Optimization of Fabrication Parameters to Prepare Tea Catechin-Loaded Liposomes using Response Surface Methodology

The purpose of this study was to optimize the formulation of tea catechin-loaded nano-liposomes using response surface methodology. Response surface methodology based on central composite rotatable design has been successfully used to model and optimize biochemical and biotechnological processes. The mass ratio of phosphatidylcholine and cholesterol (1-3), catechin concentration (3-5 mg/mL), pH values of phosphate buffer solution (6-7) and the volume ratio of organic phase and aqueous phase (2-4) were selected as independent variables with encapsulation efficiency and particle size as dependent variables. For each response, a second-order polynomial model was developed using multiple linear regression analysis. Applying a desirability function method the optimum parameters were: phosphatidylcholine to cholesterol mass ratio of 2.17, catechin concentration of 5 mg/mL, pH values of phosphate buffer solution of 6.62 and organic phase to aqueous phase volume ratio of 3.05. At this optimum point, particle size and encapsulation efficiency were found to be 220 nm and 60.18%, respectively. Furthermore, leakage ratio of nano-liposomes was used to determine the influence of storage period.


INTRODUCTION
Catechin, a major group of polyphenols extracted from green tea leaves, which exhibit a strong and powerful antioxidative, anti-obesity, hypolipidemic and anticarcinogenic activities and therapeutic potential in several chronic inflammatory diseases, including cancer (Anand et al., 2008;Middleton et al., 2000).However, its physicochemical properties generally result in poor chemical stability and lack of in vivo bioavailability (Cai et al., 2002;Dvorakova et al., 1999).
There has been considerable interest in liposomes (Rongen et al., 1997), as they may be used for protection in food and pharmacy system (Felnerova et al., 2004;Leserman, 2004;Torchilin, 2005).Liposomes are spherical vesicles with a diameter ranging from 20 nm to a few thousands nm, which are composed of a lipid bilayer with the hydrophobic chains of the lipids forming the bilayer and the polar head groups of the lipids orienting towards the extra vesicular solution and inner cavity (Edwards and Baeumner, 2006;Lorin et al., 2004).The liposomes enhance the stability of the encapsulated material by protecting them from the environment (Mozafari et al., 2008;Fathia et al., 2012).
Response Surface Methodology (RSM) is a collection of statistical and mathematical techniques by analyzing the response surface contour to find optimal process parameters and using multiple quadratic regression equation to fit between the factors and the response function.RSM is a useful technology in developing processes and optimizing their performance (Myers and Montgomery, 1995;Raissi, 2009).Besides, response surface methodology has been successfully used to model and optimize biochemical and biotechnological processes related to food (Liyana-Pathirana and Shahidi, 2005;Pompeu et al., 2009;Wang et al., 2007).
The main objective of this study was to evaluate effect of the mole ratio of phosphatidylcholine and cholesterol, catechin concentration (w/v), pH and the ratio of organic phase and aqueous phase (v/v) on the Encapsulation Efficiency (EE) and Particle Size (PS) and to find out the optimal conditions for preparing the catechin nano-liposomes using RSM.Furthermore, leakage ratio of nano-liposomes was used to determine the influence of storage period.

MATERIALS AND METHODS
Materials: Phosphatidylcholine (PC) was purchased from Beijing Shuangxuan Microbe Culture Medium Products Factory (Beijing, China).Cholesterol (CH) and Catechin were obtained from Shanghai Chemical Reagent Co. (Shanghai, China).Chloroform, diethyl ether was obtained from Hangzhou Jiachen Chemical Company.All chemicals were of reagent grade and used without further purification.
Preparation of liposomes: Catechin loaded liposomes were prepared by the reverse-phase evaporation (Szoka and Papahadjopoulos, 1978;Ming-Hui and Shi-Ying, 2007;Shah et al., 2012).Routinely, An appropriate amount of the lipid mixture of Soya Phosphatidylcholine (SPC) and Cholesterol (CS) was dissolved in a minimum amount of a mixed solvent of chloroform/ethanol (2:1, v/v) and then was dried to a thin film using a rotary evaporator under reduced pressure at a temp of 35°C and flask was rotated at 80 rpm.The residual solvent in the lipid film was removed by N 2 for 20 min and then was re-dissolved in ether.Then 3 mL of the aqueous phase solution containing phosphate-buffered saline (0.10 M, pH7, PBS) and catechin was added drop wise.The suspension was additionally sonicated in a bath-type sonicator for 10 min at 5°C.The mixture was again placed on a rotary evaporator and the organic solvent was removed under slightly reduced pressure, until the suspension became a gel, followed by continued evaporation under great vacuum.Eight mL of phosphate-buffered saline was added to the thin layer of round bottom flask to hydrate  ------------------------------------------------------- the layer and the suspension evaporated for an additional 25 min at 35°C to remove traces of solvent.
Particle size: The particle size was measured by Mastersizer 2000 instrument (Malvern) (Sadowski et al., 2008), equipped with HydroMu dispersing unit (Malvern).Measurements were taken in the range between 0.1 and 1000 μm, under the following conditions: water refractive index 1.33 and general calculation model for irregular particles.The data obtained were averaged by software.

Encapsulation Efficiency determination (EE):
The encapsulation efficiency was determined by centrifuge-UV method.Take lipsomes suspension (1.25mL) by spinning at 20000g for 1h using centrifuge, the catechin content of the supernatant was measured by UV spectrophotometry.The same suspension was ruptured using sufficient volume of ethanol and the total amount of Catechin was determined spectrophotometrically.Encapsulation efficiency was calculated using Eq. ( 1): where, Q f = The amount of free catechin Q t = The total amount of catechin present in 1.25 mL of nano-liposomes Stability analysis: Catechin nano-liposomes were stored at 4°C in a refrigeratory.Take 0.50 mL samples at predetermine intervals.The leakage ratios of samples were calculated.Leakage ratios were calculated using Eq. ( 2): where, W EE = Encapsulation of preparation = Encapsulation of a certain period of time Experimental design and optimization: RSM as a generic method for optimization was applied to optimize the formulation of catechin liposomes.Based on the preliminary experiments and our previous studies, four formulation parameters which included PC: CH, catechin concentration, pH and the ratio of organic phase and aqueous phase were identified as key factors responsible for EE and particle size.In view of the feasibility of liposome preparation, the ranges of the four factors were determined as follows: PC: CH (1-3), catechin concentration (3-5, w/v), pH (6-7) and organic phase: aqueous phase (2-4, v/v) (Table 1).The experimental runs for CCRD were shown in Table 2.
The response could be related to the selected variables by a second-order polynomial model.In this study, a second-order polynomial Eq. ( 2) was used to generate response surfaces: : The interaction coefficients (Zhang et al., 2007) Statistical significance of the terms in the regression equations was examined.The significant terms in the model were found by Analysis of Variance (ANOVA) for each response.The adequacy of model was checked accounting for R 2 and adjusted-R 2 .The desired goals for each variables and response were chosen.All the independents variables were kept within range while the responses were either maximized or minimized.

RESULTS AND DISCUSSION
Fitting the model: Table 2 showed the combined effects of phosphatidylcholine/cholesterol ratio, catechin concentration, pH and organic phase/aqueous phase ratio on PS and EE.The second-order polynomial         2.17, 62 and le 4).bjected ys.The mposed n of 5 phase shows, ded to increase with increasing storage period.This observation suggests that the leakage of catechin nanoliposomes might be attributed to hydrolyzation and degradation of bilayer membranes and/or vesicle fusion/aggregation (Flatena et al., 2008;Hincha, 2003;Wang et al., 2011).

CONCLUSION
The effect of cholesterol to phosphatidylcholine ratio, catechin concentration, pH and aqueous phase to organic phase ratio on preparing catechin nanolipsomes were studied.Second-order polynomial models were obtained for predicting particle size and encapsulation efficiency.While increasing the cholesterol to phosphatidylcholine ratio increased the particle size and encapsulation efficiency.Numerical optimization determined the optimum preparation conditions, which were phosphatidylcholine to cholesterol ratio of 2.17, catechin concentration of 5 mg/mL, pH of 6.62 and organic phase to aqueous phase ratio of 3.05.Furthermore, leakage ratio of nanoliposomes was tested for the period of 20 days.The catechin nano-liposomes showed an acceptable stability.

Table 1 :
Levels of factors used in CCRD

Table 2 :
Scheme of CCRD with the results of responses on four independent factors Independent variable

Table 3 :
ANOVA and regression coefficients of the second-order polynomial model for the response variables (actual values)