MTX concentration in hydration medium (X1), total weight of niosomal components (X2) and surfactant: cholesterol ratio (X3). The encapsulation efficiency percent (Y1: EE%) and particle size (Y2: PS) were selected as dependent variables. The optimal formulation (F12) displayed spherical morphology under transmission electron microscopy (TEM), optimum particle size of 1375.00 nm and high EE% of 78.66%. In-vivo skin deposition study showed that the highest value of percentage drug deposited (22.45%) and AUC0–10 (1.15 mg. h/cm2) of MTX from niosomes were significantly greater than that of drug solution (13.87% and 0.49 mg .h/cm2, respectively). Moreover, invivo histopathological studies confirmed safety of topically applied niosomes. Concisely, the results showed that targeted MTX delivery might be achieved using topically applied niosomes for enhanced treatment of psoriasis. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Methotrexate Niosomes Box-Behnken In-vivo skin deposition In-vivo histopathological study
1. Introduction Psoriasis is a disorder, characterized by dense inflammatory cell infiltrates, massive proliferation, impaired differentiation of epidermis, formation of new blood vessels, and changes in lymphatic structure (Clark, 2011). Successful remedies for psoriasis lead to skin with a clinically normal state, i.e., resolution of epidermal thickness and reduced numbers of inflammatory cells (Zaba et al., 2007). The currently available topical treatments for psoriasis that have been found to be effective include: coal tar, anthralin, corticosteroids, photochemotherapy and retinoids (Weinstein et al., 1989). Methotrexate (MTX) is an effective systemic cytotoxic drug for the treatment of extensive psoriasis. Systemic treatment with MTX leads to side effects such as dizziness, nausea, diarrhea, cough, headache and mouth ulcers (Kremer et al., 2003; Yazici et al., 2005). Moreover, it can result in
* Corresponding author. Tel.: +20 1149005526. E-mail address: [email protected] (A.A. Abdelbary). http://dx.doi.org/10.1016/j.ijpharm.2015.03.020 0378-5173/ ã 2015 Elsevier B.V. All rights reserved.
hematological or hepatic toxicity (Sullivan, 2009). To avoid systemic toxicity, topical administration of MTX is beneficial (Lakshmi et al., 2007). Targeted drug delivery is advantageous for concentrating the drug in the desired tissues while reducing its relative concentration in the non target tissues. This leads to enhanced drug activity with comprehensively reduced side effects (Kazi et al., 2010). Surfactant vesicles acquired growing attention as an alternative potential drug delivery system to conventional liposomes. This kind of vesicles formed by surfactants is known as niosomes or non-ionic surfactant vesicles (Marianecci et al., 2012). In comparison to phospholipid-based vesicles, the surfactant vesicles like niosomes have several advantages such as greater stability, lower cost and lesser care in handling and storage. These advantages make surfactants more attractive than phospholipids for industrial applications in the pharmaceutical field. Moreover, niosomes have been reported as carriers for drug targeting. (Baillie et al., 1985; Di Marzio et al., 2011; Paolino et al., 2007). Response surface methodology (RSM) can be used in the development and optimization of drug delivery systems. This
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methodology involves the generation of polynomial mathematical relations from a variety of experimental designs and mapping the response over the experimental domain. Central composite, BoxBehnken and D-optimal designs are types of RSM designs available for statistical optimization of the formulations. Box-Behnken statistical design is an independent, rotatable or nearly rotatable, quadratic design. The treatment combinations are placed at the midpoints of the edges and at the center of the process space. This design requires less experimental runs and time. Therefore, it is considered a cost-effective technique than other usual processes of formulation and optimization of dosage forms (Agyralides et al., 2004; Chaudhary et al., 2013; Chopra et al., 2007). The aim of this study was to develop topical MTX loaded niosomes for management of psoriasis. Hence, optimal production parameters for a stable, highly concentrated MTX formulation with optimum particle size were adjusted using Box-Behnken statistical design. Moreover, in-vivo skin deposition of MTX from the optimum niosomal formulation was compared with MTX solution using Wistar male rats. Furthermore, in-vivo histopatholgical study was performed to assess the irritation potential of MTX loaded niosomes on rat skin. 2. Materials and methods 2.1. Materials Methotrexate (MTX) was kindly provided by EIMC United Pharma Company, Cairo, Egypt. Span 60, cholesterol and HPLC grade acetonitrile were purchased from Sigma–Aldrich, St. Louis, USA. Dicetyl Phosphate (DCP) was obtained from Fluka Chemical Co., Germany. Methanol and chloroform were purchased from Adwic, El-Nasr Pharmaceutical Chemicals Co., Cairo, Egypt. Glacial acetic acid and sodium acetate anhydrous were purchased from Merck, Darmstadt, Germany. The commercially available methotrexate solution, Unitrexate1, was manufactured by EIMC United Pharma Company, Cairo, Egypt. 2.2. Experimental design A three-level three-factor Box-Behnken (BB) design was employed to statistically optimize the formulation variables for preparing MTX niosomes, in order to obtain high encapsulation efficiency percent and optimum particle size. Generation and
Table 1 Box-Behnken design (BBD) for optimization of the MTX niosomes. Factors (independent variables)
X1: Methotrexate concentration (mg/mL) X2: Total weight of niosomal components (mg) X3: Surfactant: cholesterol ratio (weight ratio)
Levels Low (1)
Medium (0)
High (+1)
5
7.5
10
150
225
300
1:1
1.5:1
2:1
Responses (dependent variables)
Constraints
Y1: Encapsulation Efficiency (%) Y2: Particle size (nm)
Maximize Minimize
evaluation of the experimental design was carried out using Design Expert1 software (Version 7, Stat-Ease Inc. Minneapolis, MN, USA). A total of 15 experiments were run; twelve of which represent the mid-point of each edge of the multidimensional cube and the remaining three are the replicates of the cube’s center point. Three independent variables were evaluated: MTX concentration in hydration medium (X1), total weight of niosomal components (X2) and surfactant: cholesterol ratio (wt/wt) (X3). The encapsulation efficiency percent (Y1: EE%) and particle size (Y2: PS) were selected as the dependent variables. The independent (low, medium and high levels) and dependent variables are shown in Table 1. The composition of the prepared MTX loaded niosomal suspensions according to BB design is shown in Table 2. Desirability was calculated for selection of the optimized formula which was subjected for further investigations. 2.3. Preparation of MTX loaded niosomes by thin film hydration technique Niosomes were prepared by slight modification of the procedure mentioned by Udupa et al. (Udupa et al., 1993). Briefly, dicetyl phosphate (DCP) (5% w/w), Span 60 and cholesterol were accurately weighed into a long-necked round-bottom flask and dissolved in 20 mL of chloroform–methanol mixture (1:1). The organic phase was slowly evaporated at 50 C under vacuum, using a rotary evaporator (Rotavapor, Heidolph VV 2000, Burladingen,
Table 2 Compositions of the three-level three-factor BBD for formulation of MTX loaded niosomes. Run
Factors levels in actual values X1 (MTX concentration) (mg/mL)
X2 (Total weight of niosomal components) (mg)
X3 (Surfactant: cholesterol ratio)
Mid points 1 (F1) 2 (F2) 3 (F3) 4 (F4) 5 (F5) 6 (F6) 7 (F7) 8 (F8) 9 (F9) 10 (F10) 11 (F11) 12 (F12)
5 10 5 10 5 10 5 10 7.5 7.5 7.5 7.5
150 150 300 300 225 225 225 225 150 300 150 300
1.5:1 1.5:1 1.5:1 1.5:1 1:1 1:1 2:1 2:1 1:1 1:1 2:1 2:1
Center points 13 (F13) 14 (F14) 15 (F15)
7.5 7.5 7.5
225 225 225
1.5:1 1.5:1 1.5:1
A.A. Abdelbary, M.H.H. AbouGhaly / International Journal of Pharmaceutics 485 (2015) 235–243
Germany) at 90 rpm such that a thin dry film of the components was formed on the inner wall of the flask (Al-Mahallawi et al., 2014). The dried thin film was then hydrated with different volumes of phosphate buffer (pH 8), containing 25 mg MTX, by rotating the flask in a water bath at 50 C using rotary evaporator under normal pressure in order to ensure complete hydration of the film. For particle size reduction, the obtained dispersion was sonicated for 1 min in a bath sonicator (Elmasonic S40, Elma, Singen, Germany). The niosomal suspension was left to mature overnight at 4 C and used for further characterization.
2.4. Characterization of MTX niosomes 2.4.1. Particle size, polydispersity index and zeta potential Particle size (z-average), polydispersity index and zeta potential of the prepared MTX niosomal suspensions were measured by Photon Correlation Spectroscopy (PCS) using a Zetasizer Nano ZS90 instrument (Malvern instruments, Worcestershire, UK). An aliquot of the nanosuspension was diluted before the measurement. Measurements were performed in triplicate using 90 scattering angle at 25 C. The electrophoretic mobility was converted to zeta potential via the Smoluchowski equation (Ichino et al., 1990). All measurements were performed in triplicate at 25 C, dispersant viscosity of 0.89 cP and dielectric constant of 78.5. The viscosity of the samples was assumed to be that of water (Wang et al., 2007). The displayed results are the average value the standard deviation.
2.4.2. Drug encapsulation efficiency percent (EE%) The encapsulation efficiency percent (EE%) of MTX was determined indirectly by calculating the difference between the total amount of MTX added in the formulation and that remaining in the aqueous medium after separating the niosomal suspension by centrifugation at 15000 rpm for 1 h at 4 C using cooling centrifuge (Model 8880, Centurion Scientific Ltd., W. Sussex, UK). MTX contents were determined spectrophotometrically at a wavelength of 307 nm. Encapsulation efficiency percent was calculated using the equation: ðTotal amount of MTX added amount of free MTXÞ 100 EE% ¼ Total amount of MTX added (1)
2.5. Formulation optimization 1
The optimized formula was obtained using the Design Expert software by applying constraints on encapsulation efficiency percent of the niosomes to reach the maximum value and on particle size to obtain the smallest value. The suggested optimized formula was then prepared and evaluated in triplicate to check the validity of the calculated optimal formulation factors and predicted responses given by the software.
2.6. Transmission electron microscopy (TEM) The particle morphology of the optimized niosomal suspension was examined using TEM (H-600, Hitachi, Japan). The sample was dropped on copper–gold carbon grid and allowed to dry. The grid was then mounted in the instrument and photographs were taken at different magnifications (Bendas and Abdelbary, 2014).
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2.7. In-vivo evaluation of MTX loaded niosomes 2.7.1. HPLC determination of MTX An isocratic HPLC method was employed for the quantification of MTX (Begas et al., 2014). A Thermo Separation HPLC system (Fremont, California) equipped with a P4000 pump unit, an AS3000 autosampler including an injection valve with a sample loop of 50 mL volume, and a UV2000 detector was used. A Zorbax Extend-C18 column (4.6 mm 250 mm) containing 3.5 mm size adsorbent as stationary phase (Agilent technologies, Santa Clara, California) was used. The column was maintained at room temperature (25.0 2.0 C). The mobile phase consisted of a mixture of 50 mM sodium acetate buffer (pH 3.6) and acetonitrile (89:11 v/v). The flow rate and the UV detector were set at 1.0 mL/ min and 307 nm, respectively. The assay procedures were validated in terms of linearity, precision, and accuracy. 2.7.2. In-vivo skin deposition study The optimized niosomal suspension was selected for the invivo skin deposition study. The protocol of the study was approved by the ethical committee of Faculty of Pharmacy, Cairo University. Fifty-four male Wistar rats, weighing 150–200 g, were involved in the study. The animals were supplied with standard diet and tap water ad libitum. The rats were randomly divided into 3 groups with 18 animals each, where group I acted as control while animals in group II and group III received topical application of MTX solution (Unitrexate1) and optimized MTX niosomal formulation, respectively. Bottle caps that served as drug pools with an area of 4.91 cm2 were stuck to rat dorsal skin, which was shaved carefully to remove hair (Shen et al., 2014). Half mL of each of MTX formulations (MTX solution and niosomes) was added non occlusively into the drug pool. At the end of the experiment, the rats were sacrificed. One group (18 rats) was kept untreated (No application of solution or suspension in this group). After different time intervals of application of treatments (1, 2, 4, 6, 8 and 10 h), at each time point 3 animals from each group were sacrificed and the dorsal rat skin was excised and immediately washed 3 times with 5 mL of normal saline in each time. The excised skin (4.91 cm2) was cut into pieces and sonicated in 5 mL dimethyl sulfoxide (DMSO) for 30 min. The extract was then filtered through a 0.45 mm filter membrane and the concentration of MTX was determined by HPLC. These data were used to calculate the skin deposition of MTX generated by the range of formulations tested. The destruction of animal carcasses was achieved by incineration. 2.7.3. In-vivo histopathological study In- vivo histopathological study was conducted to assess the irritation potential and observe the ultrastructural changes in the skin upon exposure to MTX loaded niosomes. The rats were randomly divided into 3 groups with 4 animals each. Group I acted as control while animals in group II and group III received topical application of MTX solution (Unitrexate1) and optimized MTX niosomal suspension, respectively, onto the skin three times daily for a period of 1 week. The animals were then sacrificed and the skin was excised for histopathological investigation according to the protocol described by Bancroft et al. (Bancroft and Gamble, 2008). Briefly, autopsy samples were taken from the skin of rats in different groups and fixed in 10% formol saline for 24 h. Skin washing was done with tap water, and then, serial dilutions of alcohol (methyl, ethyl, and absolute ethyl) were used for dehydration. Specimens were cleared in xylene and embedded in paraffin at 56 C in hot air oven for 24 h. Sections from the paraffin blocks of 4 mm thickness were
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cut using a microtome (Leica Microsystems SM2400, Cambridge, England). The obtained tissue sections were collected on glass slides, deparaffinized, stained with hematoxylin and eosin and then examined under an electric light microscope (Morsi et al., 2014). 3. Results and discussion 3.1. Preparation of MTX loaded niosomes by thin film hydration technique Preliminary studies were performed to carefully select the most appropriate technique for preparing MTX loaded niosomes. Also, preliminary studies were carried out to choose the proper solvent for the drug, the most appropriate surfactant and the sonication time. The film hydration technique was adopted, where the drug can be incorporated either during film formation by dissolving the drug in an organic solvent or during film hydration by incorporating the drug in the hydration medium. The target was to incorporate 25 mg MTX in the niosomal suspension. Therefore, the solubility of MTX was tested in different organic solvent mixtures together with cholesterol and the studied non ionic surfactants. Acetone, chloroform, diethyl ether and methylene chloride failed to dissolve sufficient amount of MTX to begin the film formation procedure. On the other hand, a mixture of 30% dimethyl sulfoxide with any of the tested solvents produced the required solution. However, the drug precipitated and a non continuous film was produced during solvent evaporation. Alternatively, the approach of incorporating the drug in the hydration medium instead of incorporating it during film formation was used. The solubility of MTX is pH dependent with the solubility increasing above pH 4 (Vaidyanathan et al., 1985). Different buffers starting from pH 5.5 to 8 were tested and pH 8 provided the required solubility. This was advantageous for achieving our target as increasing the pH decreases the percutaneous absorption of MTX leading to higher deposition in the skin and lower systemic absorption (Vaidyanathan et al., 1985). Different surfactants were also tested and Span 60 was chosen as it produced the highest encapsulation efficiency percent and optimum particle size. Meanwhile, methanol, chloroform and diethyl ether were used as solvents for the film forming materials in 10 and 20 mL volumes. The solvent mixture that produced the clearest and most continuous film was the mixture of methanol and chloroform in a ratio of 1:1 using a total volume of 20 mL. Regarding the sonication time, the niosomes were prepared without sonication as well as sonicated for 1 and 2 min. The longer sonication led to smaller particle size. However, due to the high solubility of MTX in the phosphate buffer pH 8, the encapsulation
efficiency percent of the produced niosomes decreased significantly after sonication for 2 min. Therefore, the sonication time was fixed at 1 min. Moreover, the aim of our work was the topical delivery of MTX and avoidance of systemic absorption. It was reported that the large vesicles with a size 600 nm do not deliver their contents into deeper layers of the skin. Rather, these vesicles stay within the stratum corneum (Verma et al., 2003). Hence, the formulation of MTX loaded niosomes with particle size smaller than 600 nm was not advantageous to avoid penetration into deeper layers of skin and consequently a possibility of systemic absorption. However, the preliminary trials conducted in our study showed that the formulated Span 60 niosomes had a size range of 500–2000 nm. Hence, a constraint was applied on particle size to achieve the smallest value during formulation optimization based on the obtained size range in the preliminary trials. This was done using Design-Expert1 software, to obtain the formula with optimum particle size for skin deposition. 3.2. MTX loaded niosomes characterization 3.2.1. Effect of formulation variables on encapsulation efficiency percent The ability of niosomes to encapsulate significant amount of MTX is essential for its targeted use for management of psoriasis topically. Values of the encapsulation efficiency are presented in Table 3. Fig. 1 illustrates the response surface and cube plots for the effects of the concentration of MTX in hydration medium (X1), the total weight of niosomal components (X2) as well as Span 60: cholesterol ratio (X3) on the encapsulation efficiency percent. ANOVA test for the observed encapsulation efficiency percent data indicates that the linear model was significant and fitting for the data. The resulting equation in terms of coded values was as follows: EE% ¼ þ44:25 þ 25:86X 1 þ 16:08X 2 þ 12:05X 3 During the preparation of MTX loaded niosomes, the drug was introduced in the hydration medium. Therefore, MTX will probably be entrapped in the inner aqueous core of the vesicles. The equation reveals that there was a significant synergistic effect (p = 0.0001) of the concentration of MTX concentration on the percent encapsulation efficiency. A fixed amount of MTX was used in each preparation (25 mg) and the concentration was increased by decreasing the volume used to dissolve MTX. The increased amount of encapsulated MTX with increasing its concentration could be due to the saturation of the hydration medium with MTX that forces the drug to be encapsulated into niosomes (Balakrishnan et al., 2009; El-Samaligy et al., 2006; Mokhtar et al., 2008). Moreover, when comparing two formulae, considering that the same volume of the hydration was entrapped inside the vesicles,
Table 3 Encapsulation efficiency percent, particle size, polydispersity index and zeta potential of MTX loaded niosomes (n = 3). Formula
Encapsulation efficiency (% SD)
Particle size (nm SD)
PDI (values SD)
Zeta potential (mV SD)
F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13 F14 F15
12.52 4.57 52.73 5.01 46.36 8.18 79.00 4.37 1.98 2.80 66.25 1.75 4.47 6.32 74.26 1.45 1.42 0.32 42.13 10.12 50.81 0.55 78.66 5.64 53.32 1.25 54.50 1.45 45.30 10.44
558.70 16.54 1113.00 7.07 976.00 42.64 2200.00 164.75 584.80 2.97 1560.50 67.17 1280.00 63.64 1380.00 25.45 518.80 8.62 1865.00 56.73 1422.00 77.78 1375.00 44.66 1471.00 34.76 1214.25 53.03 1220.50 36.10
0.19 0.09 0.43 0.14 0.27 0.18 0.31 0.21 0.48 0.09 0.22 0.17 0.40 0.00 0.32 0.20 0.36 0.12 0.39 0.18 0.44 0.00 0.42 0.22 0.38 0.11 0.21 0.12 0.35 0.04
-63.90 1.41 -58.40 1.55 -58.85 0.07 -61.65 0.07 -46.35 5.58 -57.95 2.75 -55.40 0.07 -62.50 0.99 -54.55 2.33 -51.10 4.10 -60.30 0.42 -59.00 0.84 -60.55 1.48 -66.05 0.21 -61.45 0.21
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Fig. 1. (a) Response 3D plots and (b) cube plot for the effect of MTX concentration in hydration medium (X1), total weight of niosomal components (X2) and Span 60: cholesterol ratio (X3) on encapsulation efficiency percent.
then, the increased concentration of drug in this medium will mean that more amount of the drug will be entrapped inside the vesicles. In addition, the increase in hydration volume was reported to increase the drug leakage and consequently decrease percent encapsulation efficiency (Ruckmani and Sankar, 2010). The positive coefficient of the term, X2, indicates that the total weight of the niosomal components led to a significant synergistic effect on the EE% of the prepared niosomes (p = 0.0039). Higher amount of the film forming materials (niosomal components) increased the total lipid available for hydration (Abdelkader et al., 2010). This resulted in higher number of vesicles produced engulfing inside them more volume of the hydration medium and consequently higher amount of dissolved drug will be encapsulated. In other words, the increased percent encapsulation efficiency may be attributed to the increase in the availability of lipophilic ambience, which can accommodate the drug molecules to a higher extent (Agarwal et al., 2001). The increase in EE% with increase in total lipid content was also reported by Abdelkader et al., working on naltrexone niosomes (Abdelkader et al., 2010). Finally, the equation also reveals significant synergistic effect of the Span 60: cholesterol ratio on the percent encapsulation efficiency (p = 0.0196). Increasing Span 60 concentration resulted in a less permeable niosomal membrane and hence promoted the percent encapsulation efficiency (Zaki et al., 2014). The results also showed that decreasing cholesterol concentration led to a significant increase in EE%. This might be due to the fact that
the increase in cholesterol in certain cases may disrupt the regular linear structure of the vesicle membrane, and it does not encourage drug entrapment (Abdelbary, 2011; Al-Mahallawi et al., 2014; Moribe et al., 1999). 3.2.2. Effect of formulation variables on particle size and polydispersity index The particle size of the prepared MTX loaded niosomes is shown in Table 3. The PDI of all the niosomes formulations showed narrow size distribution and good homogeneity (Table 3). As previously mentioned, the very small size (less than 600 nm) was not required in our formulation process to avoid penetration into deeper layers of skin and systemic absorption. Rather, vesicles with a size 600 nm that do not deliver their contents into deeper layers of the skin and stay within the stratum corneum were beneficial for our aim (Verma et al., 2003). Fig. 2 illustrates the response surface and cube plots for the effects of the concentration of MTX in hydration medium (X1), the total weight of niosomal components (X2) as well as Span 60: cholesterol ratio (X3) on the particle size. ANOVA test for the observed particle size data indicates that the two factor interaction model was significant and fitting for the data. The resulting equation in terms of coded values was as follows: Particle size = +1248.89 + 356.75 X1 + 350.44 X2 + 115.99 X3 + 167.43 X1X2 218.93 X1X3 348.30 X2X3
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Fig. 2. (a) Response 3D plots and (b) cube plot for the effect of MTX concentration in hydration medium (X1), total weight of niosomal components (X2) and Span 60: cholesterol ratio (X3) on particle size.
The positive coefficient of the term, X1, indicates that MTX concentration had a synergistic effect on the particle size of the prepared niosomes (p < 0.0001). Increasing the concentration of MTX was achieved by decreasing the volume of the hydration medium. The lower volume of hydration medium may have led to incomplete or distorted formation of bilayer due to excess of material and low space. In order to accommodate the scarcity of water, the surface area of the produced vesicles was decreased and consequently the particle size increased (Sankhyan and Pawar, 2013). The ANOVA results also revealed the synergistic effect of the total weight of the niosomal components on the particle size (p < 0.0001). This may be attributed to the presence of large amount of film forming materials relative to the hydration medium. This may have resulted in multiple layers accumulating over each other and hence particle size increased. Furthermore, the presence of large amount of niosomal components leads to an increase in the number of lipid particles inside each vesicle which has a linear relation with the vesicle size (Wang and He, 2009). The coefficient of the third term, X3, shows that the Span 60: cholesterol ratio also had a synergistic effect on particle size (p = 0.0397). These results can be well correlated with the previously noticed increase in the percent encapsulation efficiency with increasing the Span 60: cholesterol ratio. The higher amount of MTX enclosed inside the vesicles might be the reason for the increase in the particle size. Moreover, the increase in particle size with increasing Span 60 concentration was also observed by Zaki et al. working on diacerein loaded niosomes (Zaki et al., 2014). 3.2.3. Effect of formulation variables on zeta potential Zeta potential is the measure of the overall charges acquired by vesicles and can be used to evaluate the stability of colloidal dispersions. In general, the system is considered stable when zeta potential value is around 30 mV due to electrical repulsion
between particles (Muller et al., 2001). The values of zeta potential for the prepared MTX loaded niosomes are presented in Table 3. Dicetyl phosphate (DCP), a negative charge inducer, was added to all formulae to increase the stability of the niosomal suspension and to avoid aggregation of the vesicles. The small differences in zeta potential absolute values of the prepared niosomes (all values > 30 mV) might be due to the presence of DCP with the same amount (5%) in all prepared niosomal formulations (Shatalebi et al., 2010). 3.3. Formulation optimization and analysis of the Box-Behnken (BB) design The BB design was used for designing and analysis of the experimental trials. BB requires much fewer experiments than a full-factorial design (Goyal et al., 2013). Adequate precision measured the signal to noise ratio to ensure that the model can be used to navigate the design space (de Lima et al., 2011). A ratio greater than 4 (the desirable value) was observed in both responses as shown in Table 4. On the other hand, Predicted R2 was calculated as a measure of how good the model predicts a response value (Chauhan and Gupta, 2004; Kaushik et al., 2006). The adjusted R2 and predicted R2 should be within approximately 0.20 of each other to be in reasonable agreement (Annadurai et al., 2008). If they are not, there might be a problem with either the data or the model. It is worthy to say that the predicted R2 values were in a reasonable agreement with the adjusted R2 in both responses. After applying constraints on the particle size and encapsulation efficiency percent (Table 1), the Design Expert1 software suggested an optimized formula to be prepared whose overall desirability was 0.673. The suggested formula (F12) had a MTX concentration of 7.5 mg/mL, total weight of niosomal components of 300 mg and Span 60: cholesterol ratio of 2:1. The suggested formula was prepared and evaluated, and the residual between the
A.A. Abdelbary, M.H.H. AbouGhaly / International Journal of Pharmaceutics 485 (2015) 235–243
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Table 4 Results of regression analysis for responses Y1 (EE%) and Y2 (PS).
Y1: EE% Y2: PS
Model
Adequate Precision
R2
Adjusted R2
Predicted R2
SD
% CV
p-value
Linear 2FI
13.008 18.491
0.8834 0.9529
0.7879 0.9176
0.6614 0.8205
12.49 133.73
28.23 10.71
0.0001
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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical nanotechnology
Design and optimization of topical methotrexate loaded niosomes for enhanced management of psoriasis: Application of Box–Behnken design, in-vitro evaluation and in-vivo skin deposition study Aly A. Abdelbary * , Mohamed H.H. AbouGhaly Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Cairo University, Cairo, Egypt
A R T I C L E I N F O
A B S T R A C T
Article history: Received 5 February 2015 Received in revised form 6 March 2015 Accepted 9 March 2015 Available online 12 March 2015
Psoriasis, a skin disorder characterized by impaired epidermal differentiation, is regularly treated by systemic methotrexate (MTX), an effective cytotoxic drug but with numerous side effects. The aim of this work was to design topical MTX loaded niosomes for management of psoriasis to avoid systemic toxicity. To achieve this goal, MTX niosomes were prepared by thin film hydration technique. A Box-Behnken (BB) design, using Design-Expert1 software, was employed to statistically optimize formulation variables. Three independent variables were evaluated: MTX concentration in hydration medium (X1), total weight of niosomal components (X2) and surfactant: cholesterol ratio (X3). The encapsulation efficiency percent (Y1: EE%) and particle size (Y2: PS) were selected as dependent variables. The optimal formulation (F12) displayed spherical morphology under transmission electron microscopy (TEM), optimum particle size of 1375.00 nm and high EE% of 78.66%. In-vivo skin deposition study showed that the highest value of percentage drug deposited (22.45%) and AUC0–10 (1.15 mg. h/cm2) of MTX from niosomes were significantly greater than that of drug solution (13.87% and 0.49 mg .h/cm2, respectively). Moreover, invivo histopathological studies confirmed safety of topically applied niosomes. Concisely, the results showed that targeted MTX delivery might be achieved using topically applied niosomes for enhanced treatment of psoriasis. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Methotrexate Niosomes Box-Behnken In-vivo skin deposition In-vivo histopathological study
1. Introduction Psoriasis is a disorder, characterized by dense inflammatory cell infiltrates, massive proliferation, impaired differentiation of epidermis, formation of new blood vessels, and changes in lymphatic structure (Clark, 2011). Successful remedies for psoriasis lead to skin with a clinically normal state, i.e., resolution of epidermal thickness and reduced numbers of inflammatory cells (Zaba et al., 2007). The currently available topical treatments for psoriasis that have been found to be effective include: coal tar, anthralin, corticosteroids, photochemotherapy and retinoids (Weinstein et al., 1989). Methotrexate (MTX) is an effective systemic cytotoxic drug for the treatment of extensive psoriasis. Systemic treatment with MTX leads to side effects such as dizziness, nausea, diarrhea, cough, headache and mouth ulcers (Kremer et al., 2003; Yazici et al., 2005). Moreover, it can result in
* Corresponding author. Tel.: +20 1149005526. E-mail address: [email protected] (A.A. Abdelbary). http://dx.doi.org/10.1016/j.ijpharm.2015.03.020 0378-5173/ ã 2015 Elsevier B.V. All rights reserved.
hematological or hepatic toxicity (Sullivan, 2009). To avoid systemic toxicity, topical administration of MTX is beneficial (Lakshmi et al., 2007). Targeted drug delivery is advantageous for concentrating the drug in the desired tissues while reducing its relative concentration in the non target tissues. This leads to enhanced drug activity with comprehensively reduced side effects (Kazi et al., 2010). Surfactant vesicles acquired growing attention as an alternative potential drug delivery system to conventional liposomes. This kind of vesicles formed by surfactants is known as niosomes or non-ionic surfactant vesicles (Marianecci et al., 2012). In comparison to phospholipid-based vesicles, the surfactant vesicles like niosomes have several advantages such as greater stability, lower cost and lesser care in handling and storage. These advantages make surfactants more attractive than phospholipids for industrial applications in the pharmaceutical field. Moreover, niosomes have been reported as carriers for drug targeting. (Baillie et al., 1985; Di Marzio et al., 2011; Paolino et al., 2007). Response surface methodology (RSM) can be used in the development and optimization of drug delivery systems. This
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methodology involves the generation of polynomial mathematical relations from a variety of experimental designs and mapping the response over the experimental domain. Central composite, BoxBehnken and D-optimal designs are types of RSM designs available for statistical optimization of the formulations. Box-Behnken statistical design is an independent, rotatable or nearly rotatable, quadratic design. The treatment combinations are placed at the midpoints of the edges and at the center of the process space. This design requires less experimental runs and time. Therefore, it is considered a cost-effective technique than other usual processes of formulation and optimization of dosage forms (Agyralides et al., 2004; Chaudhary et al., 2013; Chopra et al., 2007). The aim of this study was to develop topical MTX loaded niosomes for management of psoriasis. Hence, optimal production parameters for a stable, highly concentrated MTX formulation with optimum particle size were adjusted using Box-Behnken statistical design. Moreover, in-vivo skin deposition of MTX from the optimum niosomal formulation was compared with MTX solution using Wistar male rats. Furthermore, in-vivo histopatholgical study was performed to assess the irritation potential of MTX loaded niosomes on rat skin. 2. Materials and methods 2.1. Materials Methotrexate (MTX) was kindly provided by EIMC United Pharma Company, Cairo, Egypt. Span 60, cholesterol and HPLC grade acetonitrile were purchased from Sigma–Aldrich, St. Louis, USA. Dicetyl Phosphate (DCP) was obtained from Fluka Chemical Co., Germany. Methanol and chloroform were purchased from Adwic, El-Nasr Pharmaceutical Chemicals Co., Cairo, Egypt. Glacial acetic acid and sodium acetate anhydrous were purchased from Merck, Darmstadt, Germany. The commercially available methotrexate solution, Unitrexate1, was manufactured by EIMC United Pharma Company, Cairo, Egypt. 2.2. Experimental design A three-level three-factor Box-Behnken (BB) design was employed to statistically optimize the formulation variables for preparing MTX niosomes, in order to obtain high encapsulation efficiency percent and optimum particle size. Generation and
Table 1 Box-Behnken design (BBD) for optimization of the MTX niosomes. Factors (independent variables)
X1: Methotrexate concentration (mg/mL) X2: Total weight of niosomal components (mg) X3: Surfactant: cholesterol ratio (weight ratio)
Levels Low (1)
Medium (0)
High (+1)
5
7.5
10
150
225
300
1:1
1.5:1
2:1
Responses (dependent variables)
Constraints
Y1: Encapsulation Efficiency (%) Y2: Particle size (nm)
Maximize Minimize
evaluation of the experimental design was carried out using Design Expert1 software (Version 7, Stat-Ease Inc. Minneapolis, MN, USA). A total of 15 experiments were run; twelve of which represent the mid-point of each edge of the multidimensional cube and the remaining three are the replicates of the cube’s center point. Three independent variables were evaluated: MTX concentration in hydration medium (X1), total weight of niosomal components (X2) and surfactant: cholesterol ratio (wt/wt) (X3). The encapsulation efficiency percent (Y1: EE%) and particle size (Y2: PS) were selected as the dependent variables. The independent (low, medium and high levels) and dependent variables are shown in Table 1. The composition of the prepared MTX loaded niosomal suspensions according to BB design is shown in Table 2. Desirability was calculated for selection of the optimized formula which was subjected for further investigations. 2.3. Preparation of MTX loaded niosomes by thin film hydration technique Niosomes were prepared by slight modification of the procedure mentioned by Udupa et al. (Udupa et al., 1993). Briefly, dicetyl phosphate (DCP) (5% w/w), Span 60 and cholesterol were accurately weighed into a long-necked round-bottom flask and dissolved in 20 mL of chloroform–methanol mixture (1:1). The organic phase was slowly evaporated at 50 C under vacuum, using a rotary evaporator (Rotavapor, Heidolph VV 2000, Burladingen,
Table 2 Compositions of the three-level three-factor BBD for formulation of MTX loaded niosomes. Run
Factors levels in actual values X1 (MTX concentration) (mg/mL)
X2 (Total weight of niosomal components) (mg)
X3 (Surfactant: cholesterol ratio)
Mid points 1 (F1) 2 (F2) 3 (F3) 4 (F4) 5 (F5) 6 (F6) 7 (F7) 8 (F8) 9 (F9) 10 (F10) 11 (F11) 12 (F12)
5 10 5 10 5 10 5 10 7.5 7.5 7.5 7.5
150 150 300 300 225 225 225 225 150 300 150 300
1.5:1 1.5:1 1.5:1 1.5:1 1:1 1:1 2:1 2:1 1:1 1:1 2:1 2:1
Center points 13 (F13) 14 (F14) 15 (F15)
7.5 7.5 7.5
225 225 225
1.5:1 1.5:1 1.5:1
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Germany) at 90 rpm such that a thin dry film of the components was formed on the inner wall of the flask (Al-Mahallawi et al., 2014). The dried thin film was then hydrated with different volumes of phosphate buffer (pH 8), containing 25 mg MTX, by rotating the flask in a water bath at 50 C using rotary evaporator under normal pressure in order to ensure complete hydration of the film. For particle size reduction, the obtained dispersion was sonicated for 1 min in a bath sonicator (Elmasonic S40, Elma, Singen, Germany). The niosomal suspension was left to mature overnight at 4 C and used for further characterization.
2.4. Characterization of MTX niosomes 2.4.1. Particle size, polydispersity index and zeta potential Particle size (z-average), polydispersity index and zeta potential of the prepared MTX niosomal suspensions were measured by Photon Correlation Spectroscopy (PCS) using a Zetasizer Nano ZS90 instrument (Malvern instruments, Worcestershire, UK). An aliquot of the nanosuspension was diluted before the measurement. Measurements were performed in triplicate using 90 scattering angle at 25 C. The electrophoretic mobility was converted to zeta potential via the Smoluchowski equation (Ichino et al., 1990). All measurements were performed in triplicate at 25 C, dispersant viscosity of 0.89 cP and dielectric constant of 78.5. The viscosity of the samples was assumed to be that of water (Wang et al., 2007). The displayed results are the average value the standard deviation.
2.4.2. Drug encapsulation efficiency percent (EE%) The encapsulation efficiency percent (EE%) of MTX was determined indirectly by calculating the difference between the total amount of MTX added in the formulation and that remaining in the aqueous medium after separating the niosomal suspension by centrifugation at 15000 rpm for 1 h at 4 C using cooling centrifuge (Model 8880, Centurion Scientific Ltd., W. Sussex, UK). MTX contents were determined spectrophotometrically at a wavelength of 307 nm. Encapsulation efficiency percent was calculated using the equation: ðTotal amount of MTX added amount of free MTXÞ 100 EE% ¼ Total amount of MTX added (1)
2.5. Formulation optimization 1
The optimized formula was obtained using the Design Expert software by applying constraints on encapsulation efficiency percent of the niosomes to reach the maximum value and on particle size to obtain the smallest value. The suggested optimized formula was then prepared and evaluated in triplicate to check the validity of the calculated optimal formulation factors and predicted responses given by the software.
2.6. Transmission electron microscopy (TEM) The particle morphology of the optimized niosomal suspension was examined using TEM (H-600, Hitachi, Japan). The sample was dropped on copper–gold carbon grid and allowed to dry. The grid was then mounted in the instrument and photographs were taken at different magnifications (Bendas and Abdelbary, 2014).
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2.7. In-vivo evaluation of MTX loaded niosomes 2.7.1. HPLC determination of MTX An isocratic HPLC method was employed for the quantification of MTX (Begas et al., 2014). A Thermo Separation HPLC system (Fremont, California) equipped with a P4000 pump unit, an AS3000 autosampler including an injection valve with a sample loop of 50 mL volume, and a UV2000 detector was used. A Zorbax Extend-C18 column (4.6 mm 250 mm) containing 3.5 mm size adsorbent as stationary phase (Agilent technologies, Santa Clara, California) was used. The column was maintained at room temperature (25.0 2.0 C). The mobile phase consisted of a mixture of 50 mM sodium acetate buffer (pH 3.6) and acetonitrile (89:11 v/v). The flow rate and the UV detector were set at 1.0 mL/ min and 307 nm, respectively. The assay procedures were validated in terms of linearity, precision, and accuracy. 2.7.2. In-vivo skin deposition study The optimized niosomal suspension was selected for the invivo skin deposition study. The protocol of the study was approved by the ethical committee of Faculty of Pharmacy, Cairo University. Fifty-four male Wistar rats, weighing 150–200 g, were involved in the study. The animals were supplied with standard diet and tap water ad libitum. The rats were randomly divided into 3 groups with 18 animals each, where group I acted as control while animals in group II and group III received topical application of MTX solution (Unitrexate1) and optimized MTX niosomal formulation, respectively. Bottle caps that served as drug pools with an area of 4.91 cm2 were stuck to rat dorsal skin, which was shaved carefully to remove hair (Shen et al., 2014). Half mL of each of MTX formulations (MTX solution and niosomes) was added non occlusively into the drug pool. At the end of the experiment, the rats were sacrificed. One group (18 rats) was kept untreated (No application of solution or suspension in this group). After different time intervals of application of treatments (1, 2, 4, 6, 8 and 10 h), at each time point 3 animals from each group were sacrificed and the dorsal rat skin was excised and immediately washed 3 times with 5 mL of normal saline in each time. The excised skin (4.91 cm2) was cut into pieces and sonicated in 5 mL dimethyl sulfoxide (DMSO) for 30 min. The extract was then filtered through a 0.45 mm filter membrane and the concentration of MTX was determined by HPLC. These data were used to calculate the skin deposition of MTX generated by the range of formulations tested. The destruction of animal carcasses was achieved by incineration. 2.7.3. In-vivo histopathological study In- vivo histopathological study was conducted to assess the irritation potential and observe the ultrastructural changes in the skin upon exposure to MTX loaded niosomes. The rats were randomly divided into 3 groups with 4 animals each. Group I acted as control while animals in group II and group III received topical application of MTX solution (Unitrexate1) and optimized MTX niosomal suspension, respectively, onto the skin three times daily for a period of 1 week. The animals were then sacrificed and the skin was excised for histopathological investigation according to the protocol described by Bancroft et al. (Bancroft and Gamble, 2008). Briefly, autopsy samples were taken from the skin of rats in different groups and fixed in 10% formol saline for 24 h. Skin washing was done with tap water, and then, serial dilutions of alcohol (methyl, ethyl, and absolute ethyl) were used for dehydration. Specimens were cleared in xylene and embedded in paraffin at 56 C in hot air oven for 24 h. Sections from the paraffin blocks of 4 mm thickness were
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cut using a microtome (Leica Microsystems SM2400, Cambridge, England). The obtained tissue sections were collected on glass slides, deparaffinized, stained with hematoxylin and eosin and then examined under an electric light microscope (Morsi et al., 2014). 3. Results and discussion 3.1. Preparation of MTX loaded niosomes by thin film hydration technique Preliminary studies were performed to carefully select the most appropriate technique for preparing MTX loaded niosomes. Also, preliminary studies were carried out to choose the proper solvent for the drug, the most appropriate surfactant and the sonication time. The film hydration technique was adopted, where the drug can be incorporated either during film formation by dissolving the drug in an organic solvent or during film hydration by incorporating the drug in the hydration medium. The target was to incorporate 25 mg MTX in the niosomal suspension. Therefore, the solubility of MTX was tested in different organic solvent mixtures together with cholesterol and the studied non ionic surfactants. Acetone, chloroform, diethyl ether and methylene chloride failed to dissolve sufficient amount of MTX to begin the film formation procedure. On the other hand, a mixture of 30% dimethyl sulfoxide with any of the tested solvents produced the required solution. However, the drug precipitated and a non continuous film was produced during solvent evaporation. Alternatively, the approach of incorporating the drug in the hydration medium instead of incorporating it during film formation was used. The solubility of MTX is pH dependent with the solubility increasing above pH 4 (Vaidyanathan et al., 1985). Different buffers starting from pH 5.5 to 8 were tested and pH 8 provided the required solubility. This was advantageous for achieving our target as increasing the pH decreases the percutaneous absorption of MTX leading to higher deposition in the skin and lower systemic absorption (Vaidyanathan et al., 1985). Different surfactants were also tested and Span 60 was chosen as it produced the highest encapsulation efficiency percent and optimum particle size. Meanwhile, methanol, chloroform and diethyl ether were used as solvents for the film forming materials in 10 and 20 mL volumes. The solvent mixture that produced the clearest and most continuous film was the mixture of methanol and chloroform in a ratio of 1:1 using a total volume of 20 mL. Regarding the sonication time, the niosomes were prepared without sonication as well as sonicated for 1 and 2 min. The longer sonication led to smaller particle size. However, due to the high solubility of MTX in the phosphate buffer pH 8, the encapsulation
efficiency percent of the produced niosomes decreased significantly after sonication for 2 min. Therefore, the sonication time was fixed at 1 min. Moreover, the aim of our work was the topical delivery of MTX and avoidance of systemic absorption. It was reported that the large vesicles with a size 600 nm do not deliver their contents into deeper layers of the skin. Rather, these vesicles stay within the stratum corneum (Verma et al., 2003). Hence, the formulation of MTX loaded niosomes with particle size smaller than 600 nm was not advantageous to avoid penetration into deeper layers of skin and consequently a possibility of systemic absorption. However, the preliminary trials conducted in our study showed that the formulated Span 60 niosomes had a size range of 500–2000 nm. Hence, a constraint was applied on particle size to achieve the smallest value during formulation optimization based on the obtained size range in the preliminary trials. This was done using Design-Expert1 software, to obtain the formula with optimum particle size for skin deposition. 3.2. MTX loaded niosomes characterization 3.2.1. Effect of formulation variables on encapsulation efficiency percent The ability of niosomes to encapsulate significant amount of MTX is essential for its targeted use for management of psoriasis topically. Values of the encapsulation efficiency are presented in Table 3. Fig. 1 illustrates the response surface and cube plots for the effects of the concentration of MTX in hydration medium (X1), the total weight of niosomal components (X2) as well as Span 60: cholesterol ratio (X3) on the encapsulation efficiency percent. ANOVA test for the observed encapsulation efficiency percent data indicates that the linear model was significant and fitting for the data. The resulting equation in terms of coded values was as follows: EE% ¼ þ44:25 þ 25:86X 1 þ 16:08X 2 þ 12:05X 3 During the preparation of MTX loaded niosomes, the drug was introduced in the hydration medium. Therefore, MTX will probably be entrapped in the inner aqueous core of the vesicles. The equation reveals that there was a significant synergistic effect (p = 0.0001) of the concentration of MTX concentration on the percent encapsulation efficiency. A fixed amount of MTX was used in each preparation (25 mg) and the concentration was increased by decreasing the volume used to dissolve MTX. The increased amount of encapsulated MTX with increasing its concentration could be due to the saturation of the hydration medium with MTX that forces the drug to be encapsulated into niosomes (Balakrishnan et al., 2009; El-Samaligy et al., 2006; Mokhtar et al., 2008). Moreover, when comparing two formulae, considering that the same volume of the hydration was entrapped inside the vesicles,
Table 3 Encapsulation efficiency percent, particle size, polydispersity index and zeta potential of MTX loaded niosomes (n = 3). Formula
Encapsulation efficiency (% SD)
Particle size (nm SD)
PDI (values SD)
Zeta potential (mV SD)
F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13 F14 F15
12.52 4.57 52.73 5.01 46.36 8.18 79.00 4.37 1.98 2.80 66.25 1.75 4.47 6.32 74.26 1.45 1.42 0.32 42.13 10.12 50.81 0.55 78.66 5.64 53.32 1.25 54.50 1.45 45.30 10.44
558.70 16.54 1113.00 7.07 976.00 42.64 2200.00 164.75 584.80 2.97 1560.50 67.17 1280.00 63.64 1380.00 25.45 518.80 8.62 1865.00 56.73 1422.00 77.78 1375.00 44.66 1471.00 34.76 1214.25 53.03 1220.50 36.10
0.19 0.09 0.43 0.14 0.27 0.18 0.31 0.21 0.48 0.09 0.22 0.17 0.40 0.00 0.32 0.20 0.36 0.12 0.39 0.18 0.44 0.00 0.42 0.22 0.38 0.11 0.21 0.12 0.35 0.04
-63.90 1.41 -58.40 1.55 -58.85 0.07 -61.65 0.07 -46.35 5.58 -57.95 2.75 -55.40 0.07 -62.50 0.99 -54.55 2.33 -51.10 4.10 -60.30 0.42 -59.00 0.84 -60.55 1.48 -66.05 0.21 -61.45 0.21
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Fig. 1. (a) Response 3D plots and (b) cube plot for the effect of MTX concentration in hydration medium (X1), total weight of niosomal components (X2) and Span 60: cholesterol ratio (X3) on encapsulation efficiency percent.
then, the increased concentration of drug in this medium will mean that more amount of the drug will be entrapped inside the vesicles. In addition, the increase in hydration volume was reported to increase the drug leakage and consequently decrease percent encapsulation efficiency (Ruckmani and Sankar, 2010). The positive coefficient of the term, X2, indicates that the total weight of the niosomal components led to a significant synergistic effect on the EE% of the prepared niosomes (p = 0.0039). Higher amount of the film forming materials (niosomal components) increased the total lipid available for hydration (Abdelkader et al., 2010). This resulted in higher number of vesicles produced engulfing inside them more volume of the hydration medium and consequently higher amount of dissolved drug will be encapsulated. In other words, the increased percent encapsulation efficiency may be attributed to the increase in the availability of lipophilic ambience, which can accommodate the drug molecules to a higher extent (Agarwal et al., 2001). The increase in EE% with increase in total lipid content was also reported by Abdelkader et al., working on naltrexone niosomes (Abdelkader et al., 2010). Finally, the equation also reveals significant synergistic effect of the Span 60: cholesterol ratio on the percent encapsulation efficiency (p = 0.0196). Increasing Span 60 concentration resulted in a less permeable niosomal membrane and hence promoted the percent encapsulation efficiency (Zaki et al., 2014). The results also showed that decreasing cholesterol concentration led to a significant increase in EE%. This might be due to the fact that
the increase in cholesterol in certain cases may disrupt the regular linear structure of the vesicle membrane, and it does not encourage drug entrapment (Abdelbary, 2011; Al-Mahallawi et al., 2014; Moribe et al., 1999). 3.2.2. Effect of formulation variables on particle size and polydispersity index The particle size of the prepared MTX loaded niosomes is shown in Table 3. The PDI of all the niosomes formulations showed narrow size distribution and good homogeneity (Table 3). As previously mentioned, the very small size (less than 600 nm) was not required in our formulation process to avoid penetration into deeper layers of skin and systemic absorption. Rather, vesicles with a size 600 nm that do not deliver their contents into deeper layers of the skin and stay within the stratum corneum were beneficial for our aim (Verma et al., 2003). Fig. 2 illustrates the response surface and cube plots for the effects of the concentration of MTX in hydration medium (X1), the total weight of niosomal components (X2) as well as Span 60: cholesterol ratio (X3) on the particle size. ANOVA test for the observed particle size data indicates that the two factor interaction model was significant and fitting for the data. The resulting equation in terms of coded values was as follows: Particle size = +1248.89 + 356.75 X1 + 350.44 X2 + 115.99 X3 + 167.43 X1X2 218.93 X1X3 348.30 X2X3
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Fig. 2. (a) Response 3D plots and (b) cube plot for the effect of MTX concentration in hydration medium (X1), total weight of niosomal components (X2) and Span 60: cholesterol ratio (X3) on particle size.
The positive coefficient of the term, X1, indicates that MTX concentration had a synergistic effect on the particle size of the prepared niosomes (p < 0.0001). Increasing the concentration of MTX was achieved by decreasing the volume of the hydration medium. The lower volume of hydration medium may have led to incomplete or distorted formation of bilayer due to excess of material and low space. In order to accommodate the scarcity of water, the surface area of the produced vesicles was decreased and consequently the particle size increased (Sankhyan and Pawar, 2013). The ANOVA results also revealed the synergistic effect of the total weight of the niosomal components on the particle size (p < 0.0001). This may be attributed to the presence of large amount of film forming materials relative to the hydration medium. This may have resulted in multiple layers accumulating over each other and hence particle size increased. Furthermore, the presence of large amount of niosomal components leads to an increase in the number of lipid particles inside each vesicle which has a linear relation with the vesicle size (Wang and He, 2009). The coefficient of the third term, X3, shows that the Span 60: cholesterol ratio also had a synergistic effect on particle size (p = 0.0397). These results can be well correlated with the previously noticed increase in the percent encapsulation efficiency with increasing the Span 60: cholesterol ratio. The higher amount of MTX enclosed inside the vesicles might be the reason for the increase in the particle size. Moreover, the increase in particle size with increasing Span 60 concentration was also observed by Zaki et al. working on diacerein loaded niosomes (Zaki et al., 2014). 3.2.3. Effect of formulation variables on zeta potential Zeta potential is the measure of the overall charges acquired by vesicles and can be used to evaluate the stability of colloidal dispersions. In general, the system is considered stable when zeta potential value is around 30 mV due to electrical repulsion
between particles (Muller et al., 2001). The values of zeta potential for the prepared MTX loaded niosomes are presented in Table 3. Dicetyl phosphate (DCP), a negative charge inducer, was added to all formulae to increase the stability of the niosomal suspension and to avoid aggregation of the vesicles. The small differences in zeta potential absolute values of the prepared niosomes (all values > 30 mV) might be due to the presence of DCP with the same amount (5%) in all prepared niosomal formulations (Shatalebi et al., 2010). 3.3. Formulation optimization and analysis of the Box-Behnken (BB) design The BB design was used for designing and analysis of the experimental trials. BB requires much fewer experiments than a full-factorial design (Goyal et al., 2013). Adequate precision measured the signal to noise ratio to ensure that the model can be used to navigate the design space (de Lima et al., 2011). A ratio greater than 4 (the desirable value) was observed in both responses as shown in Table 4. On the other hand, Predicted R2 was calculated as a measure of how good the model predicts a response value (Chauhan and Gupta, 2004; Kaushik et al., 2006). The adjusted R2 and predicted R2 should be within approximately 0.20 of each other to be in reasonable agreement (Annadurai et al., 2008). If they are not, there might be a problem with either the data or the model. It is worthy to say that the predicted R2 values were in a reasonable agreement with the adjusted R2 in both responses. After applying constraints on the particle size and encapsulation efficiency percent (Table 1), the Design Expert1 software suggested an optimized formula to be prepared whose overall desirability was 0.673. The suggested formula (F12) had a MTX concentration of 7.5 mg/mL, total weight of niosomal components of 300 mg and Span 60: cholesterol ratio of 2:1. The suggested formula was prepared and evaluated, and the residual between the
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Table 4 Results of regression analysis for responses Y1 (EE%) and Y2 (PS).
Y1: EE% Y2: PS
Model
Adequate Precision
R2
Adjusted R2
Predicted R2
SD
% CV
p-value
Linear 2FI
13.008 18.491
0.8834 0.9529
0.7879 0.9176
0.6614 0.8205
12.49 133.73
28.23 10.71
0.0001
