Accepted Manuscript

Process optimization by use of design of experiments: Application

for liposomalization of FK506

Hiroyasu Toyota, Tomohiro Asai, Naoto Oku

PII: S0928-0987(17)30140-9
DOI: doi: 10.1016/j.ejps.2017.03.007
Reference: PHASCI 3947
To appear in: European Journal of Pharmaceutical Sciences
Received date: 14 December 2016
Revised date: 28 February 2017
Accepted date: 5 March 2017

Please cite this article as: Hiroyasu Toyota, Tomohiro Asai, Naoto Oku , Process
optimization by use of design of experiments: Application for liposomalization of FK506.
The address for the corresponding author was captured as affiliation for all authors. Please
check if appropriate. Phasci(2017), doi: 10.1016/j.ejps.2017.03.007

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 ACCEPTED MANUSCRIPT

Title:

Process optimization by use of design of experiments: Application for liposomalization of FK506.

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Author names and affiliations:

Hiroyasu Toyota,a,b Tomohiro Asai,a and Naoto Okua

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a
Department of Medical Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka,
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52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan
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b
Pharmaceutical Research and Technology Labs., Technology, Astellas Pharma Inc., 180 Ozumi,

Yaizu, Shizuoka 425-0072, Japan

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Corresponding author:
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Naoto Oku
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Department of Medical Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka,

52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan

oku@u-shizuoka-ken.ac.jp

Tel.: + 81 54 264 5701; fax: + 81 54 264 5705

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Abstract

Design of experiments (DoE) can accelerate the optimization of drug formulations, especially

complexed formulas such as those of drugs, using delivery systems. Administration of FK506

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encapsulated in liposomes (FK506 liposomes) is an effective approach to treat acute stroke in animal

studies. To provide FK506 liposomes as a brain protective agent, it is necessary to manufacture these

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liposomes with good reproducibility. The objective of this study was to confirm the usefulness of

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DoE for the process-optimization study of FK506 liposomes. The Box-Behnken design was used to
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evaluate the effect of the process parameters on the properties of FK506 liposomes. The results of
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multiple regression analysis showed that there was interaction between the hydration temperature

and the freeze-thaw cycle on both the particle size and encapsulation efficiency. An increase in the
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PBS hydration volume resulted in an increase in encapsulation efficiency. Process parameters had no
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effect on the -potential. The multiple regression equation showed good predictability of the particle
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size and the encapsulation efficiency. These results indicated that manufacturing conditions must be
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taken into consideration to prepare liposomes with desirable properties. DoE would thus be

promising approach to optimize the conditions for the manufacturing of liposomes.

Keywords: Liposomes; Design of experiments; Box-Behnken design; Process optimization

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Abbreviations:

ANN, artificial neural networks; ANOVA, analysis of variance; DoE, design of experiments; DPPC,

dipalmitoylphosphatidylcholine; DSPE, distearoylphosphatidylethanolamine; FK506 liposomes,

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polyethylene glycol-modified liposomes encapsulating FK506; LUV, large unilamellar vesicles;

MLR, multiple linear regression; MLV, multilamellar vesicles; ODS, octadecylsilane; PCR, principal

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component regression; PEG, polyethylene glycol; PLSR, partial least square regression; s.e.m,

standard error of mean; SUV, small unilamellar vesicles

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1. Introduction

Liposomes are biodegradable vesicular structures composed of a lipid bilayer and are used as a drug

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delivery system (Bangham and Horne, 1964; Gregoriadis and Florence, 1993). Liposomes have been

used to improve therapeutic efficiency and to decrease the adverse side effects of drugs. These

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effects are manifested because liposomalization changes the pharmacokinetics of drugs (Samad et al.,

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2007). Polyethylene glycol (PEG) modification and conjugation of targeting ligands to the surface of
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liposomes are used for further improvement of therapeutic efficacy (Blume and Cevc,1990; Blume et
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al., 1993).

FK506 is an immunosuppressant and mainly used to prevent rejection of organ transplants. FK506
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binds to FK506-binding protein, and this complex interacts with calcineurin to inhibit the production
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of inflammatory cytokines (Liu et al., 1991). It was reported that treatment with FK506 has positive
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effect on acute stroke in animal studies. Even though a high dose of FK506 induces adverse side
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effects, polyethylene glycol-modified liposomes encapsulating FK506 (FK506 liposomes) show

therapeutic efficacy at a lower dose compared with free FK506 (Ishii et al., 2013).

The thin-film method is used for manufacturing FK506 liposomes. This method consists of the

following processes: dissolution, evaporation, hydration, and extrusion (Samad et al., 2007). The

International Conference on Harmonization Q8 guideline has recommended gaining information and

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knowledge of drug products from pharmaceutical development studies to support manufacturing

controls. One of the disadvantages of the usage of liposomes is the batch-to-batch variation (Muthu

et al., 2014). In order to manufacture FK506 liposomes with desirable properties, it is necessary to

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confirm the relationship between manufacturing conditions and drug product quality and then

establish the optimal manufacturing conditions.

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In order to optimize manufacturing conditions, design of experiments (DoE) is used in many areas

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(Singh et al., 2005b). In DoE, at first a study design is prepared to collect appropriate data and then
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the data obtained from the experiments is analyzed by using a statistical method to extract the
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maximum amount of information (Singh et al., 2005a). The most common experimental design is the

full-factorial design. In this design, all combinations of variables are examined. When a two-level
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full-factorial design is selected, the number of runs is given by 2k, where k is the number of the
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variables. This full-factorial design is not useful when the number of the variables is increased
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because the number of runs goes on increasing exponentially (Adenso-Diaz and Laguna, 2006).
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Instead of the full-factorial design, the fractional-factorial design or Box-Behnken design is used in

DoE. The fractional-factorial design is able to examine the effects of a large number of variables

with relatively few experimental runs by ignoring interaction effects (Gunst and Mason, 2009),

whereas the Box-Behnken design is used for optimization studies. This design is suitable to create a

quadratic model. A three-factor Box-Behnken design is almost rotatable, which means that all design

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points are at the same distance from the center of the design. This property is preferable to create a

response surface plot, because the prediction error is the same for all design points (Box and

Behnken, 1960). The number of runs required for this Box-Behnken design is given by 2k(k-1)+C0,

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where k is the number of variables and C0 is the number of center points. This design is more

efficient compared with the three-level full-factorial design (Ferreira et al., 2007). Even though DoE

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is a useful tool for process optimization, few studies using DoE have been reported regarding

process optimization for liposomes (Singh et al., 2005b).

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In this present study, DoE was used to confirm the relationship between the process parameters of
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the preparation process and the properties of FK506 liposomes. Prediction models for the properties

of FK506 liposomes were generated by DoE and were validated to confirm the usefulness of DoE.
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This study should help to establish optimal conditions for the manufacturing of liposomes.
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2. Materials and methods

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2.1. Materials

Dipalmitoylphosphatidylcholine (DPPC) and distearoylphosphatidylethanolamine (DSPE)-PEG

(molecular weight of PEG: 2000) were gifts from Nippon Fine Chemical Co., Ltd. (Hyogo, Japan).

FK506 was obtained from Astellas Pharma Inc. (Tokyo, Japan). Chloroform, methanol, and

tert-butanol were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan).

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2.2. Design of Experiment

The three-factor Box-Behnken design was used to generate a quadratic model. In this design, all

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factors have 3 levels: low, center, and high. Three center samples were included in this design and

used as a source for error estimation. The quadratic model is calculated by using multiple regression

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analysis, and the model is described by the following formula:

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where Y is the response, X is a variable, and a is a regression coefficient. Analysis of variance
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(ANOVA) was conducted to identify the statistically significant term of the model. Response surface

plots were generated by using this model.

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2.3. Preparation of Liposomes

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FK506 liposomes were composed of FK506, DPPC, and DSEP-PEG. DPPC (20.0 mol) and
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DSEP-PEG (1.0 mol) were dissolved in chloroform, whereas FK506 (1.0 mol) was dissolved in

methanol. The lipid solutions, FK506 solution, and tert-butanol were mixed in a flask. After mixing,

the solution was evaporated to obtain a thin lipid film. This film was then hydrated with PBS (pH

7.4) and freeze-thawed with liquid nitrogen. The liposomes were sized by 5 extrusions through

100-nm pore-size polycarbonate filters (Nuclepore, Cambridge, MA, USA). Unencapsulated FK506

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was removed by ultracentrifugation at 604,000g for 15 min (CS120GXL, Hitachi, Tokyo, Japan),

and the obtained liposomes were suspended in 2 mL of PBS.

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2.4. Determination of FK506 amount encapsulated in liposomes

FK506 was measured by HPLC (Hitachi, Tokyo, Japan) as described earlier (Ishii et al., 2013).

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FK506 liposomes were dissolved in tetrahydrofuran, and 20 l of the solution was applied to an

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octadecylsilane (ODS) column (TSK gel ODS-80TM, 4.6150 mm, Tosoh Corporation, Tokyo,
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Japan). The mobile phase of acetonitrile:water (60:40, v/v), the flow rate of 1 mL/min, column
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temperature of 60C, and UV detection at 214 nm were used. This method has been validated. The

FK506 peak was well separated from placebo peaks. The linearity was confirmed within the range
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100300 g/mL and correlation coefficient was 1.00. The average recovery was 100.2 % with RSD
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1.2% (n=5). The sample solution and standard solution were stable at room temperature for 24 h.
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2.5. Particle size and -potential measurement

The particle size and the -potential of FK506 liposomes were determined by using a Zeta Sizer

Nano ZS (Malvern, Worcestershire, UK). To measure the particle size and the -potential, 10 L of

liposomes was diluted to 1 mL with PBS.

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2.6. Statistical analysis

The Unscrambler (CAMO Software, NJ, USA) was used for multiple regression analysis, ANOVA,

and response surface methodology.

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3. Results

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The Box-Behnken design was used to optimize the hydration process. This design is suitable for

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second-order models. The manufacturing conditions and measurement results are shown in Table 1.
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The properties of FK506 liposomes were affected by some of the process parameters.
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3.1. Particle size

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The effects of process parameters on the particle size of FK506 liposomes are shown in Figure 1. An
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increase in the number of freeze-thaw cycles resulted in decreased particle size. The results of
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multiple regression analysis are shown in Table 2. Non-significant terms (p>0.05) were removed in a
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hierarchical manner. The following formula was used for this model:

(1)

where Y1 is particle size, X2 is hydration temperature, and X3 is number of freeze-thaw cycles. This

model was statistically significant, and the interaction of the hydration temperature with the

freeze-thaw cycle was a significant term in this model. To confirm the relationship between the

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particle size and the process parameters, we prepared a response surface plot for the particle size of

the FK506 liposomes (Figure 2). At a high hydration temperature, the particle size was decreased

when the number of freeze-thaw cycles was increased. In contrast, variation of the particle size was

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small at a low hydration temperature. The theoretical values calculated from this model were

compared with the observed values. As shown in Table 3, some degree of correlation was observed.

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3.2. -potential
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The effects of process parameters on the -potential of FK506 liposomes are shown in Figure 3. No
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effects of the process parameters on the -potential were observed. From the results of multiple

regression analysis, the model was not statistically significant (p>0.05). This result also indicated
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that the process parameters did not affect the -potential of FK506 liposomes.
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3.3. Encapsulation efficiency

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The effects of the process parameters on the encapsulation efficiency of FK506 liposomes are shown

in Figure 4. Some process parameters affected this efficiency. The results of multiple regression

analysis are shown in Table 4. The following formula was used for this model:

(2)

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where Y2 is encapsulation efficiency, X1 is PBS volume, X2 is hydration temperature, and X3 is

number of freeze-thaw cycles. Main effects, interaction, and quadratic terms had statistically

significant effects on the encapsulation efficiency of FK506 liposomes. To show the interaction

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between the hydration temperature and the number of freeze-thaw cycles, we prepared the response

surface plot for encapsulation efficiency of FK506 liposomes (Figure 5). An increase in the number

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of freeze-thaw cycles resulted in a decrease in encapsulation efficiency. When the hydration

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temperature was high, the freeze-thaw cycle had a large influence on the encapsulation efficiency.
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The theoretical values calculated from this model were compared with the observed values, and a
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good prediction was observed (Table 5).

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3.4. Confirmation study

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In order to confirm the predictive ability of the model, we conducted a confirmation study. Predicted
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values were calculated by using the above equations 1 and 2. The predicted values and observed
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values for the manufacturing conditions are shown in Table 6. The predicted values were close to the

measured ones, thus indicating that the predictive ability of the model was high.

4. Discussion

In this study, we confirmed that the process parameters had effects on the particle size and

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encapsulation efficiency of FK506 liposomes. In contrast, there was no change in the -potential of

the FK506 liposomes.

The particle size of liposomes is an important property for the pharmacokinetics of liposomes (Allen

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and Everest, 1983). In general, the extrusion process is an important process for the particle size of

liposomes, and the particle size is adjusted by the pore size of the filters and the extrusion-cycle

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number. The multilamellar vesicles (MLV) obtained in the hydration process are sized in the

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extrusion process, and the small unilamellar vesicles (SUV) or the large unilamellar vesicles (LUV)
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are obtained by extrusion (Mayer et al., 1986; Berger et al., 2001). Our results indicated that the
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hydration process also affected the particle size of the FK506 liposomes. There was interaction

between the hydration temperature and the number of freeze-thaw cycles on the particle size of
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FK506 liposomes (Figure 2). It was earlier reported that the particle size and the lamellarity of the
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liposomes decrease as the number of freeze-thaw cycles is increased (Trakia et al., 2000). Indeed,
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freeze-thaw cycle affected particle size before extrusion process (Figure S1 in the Supplementary
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material). Therefore process parameters affected the particle size of MLV before extrusion and

thereby caused variation in the particle size after extrusion. The response surface plot showed that

the particle size increased with an increase in the hydration temperature when the number of

freeze-thaw cycles was small. High temperature is known to induce fusion of liposomes (Ellens et al.,

1989). Fusion of FK506 liposomes at high temperature would result in an increased particle size.

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Comparison of the predicted and observed values of the particle size is shown in Table 3, and some

degree of correlation was observed. Although this model was statistically significant, variation in the

particle size with respect to the manufacturing conditions was small. We assume that the particle size

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of the liposomes was mainly adjusted by the extrusion process and that the prediction model

considering the hydration and extrusion processes is necessary to prepare liposomes of desirable

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particle size.

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The surface charge of liposomes is also an important property for the pharmacokinetics of liposomes
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(Webb et al.,1998). In addition, the surface charge affects the stability of liposomes, because the
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proper surface charge prevents the phenomenon of liposome aggregation (Nakamori et al., 1993).

Regarding the -potential, the process parameters had no effect on it. The formulation of liposomes
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is an important factor for the -potential of liposomes, and the amount of cationic and anionic lipids
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decide the -potential of liposomes (Soema et al., 2015; Taylor et al., 2007). Therefore, there was no
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critical process step with respect to the -potential of the FK506 liposomes.
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The encapsulation efficiency is the ratio of the amount of drug entrapped in liposomes to the amount

of the drug presented. Therapeutic effects depend on the amount of drugs entrapped by liposomes

(Mainardes and Silva, 2004). The properties of encapsulated drugs and liposomes are important

factors for encapsulation efficiency. Hydrophilic drugs are trapped in the aqueous part of liposomes;

and lipophilic drugs, in the liposomal membrane. The log Poctanol/water of drugs correlates with the

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encapsulation efficiency (Nii and Ishii, 2005). In term of liposome properties, the aqueous volume,

particle size, formulation, and preparation method of liposomes all affect the encapsulation

efficiency (Kulkarni et al., 1995). FK506 is a lipophilic drug and is thus considered to be entrapped

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by the membrane of liposomes (Jan, D., 2003). Therefore, the hydration process is considered to be a

critical step affecting encapsulation efficiency. Regarding manufacturing conditions, hydration time,

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volume of water used for hydration, and agitation method are important factors for encapsulation

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efficiency (Szoka and Papahadjopoulos, 1980). In our present study, the encapsulation efficiency
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was found to correlate with the hydration process of the manufacturing conditions (Table 4) and the
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PBS volume had a positive effect on encapsulation efficiency (Figure 4). There is dead volume in

manufacturing equipment, which affects the yield of liposomes (Jousma et al., 1987; Berger et al.,
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2001). A high volume of PBS would decrease the loss of FK506 liposomes in the manufacturing of
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liposomes and increase encapsulation efficiency. The response surface plot of encapsulation
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efficiency showed that there was interaction between the hydration temperature and the number of
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freeze-thaw cycles with regards to the encapsulation efficiency (Figure 5). Phospholipids dispersed

in an aqueous solution have a phase-transition temperature. Above this temperature, the assembly of

phospholipids changes from gel to liquid crystalline phase. An increase in the hydration temperature

enhances the fluidity of the liposomal membrane and thus improves encapsulation efficiency (Ma et

al., 1991). This same tendency was observed when the number of freeze-thaw cycles was small. In

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contrast, the response surface plot showed that encapsulation efficiency decreased with an increase

in the hydration temperature and number of freeze-thaw cycles. Freezing and thawing at high

temperature did not induce degradation of FK506 and process parameters did not affect the assay

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value before extrusion process (Figure S2 in the Supplementary material). Freezing and thawing of

liposomes changes the structure of liposomes (Trakia et al., 2000) and therefore repetition of the

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freeze-thaw cycle at high temperature might induce leakage of FK506 from the membrane of the

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liposomes and free FK506 would be trapped by the filters during extrusion process.
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There are some methods to calculate the model from the data. Multiple linear regression (MLR),
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principal component regression (PCR), partial least square regression (PLSR) or artificial neural

networks (ANN) have been proposed as multivariate analysis method (Cheng and Sun, 2015). In this
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study, the prediction model was calculated using MLR based on the data of DoE. MLR is the
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simplest method for model creation, but the model would be unstable when there is collinearity
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between variables (Cheng and Sun, 2015; Balabin et al., 2007). Since Box-Behnken designs is
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orthogonal, the MLR is stable and collinearity does not affect the model (Yetilmezsoy et al., 2009).

In order to confirm the relationship between process parameters and properties of FK506 liposomes,

MLR would be suitable because it is easy to interpret the regression equation.

DoE is often used for pharmaceutical development studies on drug products (Singh et al., 2005b). By

conducting a confirmation study, we confirmed a high predictive ability of the model produced by

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MLR (Table 6). If the full-factorial design had been applied in this study, 27 runs would have been

needed. In contrast, the Box-Behnken design required only 15 runs for this study. As such, DoE is a

useful tool for optimizing the manufacturing conditions of not only oral drug products but also

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liposomes or other lipid complexes.

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5. Conclusions

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The manufacturing condition of the hydration process affected the particle size and encapsulation
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efficiency of FK506 liposomes. DoE is a useful tool to understand the relationship between process
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parameters and quality of FK506 liposomes. The derived multiple regression equation helps in

preparing FK506 liposomes with desirable properties.

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Acknowledgements
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The authors wish to thank Astellas Pharma Inc. (Tokyo, Japan) for the gift of the FK506 and the use
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of The Unscrambler.

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Szoka Jr, F., Papahadjopoulos, D., 1980. Comparative properties and methods of preparation of lipid

vesicles (liposomes). Annu. Rev. Biophys. Bioeng. 9, 467-508.

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Taylor, T. M., Gaysinsky, S., Davidson, P. M., Bruce, B. D., Weiss, J., 2007. Characterization of

antimicrobial-bearing liposomes by -potential, vesicle size, and encapsulation efficiency. Food.

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Biophys. 2, 1-9.

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Trakia, M., Warschawski, D. E., Recouvreur, M., Cartaud, J., Devaux, P. F., 2000. Formation of

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unilamellar vesicles by repetitive freeze-thaw cycles: characterization by electron microscopy and
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P-nuclear magnetic resonance. Eur. Biophys. J. 29, 184-195.
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Webb, M. S., Saxon, D., Wong, F. M., Lim, H. J., Wang, Z., Bally, M. B., Mayer, L. D., 1998.
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Comparison of different hydrophobic anchors conjugated to poly (ethylene glycol): effects on the
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pharmacokinetics of liposomal vincristine. Biochim. Biophys. Acta, Biomembr. 1372, 272-282.

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Yetilmezsoy, K., Demirel, S. and Vanderbei, R. J., 2009. Response surface modeling of Pb (II)

removal from aqueous solution by Pistacia vera L.: BoxBehnken experimental design. J. Hazard.

Mater. 171, 551-562.

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Table 1 Experimental design and observed results of the optimization study.

Hydration Process
Percent
Number of Particle size -potential
No. PBS volume Hydration entrapped
freeze-thaw (nm) (mV)
(mL) temperature (C) FK506 (%)
cycles
1 1 45 3 107.1 -4.32 44.2

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2 1 55 3 102.9 -4.04 45.3
3 5 45 3 104.7 -4.26 57.8

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4 5 55 3 107.9 -3.20 55.7
5 1 50 1 113.1 -3.86 41.1

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6 1 50 5 102.0 -7.46 36.0
7 5 50 1 107.9 -4.51 53.6
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8 5 50 5 108.0 -4.48 51.9
9 3 45 1 100.1 -6.29 53.4
10 3 45 5 106.1 -3.89 55.0
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11 3 55 1 113.3 -3.04 58.3

12 3 55 5 101.0 -4.58 50.7
13 3 50 3 104.3 -3.40 57.0
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14 3 50 3 112.0 -3.17 56.7

15 3 50 3 104.1 -4.23 54.5
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Table 2 Results of ANOVA for the particle size of FK506 liposomes.

Input variables Sum of squares Degree of freedom P value

a a
PBS volume - - -a
Hydration temperature 6.301 1 0.458
Freeze-thaw cycle 37.411 1 0.087
a a
PBS volume Hydration temperature - - -a

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Hydration temperature Freeze-thaw cycle 83.723 1 0.017*
a a
PBS volume Freeze-thaw cycle - - -a

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Hydration temperature Hydration temperature -a -a -a

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Freeze-thaw cycle Freeze-thaw cycle -a -a -a
Model 127.435 3 0.038*
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Error 116.965 11
Adjusted total 244.400 14

*p <0.05
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Table 3 Observed and predicted particle sizes of FK506 liposomes.

No. Observed (nm) Predicted (nm) Residuals

1 107.1 105.4 1.7
2 102.9 107.2 -4.3
3 104.7 105.4 -0.7
4 107.9 107.2 0.7

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5 113.1 108.5 4.6
6 102.0 104.1 -2.1

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7 107.9 108.5 -0.5
8 108.0 104.1 3.8

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9 100.1 103.0 -2.9
10 106.1 107.8 -1.7
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11 113.3 113.9 -0.6
12 101.0 100.5 0.6
13 104.3 106.3 -2.0
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14 112.0 106.3 5.7

15 104.1 106.3 -2.2
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Table 4 Results of ANOVA for encapsulation efficiency of FK506 liposomes.

Input variables Sum of squares Degree of freedom P value

PBS volume 343.220 1 0.000**
Hydration temperature 0.020 1 0.917
Freeze-thaw cycle 20.480 1 0.011*
a a
PBS volume Hydration temperature - - -a

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Hydration temperature Freeze-thaw cycle 21.160 1 0.010*
a a
PBS volume Freeze-thaw cycle - - -a

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Hydration temperature Hydration temperature 10.566 1 0.042*

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Freeze-thaw cycle Freeze-thaw cycle 42.893 1 0.002**
Model 618.961 7 0.000**
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Error 11.997 7
Adjusted total 630.957 14

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Table 5 Observed and predicted encapsulation efficiencies of FK506 liposomes.

No. Observed (%) Predicted (%) Residuals

1 44.2 44.3 -0.1
2 45.3 44.2 1.1
3 57.8 57.4 0.4
4 55.7 57.3 -1.6

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5 41.1 40.7 0.4
6 36.0 37.5 -1.5

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7 53.6 53.8 -0.2
8 51.9 50.6 1.3

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9 53.4 53.7 -0.3
10 55.0 55.1 -0.1
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11 58.3 58.2 0.1
12 50.7 50.4 0.3
13 57.0 56.1 0.9
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14 56.7 56.1 0.6

15 54.5 56.1 -1.6
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Table 6 Experimental design and observed results of the confirmation study.

PBS Hydration Number of Percent entrapped

Particle size (nm)
No. volume temperature freeze-thaw FK506 (%)
(mL) (C) cycles Predicted Observed Predicted Observed
1 3 47.5 4 55.4 53.3 105.9 104.8
2 2 50.0 2 51.0 51.2 107.4 102.4

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Figure legends

Figure 1 Factorial effects on the particle size of FK506 liposomes. (A) Effects of the PBS volume

at 1 mL (n=4), 3 mL (n=3) and 5 mL (n=4). (B) Effects of the hydration temperature at 45C (n=4),

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50C (n=3), and 55C (n=4). (C) Effects of the number of freeze-thaw cycles at 1 cycle (n=4), 3

cycles (n=3), and 5 cycles (n=4). Each plot shows the average value s.e.m.

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Figure 2 Response surface plot of the particle size showing the effects of the hydration temperature
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(X2) and the number of freeze-thaw cycles (X3) when the PBS volume was 3 mL. This plot was
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generated by using the regression model. Contour lines show the same predicted value.
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Figure 3 Factorial effects on the -potential of FK506 liposomes. (A) Effects of the PBS volume
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at 1 mL (n=4), 3 mL (n=3), and 5 mL (n=4). (B) Effects of the hydration temperature at 45C (n=4),
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50C (n=3), and 55C (n=4). (C) Effects of the number of freeze-thaw cycles at 1 cycle (n=4), 3
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cycles (n=3), and 5 cycles (n=4). Each plot shows the average value s.e.m.

Figure 4 Factorial effects on encapsulation efficiency of FK506 liposomes. (A) Effects of the PBS

volume at 1 mL (n=4), 3 mL (n=3), and 5 mL (n=4). (B) Effects of the hydration temperature at

45C (n=4), 50C (n=3), and 55C (n=4). (C) Effects of the number of freeze-thaw cycles at 1

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cycle (n=4), 3 cycles (n=3), and 5 cycles (n=4). Each plot gives the average value s.e.m.

Figure 5 Response surface plot of the encapsulation efficiency showing the effects of the hydration

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plot was generated by using the regression model. Contour lines show the same predicted value.

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