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w emulsion as carriers for lipophilic drugs
Colloids and Surfaces B: Biointerfaces 15 (1999) 117 – 125 www.elsevier.nl/locate/colsurfb
Study on the formulation of o/w emulsion as carriers for lipophilic drugs Pei Kan, Zhi-Beng Chen, Rou-Yen Kung, Chau-Jen Lee, I-Ming Chu * Department of Chemical Engineering, National Tsing Hua Uni6ersity, Hsinchu, Taiwan 300, ROC Received 5 October 1998; accepted 23 April 1999
Abstract Soybean oil, white mineral oil and tricaprylin were employed with various emulsifiers to create o/w (oil-in-water) emulsion with particle size ranging from 70 to 200 nm for potential drug delivery applications. Suitable formulation of this o/w emulsion was sought for, with aims of good shelf stability (in particle size and drug retention), and high drug loading capacity. Nonionic surfactants, including Cremophore EL, Pluronic F68 and Tween 80, were compared when used in combination with egg phosphatidylcholine (EPC) as the emulsifiers in this study. The effect of types of emulsifiers and oils used on the particle size and shelf stability was investigated. The results revealed a synergistic effect of surfactants EPC and Tween 80 on emulsion stability, with an optimum ratio of ca. 1:1 wt/wt. Emulsion made of the following formulation was found to posses small size around 80 – 120 nm and good storage stability: tircaprylin/Tween 80/EPC (1 g/0.3 g/0.4 g) in 40 ml of 2.25% glycerol aqueous solution. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Lipophilic drug carrier; O/w emulsion; Triacylglycerol; Mixed surfactant
1. Introduction Lipophilic drugs, such as paclitaxel, usually have low aqueous solubility. Development of suitable formulation for their administration has been a key point to wider application and better therapeutic effect. Currently, several types of systems, e.g. liposomes and o/w emulsion [1] have been suggested as candidates for those lipophilic drugs in order to raise the aqueous concentration of the * Corresponding author. Tel.: + 886-3-5713704; fax: + 8863-5715408. E-mail address: [email protected] (I.-M. Chu)
drug as well as to provide better pharmacokinetic properties. Although liposomes are very biocompatible, they suffer from poor shelf stability, low reproducibility and insufficient (for lipophilic drugs) loading [2]. In contrast, o/w emulsion provides not only good biocompatibility, longer shelf life, but has already been used for commercial fat nutrition for years. Thus, delivery systems based on this kind of fat/water emulsion may be a promising alternative [3]. There are only a few studies using the nonionic surfactant/phospholipid system to stabilize the emulsion in submicron size [4]. This study is focused on the effect of the nonionic surfactant/
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EPC mixed emulsifier formulation on particle size and shelf stability. Among the surfactants studied are: Pluronic F68 (poloxamer), EPC, Tween 80 and Cremophore EL, the latter two were reported [5–11] to be possible agents that may promote or contribute to reverse the multidrug resistance (MDR) of tumor cells, and as a result, we studied Tween 80 in much more detail than the other surfactants, since Cremophor EL has well known side effects on humans [12]. Also, the particle size for this study was aimed around 100 nm. The selected size range was based on findings [13–17] of optimum tumor site penetration of these particles. For example, Liu et al. [17] reported a bellshaped curve of blood level of glycolipidcontaining liposomes and implied that the optimal size range to maintain high blood level was from 70 to 200 nm. According to that report the optimal particle size range for the most efficient accumulation of drug carriers in the tumor was between 200 and 90 nm. A plausible explanation for this phenomenon is that the permeability of tumor vascular wall was in general larger as compared to normal tissues, thus particles could extravasate through leaky endothelium by passive transport [16]. Thus the objective of this study is to search for suitable formulation to make stable emulsion in the above mentioned size range.
2. Materials and methods
2.1. Materials Egg phosphatidylcholine (EPC, cat no. P9671) and Cremophor EL (polyoxyethylated castor oil) were obtained from Sigma, Tween 80 (polysorbate 80, polyoxyethylene 20 sorbitan monooleate) from Merck, and Pluronic F68 (poloxamer 188, polyoxyethylene-polyoxypropylene derivatives) from Fluka, respectively. Tricaprylin (C8:0), soybean oil and white mineral oil (light oil), which were referred to as the oil-phase carriers, were obtained from Sigma. Hexane, methanol, glycerol and sodium chloride were purchased from Merck.
2.2. Preparation of o/w emulsions A total of 0.1–0.4 g of a selected nonionic surfactant was mixed with 0–0.8 g EPC, followed by addition of 1 ml of selected oil, all of the components were dissolved in 4.0 ml hexane. After the mixture solution was added dropwise into 40 ml (unless otherwise specified), 2.25 % (w/v) glycerol solution, the solution was sonicated (Soniprep 150, MSE) at constant 20°C for 30 min. The mixture turned gradually into white and opaque appearance. The solution was followed by vacuum evaporation at 30°C to remove residual hexane. The system was set at 100 mbar or less pressure for 30 min to ensure the complete removal of hexane. Emulsion particle size of each sample was measured by a laser particle analyzer (LPA-3000, Otsuka, Electronics, Japan) immediately after the emulsification process. Membrane extrusion using a 0.2 micron membrane was used to ensure a homogeneous size distribution in the later investigation, as specified.
2.3. Shelf stability test The samples were placed at 4°C or ambient temperature, for long-term shelf stability test. Changes in particle size were monitored at predetermined time intervals. Samples were diluted ca. 500-fold with 2.25 % (w/v) glycerol solution prior to the particle diameter measurement. Mean diameters based on weight distribution were obtained.
2.4. Interfacial tension measurement Interfacial tension was measured by the Wilhelmy palte method on a surface tensiometer (model CVBP-A3, Kaimenkagaku, Tokyo, Japan) with temperature control at 30°C.
3. Results and discussion
3.1. Effect of hydrophilic-lipophilic balance of the emulsifiers used As can be inferred from Fig. 1, the differences in the physicochemical properties of the oils used
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do not affect the emulsion particle sizes to a significant degree. We decided to use tricaprylin in further formulation development because it has greater solubility for our intended drug, paclitaxel. On the other hand, the type of emulsifiers was the major determinant influencing the particle size. The results in Fig. 1 indicated that the emulsion diameter tends to increase with emulsifier systems hydrophilic – lipophilic balance (HLB) value. The HLB values of the three nonionic surfactants used are: Pluronic F68, 29; Tween 80, 15; and Cremophor EL, 13. Thus, Pluronic F68/ EPC system resulted in the largest particle size. It is known that the emulsifier with HLB between 10 and 15 is considered to be appropriate for o/w emulsification [18], and higher HLB is prone to create o/w emulsion and micelles simultaneously. The HLB value of phospholipid, such as EPC, is around 9, so the HLB values of the emulsifier systems used here (surfactant plus EPC) were estimated, based on the additive rule in term of weight fraction, approximately to be from 13 to 10 for Tween 80 and Cremophore EL, which fall into the appropriate range.
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3.2. Effect of the amount of Tween 80 and EPC Tween 80 has the advantage of being less toxic and maybe contributing to counter multidrug resistance [11], was chosen in combination with EPC as the emulsifier for further study in making o/w emulsion with tricaprylin as oil phase. The emulsion diameter changed significantly corresponding to the amount of Tween 80 and EPC added, as shown in Fig. 2. Since we intended to make the emulsions with defined diameter (70– 200 nm), proper choice of Tween 80 or EPC content is required. When Tween 80 content was fixed, the EPC content which resulted in minimum emulsion size can be determined from Fig. 2. As Tween 80 increased, this optimal EPC content rose. Also, when only EPC (0.4 g/40 ml solution) was used, the resultant emulsion diameter was 350 nm. This value is similar to the preliminary research [19] on the characterization of the commercial fat emulsion, Intralipid, where EPC and soybean PC were the main emulsifying agents. Pure phosphatidylcholine (PC) is known to tend to form bilayer structure in aqueous solu-
Fig. 1. Particle size of the emulsions formulated with a variety of oils and surfactants. Each contains 0.3 g surfactant (B: Cremophor EL, C: Tween 80, D: Pluronic F68), 0.4 g EPC and 1.0 ml oil in 40 ml, 2.25 % (w/v) glycerol solution.
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Fig. 2. Particle size of the O/W emulsion as affected by the tricaprylin/Tween 80/EPC formulation. Every sample contains 1.0 ml tricaprylin in 40 ml, 2.25 % (w/v) glycerol solution.
tions and is therefore classified as an ineffective o/w emulsifier [4]. The effect of EPC fraction in the Tween 80/ EPC emulsifier system on emulsion size is shown in Fig. 3. The data showed a V-shaped curve. It can be deduced that a synergistic effect took place in the Tween 80/EPC emulsifier system. Levy et al. [20] and Weingarten et al. [18] reported that phospholipids, oleic acid and poloxamer (Pluronic series) induce certain interactions which gave extraordinary stability in o/w emulsion formulation. The mechanism of the synergistic effect arising from the Tween 80/EPC system is unclear but speculated to resemble that of the nonionic surfactant/phospholipid models described by Levy et al. [18] and Weingarten et al. [20]. The optimal Tween 80/EPC ratio (wt/wt) in the mixed emulsifier system was estimated to be 1/1 in (wt/
wt) and 1/2 in (mol/mol). This compares to the value 1/1 (mol/mol) found in the poloxamer (Pluronic series)/EPC system [18] and 1/1.25 (mol/ mol) for Tween 80/DPPC in trioein. Measurements of the interfacial tension between tricaprylin and 2.25% glycerol aqueous solution also confirmed the synergistic effect of Tween 80 and EPC. As shown in Fig. 4 the mixed emulsifiers have greater ability in lowering the interfacial tension. The CMC values for the mixed surfactant systems are also lower than those in the single surfactant systems, indicating that a more closely packed monolayer was formed when Tween 80 and EPC were together. Ratios tested were between 0.6 and 1.5 (wt/wt) for Tween 80/ EPC, which conincided with the ratios that resulted in more stable emulsion, as shown below in Section 3.4.
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Fig. 3. Particle size versus EPC fraction of the emulsions. The emulsifier/oil ratio means the emulsifier weight/oil volume ratio (g ml − 1) regardless of the variations in oil content in the solutions.
Fig. 4. Interfacial tension between tricaprylin and 2.25% glycerol solution. The various emulsifier(s) included in the system were ( ) Tween 80; ( ) EPC; () Tween 80/EPC (wt/wt) 1.44; () Tween 80/EPC (wt/wt) 0.62; (") Tween 80/EPC (wt/wt) 1.02.
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Fig. 5. Variations in particle size of the emulsions with different emulsifier/oil ratios (wt/wt). All the samples have the same formulation of Tween 80/EPC ratio of 1/1 (wt/wt). Error bars were drawn according to the standard errors calculated from three measurements, respectively.
3.3. Emulsifier/oil ratio and oil/water ratio
3.4. Stability of the formulated emulsion
The effect of emulsifier/oil ratio was investigated at a constant Tween 80/EPC weight ratio of 1/1. Fig. 5 showed that over the ratio of 0.6 g emulsifier to 1.0 ml oil namely, 0.6 (wt/wt), the emulsion diameter fell into our desired range. Higher emulsion/oil ratio will make smaller particles (50–80 nm) and the system will approach the regime of micro – emulsion, which is out of scope of this study. In order to carry sufficient lipophilic drugs in emulsion, trials to increase oil composition from 2.5 to 10% or 20% (v/v) were conducted. The Tween 80/EPC emulsions were produced in accordance with the specific formulation with the ratios of EPC/Tween 80 around 1/1 (wt/wt) and the emulsifier/oil of 0.6 (wt/v). As shown in Fig. 6, oil/water beyond 10 % (v/v) led to an enlargement in particle size, and were found less stable (data not shown).
The physicochemical stability of emulsion during storage depends critically on the emulsion formulation. Storage temperature was chosen to be 4°C. The change of particle size in various formulas during a 3-month long storage was monitored and shown in Fig. 7. Large particles more than 1000 nm were detected from unstable formulas due to the emulsion flocculation/coalescence and Ostwald ripening process. The unstable formulas usually deviate greatly from the optimum 1/1 (wt/wt) ratio of Tween 80/EPC. Fig. 8 shows the change of emulsion diameter in 42-day storage which is similar to the 96-day results (data not shown). The plot is divided into three regions. The points in the middle region represent formulas for long-lasting stable emulsion. These formulas give emulsion of virtually unchanged size in the given time. Contrarily, the
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Fig. 6. Particle size of emulsions with various oil/water ratios. The emulsifier/oil ratio was kept at 0.7 (wt/wt), with Tween 80/EPC at 3/4 ratio.
Fig. 7. Shelf stability of emulsion in 92-day storage. Each sample is comprised of 1 g tricaprylin in 40 ml, 2.25% (w/v) glycerol solution. The amount of Tween 80 and EPC used were (g): ( ) 0.3/0.3; ( ) 0.3/0.4; () 0.4/0.4; ( ) 0.1/0.3; () 0.3/0.1; () 0.4/0.1, respectively.
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Fig. 8. Variation in particle size of the emulsions of various formulas, which was measured on the 42nd day. Each sample contains 1 g tricaprylin and various amounts of Tween 80 and EPC (0.2 to 0.8 g together) in 40 ml of 2.25% glycerol solution.
formulas pertinent to the other two regions are unstable with a few exceptions, and do not yield consistent results in producing emulsion (data not shown). As a result, the most favorable formulas for the stable emulsion require that the Tween 80/EPC be from 0.4/0.6 to 0.6/0.4 (wt/wt). If the emulsions contain elevated oil content up to 20 % (v/v), they are not as stable as those with 10 % (v/v) or less (data not shown), even with the totally same formulation. The acceptable oil/water ratio should thus be equal to or below 10 % (v/v).
vides an applicable strategy to manufacture the promising o/w emulsion for lipophilic drug delivery system in the engineering aspect.
Acknowledgements Funding from NSC87-2314-B007-002-M08 from National Science Council, Republic of China, is gratefully acknowledged.
References 4. Conclusions A specific formulation, tricaprylin:Tween 80:EPC:2.25% glycerol solution of 1 g:0.3 g:0.4 g:40 ml was determined which resulted in a sizespecific emulsion with at least 3-month storage stability under 4°C. A synergistic effect of Tween 80 and EPC was observed which resembles the behavior of poloxamer/PC system. The study pro-
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Study on the formulation of o/w emulsion as carriers for lipophilic drugs Pei Kan, Zhi-Beng Chen, Rou-Yen Kung, Chau-Jen Lee, I-Ming Chu * Department of Chemical Engineering, National Tsing Hua Uni6ersity, Hsinchu, Taiwan 300, ROC Received 5 October 1998; accepted 23 April 1999
Abstract Soybean oil, white mineral oil and tricaprylin were employed with various emulsifiers to create o/w (oil-in-water) emulsion with particle size ranging from 70 to 200 nm for potential drug delivery applications. Suitable formulation of this o/w emulsion was sought for, with aims of good shelf stability (in particle size and drug retention), and high drug loading capacity. Nonionic surfactants, including Cremophore EL, Pluronic F68 and Tween 80, were compared when used in combination with egg phosphatidylcholine (EPC) as the emulsifiers in this study. The effect of types of emulsifiers and oils used on the particle size and shelf stability was investigated. The results revealed a synergistic effect of surfactants EPC and Tween 80 on emulsion stability, with an optimum ratio of ca. 1:1 wt/wt. Emulsion made of the following formulation was found to posses small size around 80 – 120 nm and good storage stability: tircaprylin/Tween 80/EPC (1 g/0.3 g/0.4 g) in 40 ml of 2.25% glycerol aqueous solution. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Lipophilic drug carrier; O/w emulsion; Triacylglycerol; Mixed surfactant
1. Introduction Lipophilic drugs, such as paclitaxel, usually have low aqueous solubility. Development of suitable formulation for their administration has been a key point to wider application and better therapeutic effect. Currently, several types of systems, e.g. liposomes and o/w emulsion [1] have been suggested as candidates for those lipophilic drugs in order to raise the aqueous concentration of the * Corresponding author. Tel.: + 886-3-5713704; fax: + 8863-5715408. E-mail address: [email protected] (I.-M. Chu)
drug as well as to provide better pharmacokinetic properties. Although liposomes are very biocompatible, they suffer from poor shelf stability, low reproducibility and insufficient (for lipophilic drugs) loading [2]. In contrast, o/w emulsion provides not only good biocompatibility, longer shelf life, but has already been used for commercial fat nutrition for years. Thus, delivery systems based on this kind of fat/water emulsion may be a promising alternative [3]. There are only a few studies using the nonionic surfactant/phospholipid system to stabilize the emulsion in submicron size [4]. This study is focused on the effect of the nonionic surfactant/
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EPC mixed emulsifier formulation on particle size and shelf stability. Among the surfactants studied are: Pluronic F68 (poloxamer), EPC, Tween 80 and Cremophore EL, the latter two were reported [5–11] to be possible agents that may promote or contribute to reverse the multidrug resistance (MDR) of tumor cells, and as a result, we studied Tween 80 in much more detail than the other surfactants, since Cremophor EL has well known side effects on humans [12]. Also, the particle size for this study was aimed around 100 nm. The selected size range was based on findings [13–17] of optimum tumor site penetration of these particles. For example, Liu et al. [17] reported a bellshaped curve of blood level of glycolipidcontaining liposomes and implied that the optimal size range to maintain high blood level was from 70 to 200 nm. According to that report the optimal particle size range for the most efficient accumulation of drug carriers in the tumor was between 200 and 90 nm. A plausible explanation for this phenomenon is that the permeability of tumor vascular wall was in general larger as compared to normal tissues, thus particles could extravasate through leaky endothelium by passive transport [16]. Thus the objective of this study is to search for suitable formulation to make stable emulsion in the above mentioned size range.
2. Materials and methods
2.1. Materials Egg phosphatidylcholine (EPC, cat no. P9671) and Cremophor EL (polyoxyethylated castor oil) were obtained from Sigma, Tween 80 (polysorbate 80, polyoxyethylene 20 sorbitan monooleate) from Merck, and Pluronic F68 (poloxamer 188, polyoxyethylene-polyoxypropylene derivatives) from Fluka, respectively. Tricaprylin (C8:0), soybean oil and white mineral oil (light oil), which were referred to as the oil-phase carriers, were obtained from Sigma. Hexane, methanol, glycerol and sodium chloride were purchased from Merck.
2.2. Preparation of o/w emulsions A total of 0.1–0.4 g of a selected nonionic surfactant was mixed with 0–0.8 g EPC, followed by addition of 1 ml of selected oil, all of the components were dissolved in 4.0 ml hexane. After the mixture solution was added dropwise into 40 ml (unless otherwise specified), 2.25 % (w/v) glycerol solution, the solution was sonicated (Soniprep 150, MSE) at constant 20°C for 30 min. The mixture turned gradually into white and opaque appearance. The solution was followed by vacuum evaporation at 30°C to remove residual hexane. The system was set at 100 mbar or less pressure for 30 min to ensure the complete removal of hexane. Emulsion particle size of each sample was measured by a laser particle analyzer (LPA-3000, Otsuka, Electronics, Japan) immediately after the emulsification process. Membrane extrusion using a 0.2 micron membrane was used to ensure a homogeneous size distribution in the later investigation, as specified.
2.3. Shelf stability test The samples were placed at 4°C or ambient temperature, for long-term shelf stability test. Changes in particle size were monitored at predetermined time intervals. Samples were diluted ca. 500-fold with 2.25 % (w/v) glycerol solution prior to the particle diameter measurement. Mean diameters based on weight distribution were obtained.
2.4. Interfacial tension measurement Interfacial tension was measured by the Wilhelmy palte method on a surface tensiometer (model CVBP-A3, Kaimenkagaku, Tokyo, Japan) with temperature control at 30°C.
3. Results and discussion
3.1. Effect of hydrophilic-lipophilic balance of the emulsifiers used As can be inferred from Fig. 1, the differences in the physicochemical properties of the oils used
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do not affect the emulsion particle sizes to a significant degree. We decided to use tricaprylin in further formulation development because it has greater solubility for our intended drug, paclitaxel. On the other hand, the type of emulsifiers was the major determinant influencing the particle size. The results in Fig. 1 indicated that the emulsion diameter tends to increase with emulsifier systems hydrophilic – lipophilic balance (HLB) value. The HLB values of the three nonionic surfactants used are: Pluronic F68, 29; Tween 80, 15; and Cremophor EL, 13. Thus, Pluronic F68/ EPC system resulted in the largest particle size. It is known that the emulsifier with HLB between 10 and 15 is considered to be appropriate for o/w emulsification [18], and higher HLB is prone to create o/w emulsion and micelles simultaneously. The HLB value of phospholipid, such as EPC, is around 9, so the HLB values of the emulsifier systems used here (surfactant plus EPC) were estimated, based on the additive rule in term of weight fraction, approximately to be from 13 to 10 for Tween 80 and Cremophore EL, which fall into the appropriate range.
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3.2. Effect of the amount of Tween 80 and EPC Tween 80 has the advantage of being less toxic and maybe contributing to counter multidrug resistance [11], was chosen in combination with EPC as the emulsifier for further study in making o/w emulsion with tricaprylin as oil phase. The emulsion diameter changed significantly corresponding to the amount of Tween 80 and EPC added, as shown in Fig. 2. Since we intended to make the emulsions with defined diameter (70– 200 nm), proper choice of Tween 80 or EPC content is required. When Tween 80 content was fixed, the EPC content which resulted in minimum emulsion size can be determined from Fig. 2. As Tween 80 increased, this optimal EPC content rose. Also, when only EPC (0.4 g/40 ml solution) was used, the resultant emulsion diameter was 350 nm. This value is similar to the preliminary research [19] on the characterization of the commercial fat emulsion, Intralipid, where EPC and soybean PC were the main emulsifying agents. Pure phosphatidylcholine (PC) is known to tend to form bilayer structure in aqueous solu-
Fig. 1. Particle size of the emulsions formulated with a variety of oils and surfactants. Each contains 0.3 g surfactant (B: Cremophor EL, C: Tween 80, D: Pluronic F68), 0.4 g EPC and 1.0 ml oil in 40 ml, 2.25 % (w/v) glycerol solution.
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Fig. 2. Particle size of the O/W emulsion as affected by the tricaprylin/Tween 80/EPC formulation. Every sample contains 1.0 ml tricaprylin in 40 ml, 2.25 % (w/v) glycerol solution.
tions and is therefore classified as an ineffective o/w emulsifier [4]. The effect of EPC fraction in the Tween 80/ EPC emulsifier system on emulsion size is shown in Fig. 3. The data showed a V-shaped curve. It can be deduced that a synergistic effect took place in the Tween 80/EPC emulsifier system. Levy et al. [20] and Weingarten et al. [18] reported that phospholipids, oleic acid and poloxamer (Pluronic series) induce certain interactions which gave extraordinary stability in o/w emulsion formulation. The mechanism of the synergistic effect arising from the Tween 80/EPC system is unclear but speculated to resemble that of the nonionic surfactant/phospholipid models described by Levy et al. [18] and Weingarten et al. [20]. The optimal Tween 80/EPC ratio (wt/wt) in the mixed emulsifier system was estimated to be 1/1 in (wt/
wt) and 1/2 in (mol/mol). This compares to the value 1/1 (mol/mol) found in the poloxamer (Pluronic series)/EPC system [18] and 1/1.25 (mol/ mol) for Tween 80/DPPC in trioein. Measurements of the interfacial tension between tricaprylin and 2.25% glycerol aqueous solution also confirmed the synergistic effect of Tween 80 and EPC. As shown in Fig. 4 the mixed emulsifiers have greater ability in lowering the interfacial tension. The CMC values for the mixed surfactant systems are also lower than those in the single surfactant systems, indicating that a more closely packed monolayer was formed when Tween 80 and EPC were together. Ratios tested were between 0.6 and 1.5 (wt/wt) for Tween 80/ EPC, which conincided with the ratios that resulted in more stable emulsion, as shown below in Section 3.4.
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Fig. 3. Particle size versus EPC fraction of the emulsions. The emulsifier/oil ratio means the emulsifier weight/oil volume ratio (g ml − 1) regardless of the variations in oil content in the solutions.
Fig. 4. Interfacial tension between tricaprylin and 2.25% glycerol solution. The various emulsifier(s) included in the system were ( ) Tween 80; ( ) EPC; () Tween 80/EPC (wt/wt) 1.44; () Tween 80/EPC (wt/wt) 0.62; (") Tween 80/EPC (wt/wt) 1.02.
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Fig. 5. Variations in particle size of the emulsions with different emulsifier/oil ratios (wt/wt). All the samples have the same formulation of Tween 80/EPC ratio of 1/1 (wt/wt). Error bars were drawn according to the standard errors calculated from three measurements, respectively.
3.3. Emulsifier/oil ratio and oil/water ratio
3.4. Stability of the formulated emulsion
The effect of emulsifier/oil ratio was investigated at a constant Tween 80/EPC weight ratio of 1/1. Fig. 5 showed that over the ratio of 0.6 g emulsifier to 1.0 ml oil namely, 0.6 (wt/wt), the emulsion diameter fell into our desired range. Higher emulsion/oil ratio will make smaller particles (50–80 nm) and the system will approach the regime of micro – emulsion, which is out of scope of this study. In order to carry sufficient lipophilic drugs in emulsion, trials to increase oil composition from 2.5 to 10% or 20% (v/v) were conducted. The Tween 80/EPC emulsions were produced in accordance with the specific formulation with the ratios of EPC/Tween 80 around 1/1 (wt/wt) and the emulsifier/oil of 0.6 (wt/v). As shown in Fig. 6, oil/water beyond 10 % (v/v) led to an enlargement in particle size, and were found less stable (data not shown).
The physicochemical stability of emulsion during storage depends critically on the emulsion formulation. Storage temperature was chosen to be 4°C. The change of particle size in various formulas during a 3-month long storage was monitored and shown in Fig. 7. Large particles more than 1000 nm were detected from unstable formulas due to the emulsion flocculation/coalescence and Ostwald ripening process. The unstable formulas usually deviate greatly from the optimum 1/1 (wt/wt) ratio of Tween 80/EPC. Fig. 8 shows the change of emulsion diameter in 42-day storage which is similar to the 96-day results (data not shown). The plot is divided into three regions. The points in the middle region represent formulas for long-lasting stable emulsion. These formulas give emulsion of virtually unchanged size in the given time. Contrarily, the
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Fig. 6. Particle size of emulsions with various oil/water ratios. The emulsifier/oil ratio was kept at 0.7 (wt/wt), with Tween 80/EPC at 3/4 ratio.
Fig. 7. Shelf stability of emulsion in 92-day storage. Each sample is comprised of 1 g tricaprylin in 40 ml, 2.25% (w/v) glycerol solution. The amount of Tween 80 and EPC used were (g): ( ) 0.3/0.3; ( ) 0.3/0.4; () 0.4/0.4; ( ) 0.1/0.3; () 0.3/0.1; () 0.4/0.1, respectively.
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Fig. 8. Variation in particle size of the emulsions of various formulas, which was measured on the 42nd day. Each sample contains 1 g tricaprylin and various amounts of Tween 80 and EPC (0.2 to 0.8 g together) in 40 ml of 2.25% glycerol solution.
formulas pertinent to the other two regions are unstable with a few exceptions, and do not yield consistent results in producing emulsion (data not shown). As a result, the most favorable formulas for the stable emulsion require that the Tween 80/EPC be from 0.4/0.6 to 0.6/0.4 (wt/wt). If the emulsions contain elevated oil content up to 20 % (v/v), they are not as stable as those with 10 % (v/v) or less (data not shown), even with the totally same formulation. The acceptable oil/water ratio should thus be equal to or below 10 % (v/v).
vides an applicable strategy to manufacture the promising o/w emulsion for lipophilic drug delivery system in the engineering aspect.
Acknowledgements Funding from NSC87-2314-B007-002-M08 from National Science Council, Republic of China, is gratefully acknowledged.
References 4. Conclusions A specific formulation, tricaprylin:Tween 80:EPC:2.25% glycerol solution of 1 g:0.3 g:0.4 g:40 ml was determined which resulted in a sizespecific emulsion with at least 3-month storage stability under 4°C. A synergistic effect of Tween 80 and EPC was observed which resembles the behavior of poloxamer/PC system. The study pro-
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