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The application of Plackett-Burman design and response surface methodology for optimization of formulation variables to produce Piroxicam niosomes

Ahmed M.S. Ahmed1,3, Mamdouh M. Ghourab2, Shadeed Gad2, and Mona K. E. Qushawy1*
  1. Department of Pharmaceutics, Faculty of Pharmacy and Pharmaceutical Industries, Sinai University, El-Arish, North Sinai, Egypt
  2. Department of Pharmaceutics, Faculty of Pharmacy, Suez Canal University, Ismailia, Egypt
  3. Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Al-Azhar University, Nasr City, Cairo, Egypt
Corresponding Author: Mona K. E. Qushawy , E-mail: monakamel88@yahoo.com
Date of Submission: 08-04-2013 Date of Acceptance: 01-05-2013 Conflict of Interest: NIL Source of Support: NONE
Citation: Ahmed M.S. Ahmed1,3, Mamdouh M. Ghourab2, Shadeed Gad 2, and Mona K. E. Qushawy1* “The application of Plackett-Burman design and response surface methodology for optimization of formulation variables to produce Piroxicam niosomes” Int. J. Drug Dev.& Res., April-June 2013, 5(2): 121-130. doi: doi number
Copyright: © 2013 IJDDR, Mona K. E. Qushawy et al. This is an open access paper distributed under the copyright agreement with Serials Publication, which
permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Abstract

The aim of the present study were (1) to screen seven independent variables (X) including; speed of rotation (X1), amount of chloroform (X2), HLB (X3), PIR amount in buffer (X4), sonication time (X5), total lipid amount (X6), and surfactant cholesterol ratio (X7) using Plackett-Burman design to identify which variables significantly affect the entrapment efficiency (EE%) and in-vitro release (Y) of PIR niosomes; and (2) to optimize the formulation by response surface methodology (RSM). PIR niosomes were prepared by lipid evaporation method and all vesicles were evaluated for their particle size, EE%, and in-vitro drug release. The niosomes appeared as round in shape and size ranged between 97.85 and 161.25nm. EE% was found to be between 20.23% and 37.13%. The release profiles of PIR niosomes occurred in two distinct phases, an initial phase for about 8 hours, followed by slow phase for at least 4 hours. The in-vitro study suggests that higher EE% was related with slow release. The release pattern shown by these formulations were Higuchi diffusion controlled mechanism. RSM showed that X3, X7, and X6 had the main effect on the responses (Y).

Keywords

PIR (PIR); niosomes; entrapment efficiency (EE%); Plackett-Burman design; response surface methodology (RSM); optimization.

INTRODUCTION

Niosomes or non-ionic surfactants vesicles are microscopic lamellar structures formed on the admixture of a non-ionic surfactant, cholesterol and phosphate with subsequent hydration in aqueous media (13). PIR is a non-steroidal anti-inflammatory drug that has anti-pyretic activity and has been used for the treatment of rheumatoid arthritis, osteoarthritis and traumatic contusions (1). Optimization of a formulation or process is finding the best possible composition or operating conditions. Some strategies are frequently used to achieve optimization such as Box-Behnken, centralcomposite, Plackett-Burman designs, etc.

MATERIAL & METHODS

Materials

PIR was provided by El-Mehan Drug Company, (Cairo,Egypt), Span 20 and Span 60 from Sigma Chemical Co., (Germany), Cholesterol from Sigma Chemical Co., (USA), Sodium hydroxide and Potassium dihydrogen phosphate, PureLab, Madison, USA, and Chloroform from Labscan Ltd, Dublin, (Ireland). All other chemicals used were of analytical grade.

Methods

Seven variables were screened using Plackett- Burman design and twelve formulae of PIR niosomes were obtained as shown in tables (1 and 2).
Niosomes were prepared by a lipid hydration method. Surfactant and cholesterol were taken in a round bottom flask and dissolved in chloroform. The solvent was evaporated using a rotary evaporator at different speed, under low pressure at 60°C. Niosomes were formed by adding phosphate buffered saline, PBS (pH 7.4) containing different amount of PIR slowly to the dried thin film formed on the walls of the flask, with gentle agitation. The resulting niosomal suspension was sonicated with probe sonicator then left to mature overnight at 4°C.

Entrapment efficiency of niosomes (EE%)

The unentraped drug was separated from the niosomal dispersions by centrifugation at 15,000 rpm for 45 min. The supernatant was separated, diluted to 100 ml with PBS pH 7.4, filtered using a membrane filter (0.45μm pore size), and measured using a spectrophotometer at 354 nm. EE% was calculated by the following equation:
EE%=[( Ct-Cr\Ct)] ×100%
Ct is the concentration of total PIR.
Cr is the concentration of free PIR.

In-vitro release of PIR

In-vitro release of PIR This study was carried out using a USP dissolution tester (Apparatus I). Niosomal suspension (5ml) was placed in cylindrical tubes (2.5cm in diameter and 6cm in length). Each tube is tightly covered with a molecular porous membrane from one end and attached to the shafts of the USP Dissolution apparatus, instead of the baskets, from the other end. The shafts were then lowered to the vessels containing 250 ml of PBS (pH 7.4) at 37±0.5 ºC, and 50 rpm. 5ml samples were withdrawn at time intervals of 1, 2, 3, 4, 6, 8, 10, and 12 hr. followed by replacement with fresh medium. The samples were analyzed spectrophotometrically at 354 nm. The obtained data were subjected to kinetic treatment according to zero, first, and Higuchi diffusion models (7).The correlation coefficient (r) was determined in each case.

Transmission electron microscopy (TEM)

A sample drop was diluted 10-fold using de-ionized water and a drop of this diluted dispersion was applied to a collodion-coated 300 mesh copper grid and left for 5 min to allow some of the niosomes to adhere to collodion. A drop of 2% aqueous solution of uranyl acetate was applied for 1 min. The sample was air dried and examined with TEM.

RSM for optimization of PIR niosomes

A second model used for optimization. The model is of the form:Y = A0+A1X1+A2X2+A3X3+A4X4+ -------- +AnXn where Y is the response, A0 is a constant and A1–An are the regression coefficients; X1, X2 and X3 are the independent variables.

RESULTS AND DISCUSSION

Plackett-Burman design for the screening of PIR niosomes

The purpose of this first step of the optimization was to identify which variables significantly affect niosomes preparation. Seven variables were screened as shown in table (1) and twelve different formulae of PIR niosomes were obtained as was shown in table (2).

EE% of PIR niosomes (Y1)

As illustrated in figure (1), EE% was ranged between 20.23% for F6 and 37.13% for F10. Figure (2) show the effect of the different independent variables on EE% of PIR using STATGRAPHIC plus computer program. EE% decreased from 29.32 to 29.28% by increasing (X1). EE% decreased from 29.49 to 29.11% when (X2) was increased. Increasing (X3) lead to decrease in EE% from 32.87 to 25.73%. This can be explained by;
(a) Span 60 has the highest phase transition temperature (Tºc). Surfactants of higher (Tºc) are more likely in the ordered gel form forming less leaky bilayers, thus having higher EE%, while surfactants of lower (Tºc) are more likely in the less ordered liquid form(11).
(b) Increasing the alkyl chain length is leading to higher EE% (6). EE% followed the trend Span 60 > Span 40 > Span 20 > Span 80 (8).
Increasing (X4) form 10 to 20 mg, EE% was decreased from 29.88 to 28.72%. As EE% depends upon the amount of the aqueous phase enveloped in the niosomal vesicles during preparation, the result that EE% slightly changed with increasing drug concentration suggests that the enveloped aqueous phase was the same irrespective of drug concentration (12). Increasing the vinpocetine concentration led to significant decrease in EE% (4).
EE% decreased from 30.16 to 28.44% by increasing (X5). The sonication of 5- Fluorouracil loaded bolaniosomes elicited a reduction of the carrier loading capacity (11). This might be attributed to the increase in the number of formed niosomes and consequently the volume of the hydrophobic bilayer domain, the available housing for entrapment hydrophobic drug (16).
EE% increased from 27.96 to 30.64 % when (X6) was increased. In aceclofenac niosomes, as the total lipid concentration increased, drug EE% increased (15). Increasing (X7) lead to decrease in EE% from 32.11 to 26.49%. Niosomes which gave the best dispersibility and the highest EE% were those from the mixture of Tween61 and cholesterol at 1:1 molar ratio (9).

In-vitro release of PIR

Figures (3-5) showed the release profile of PIR niosomes. It was found that there was an inversely proportional relationship between EE% and in-vitro release.
Figure (6) show the effect of independent variables on in-vitro release of PIR-entrapped niosomes after one hour (Y2). Increasing (X1) lead to decrease in (Y2) from 30.29 to 30.16%. (Y2) increased from 30.18 to 30.27% by increasing (X2). Increasing (X3) lead to increase in (Y2) from 26.02 to 34.43%. (Y2) increased from 29.97 to 30.48% by increasing (X4). Increasing (X5) lead to increase in (Y2) from 29.49 to 30.96%. (Y2) decreased from 31.43 to 29.02% by increasing (X6). Increasing (X7) lead to increase in (Y2) from 27.22 to 33.22%.
Figure (7) show the effect of independent variables on in-vitro release of PIR -entrapped noisome after six hour (Y3). Increasing (X1) lead to increase in (Y3) from 65.29 to 66.24%. (Y3) decreased from 67.14 to 64.39% by increasing (X2). Increasing (X3) lead to increase in (Y3) from 62.69 to 68.83%. (Y3) increased from 64.25 to 67.28% by increasing (X4). Increasing (X5) lead to decrease in (Y3) from 66.30 to 65.20%. (Y3) decreased from 66.53 to 64.99% by increasing (X6). Increasing (X7) lead to increase in (Y3) from 63.00 to 68.50%.
Figure (8) illustrated main effect of independent variables on in-vitro release after twelve hour (Y4). Increasing (X1) lead to increase in (Y4) from 91.40 to 91.47%. (Y4) decreased from 92.15 to 90.72% by increasing (X2). Increasing (X3) lead to increase in (Y4) from 89.90 to 92.97%. (Y4) increased from 91.37 to 91.51% by increasing (X4). Increasing (X5) lead to increase in (Y4) from 91.41 to 91.46 %. (Y4) decreased from 92.77 to 90.11% by increasing (X6). Increasing (X7) lead to increase in (Y4) from 90.01 to 92.86%.
(X1) and (X2) have a negligible effect on the in-vitro release of PIR niosomes.
The rate of release was decreased by increasing HLB (X3). This can be explained by;
(a)The higher phase transition temperature (T°c) of Span 60 leads to the formation of vesicles with less permeable and less leaky rigid bilayers.
(b) Span 60 possesses a higher alkyl chain lenghth than smaller side chain length (C12) of span 20. The higher the chain length of Span, the lower the release rate (3).
The rate of release was decreased by increasing drug (X4). This may be attributed to that niosomal formulations could enhance the solubility of certain poorly soluble drugs but to a maximum limit after which any increase in the drug concentration leads to drug precipitation. The drug crystals dispersed inbetween the niosomal pellets were observed under the optical microscope, when higher amount of drug was used (2).
The rate of release was increased by increasing (X5). This may be attributed to that a reduced mean sized of noisome can be an important parameter to improve the biopharmaceutical properties.
The sonication elicited a reduction of the polydispersity index up to a value of 0.1 so the release profile of 5-FU showing a first phase (0–10 h) of faster 5-FU release rate followed by a second phase of slower release (12).
The rate of release was decreased as (X6) increased. This may be attributed to that by increasing cholesterol, the bilayer hydrophobicity increased and permeability decreased. The incorporation of cholesterol into the lipid bilayers modified the membrane fluidity by decreasing the movement of the mobile hydrocarbon chains of non-ionic surfactant leading to loss of bilayer permeability (13). The rate of release was increased as (X7) increased. This might be attributed to the increase in the number of formed niosomes. The increase of surfactant cholesterol molar ratio markedly increased the release of the drug (5).
As shown in table (3) the release of PIR from all prepared niosomal preparations followed Higuchi's diffusion model which showed higher correlation coefficient values. The diffusional release is observed for triton niosomes (10).

Characterization of niosomes

As shown in figure (9) niosomes appeared as spherical nano vesicles with particle size from 97.85 to 161.25nm as showen in table (4).
According to the particle size results, an increase in HLB (X3) increases the vesicle size and hence decreasing EE%. Also an increase in (X6) decreases the vesicle size, and hence increasing EE%. The relationship between niosome size and Span hydrophobicity has been attributed to the decrease in surface energy with increasing hydrophobicity resulting in the smaller vesicle (14).

Response surface methodology

Figures (10-13) showed the response surface plots which displayed the effect of independent variables (X) on Y1, Y2, Y3, and Y4 respectively. It was obvious that X3, X7, and X6 had the main effect on the responses (Y).Table (5) showed the combination of the independent factor levels which gave the optimized formula of PIR niosomes

CONCLUSION

Plackett-Burman design was successful in identifying that X3, X7, and X6 had the main effect on the responses (Y) of PIR niosomes; which in turn can be chosen for further and preparing an optimized formula.

Tables at a glance

Table icon Table icon Table icon Table icon Table icon
Table 1 Table 2 Table 3 Table 4 Table 5
 

Figures at a glance

Figure 1 Figure 2 Figure 3 Figure 4 Figure 5
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