Alayoubi - Vitamin E Fortified Parenteral Lipid Emulsions

1Introduction

Intravenous lipid emulsions, such as Intralipid have been in clinical use for nearly 50 years1. They were developed to prevent or reverse fatty acid deficiency and provide a source of calories for patients requiring parenteral nutrition2. The positive attributes of lipid emulsions are numerous3. In general, they are biocom-patible, biodegradable, physically stable (particularly nanoemulsions and microemulsions), and relatively easy to produce on a large scale using proven technol-ogy4. Therefore, lipid emulsions were also investigated as potential delivery vehicles for drugs with poor water

solubility by taking advantage of the large oil content in these emulsions5.

Oils typically used in lipid emulsions consist of digestible long-chain triglycerides (LCT), such as soybean oil, sesame seed oil, cottonseed oil, and safflower oil3. Until recently, triglycerides derived from soybean were the sole source of commercial parenteral lipids. The infusion of these lipids, however, was implicated in complications such as impaired function of the clotting and immunological systems6 as well as increased lipid peroxidation in vivo. Therefore, a second-generation lipid emulsions were developed in which soybean oil was

RESEARCH ARTICLE

Vitamin E fortified parenteral lipid emulsions: PlackettBurman screening of primary process and composition parameters

Alaadin Alayoubi1, Mahmoud Nazzal2, Paul W. Sylvester1, and Sami Nazzal1

1Department of Basic Pharmaceutical Sciences, College of Pharmacy, University of Louisiana at Monroe, Monroe, LA, USA and 2Department of Allied Medical Sciences, Faculty of Applied Medical Sciences, Jordan University of Science and Technology, Irbid, Jordan

AbstractThe objective of this study was to screen the effect of eight formulations and process parameters on the physical attributes and stability of Vitamin E-rich parenteral lipid emulsions. Screening was performed using a 12-run, 8-factor, 2-level PlackettBurman design. This design was employed to construct polynomial equations that identified the magnitude and direction of the linear effect of homogenization pressure, number of homogenization cycles, primary and secondary emulsifiers, pre-homogenization temperature, oil loading, and ratio of vitamin E to medium-chain triglycerides (MCT) in the oil phase on particle size, polydispersity index, short-term stability, and outlet temperature of manufactured emulsions. The viscosity of vitamin E was reduced from 3700 (100%) to 64 mPa.s (30%) by MCT addition. As viscosity is critical for efficient emulsification, vitamin/MCT ratio was the most significant contributor for the stability of emulsions. Particle size increased from 236 to 388 nm, and percentage vitamin remaining emulsified after 48 h dropped from 100 to 73% with increase in vitamin/MCT ratio from 30/70 to 70/30. Significant decrease in particle size and PI, and an increase in outlet temperature were also observed with increase in homogenization pressure and number of homogenization cycles. Emulsifiers and oil loading, however, had insignificant effect on the responses. Overall, stable submicron emulsions at vitamin/MCT ratio of 30/70 could be prepared at 25,000 psi and 25 cycles in ambient conditions. The identification of these parameters by a well-constructed design demonstrated the utility of screening studies in the Quality by Design approach to pharmaceutical product development.Keywords: vitamin E, PlackettBurman, emulsion, screening, experimental design

Address for Correspondence: Sami Nazzal, Ph.D., Associate Professor of Pharmaceutics, College of Pharmacy1800 Bienville Dr, University of Louisiana at Monroe, Monroe, LA 71201. Tel: +1 (318) 342-1726. Fax: +1 (318) 342-1737. E-mail: [email protected]

(Received 12 January 2012; revised 28 March 2012; accepted 29 March 2012)

Drug Development and Industrial Pharmacy, 2012; Early Online: 111 2012 Informa Healthcare USA, Inc.ISSN 0363-9045 print/ISSN 1520-5762 onlineDOI: 10.3109/03639045.2012.682223

Drug Development and Industrial Pharmacy

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12January2012

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10.3109/03639045.2012.682223

2012

Process development for vitamin E lipid emulsions

A. Alayoubi et al.

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2 A. Alayoubi et al.


partially substituted with medium-chain triglycerides (MCT7). Several examples have also been cited where -tocopherol (vitamin E) in small quantities, usually

Process development for vitamin E lipid emulsions 3


(response) variables using the following polynomial model20:

1 1 2 2 3 3 4 4 n n0A A A A A AY X X X X X

In this model, Y is the response, Ao is a constant, and

A1A

n are the coefficients of the independent variables

(X). When generating the model and analyzing input data, the PlackettBurman design ranks the variables based on their magnitude of effect and designates a signs (+ or ) to each effect to indicate whether an increase in the level of each variable has a positive (+) or negative () effect on the response21. For this study, eight variables were screened in random 12 blocks (runs) for their effect on the quality of vitamin E loaded emulsions. The list of independent variables (X) and their levels, and the monitored responses (Y) are listed in Table 1. This design was constructed using

STATGRAPHICS Plus software (Version 2.1; Statistical Graphics Corp., Rockville, MD).

Preparation of the emulsionsEmulsions (15 mL) were prepared by first mixing vita-min E with MCT at 30/70 or 70/30 ratios to form the oil phase. When needed, cholesterol was then dissolved in the oil phase to act as a secondary emulsifier. The primary emulsifiers [Lipoid E80 S and Tween 80] were dispersed in DI water to form the aqueous phase of the emulsion to which glycerol (2.25%, w/w of the total emulsion) was added to adjust tonicity. The con-centration of the primary and secondary emulsifiers varied according to the statistical mod-el as given in Table 2. The oil and the aqueous phases were heated separately for 5 min at the temperature specified by the design for each trial. The two phases were then mixed at 15,000 rpm for 2 min using an IKA Ultra- Turrax T8 mixer (IKA Works Inc., Wilmington, NC) to form the crude pre-emulsion. The final concentration of the oil phase in the emulsions was either 10 or 20% w/w of the total emulsion. A submicron emulsion was obtained by passing the coarse pre-emulsion through a high-pressure homogenizer (EmulsiFlex-C3, Avestin Inc, Ottawa, Canada) for several cycles and under homogenization pressure predefined by the statistical design. The temperature of the resulting emulsions was measured and the pH was adjusted to 8 0.05 using 0.1 N sodium hydroxide solution. This was essential as lipid emulsions are most stable at pH values higher than 7.522.

Physical characterization of the Vitamin E emulsionsIntensity-weighed mean particle size and population distribution (polydispersity index [PI]) of the emulsions were measured by photon correlation spectroscopy at 23C and a fixed angle of 90 using Nicomp 380 ZLS submicron particle size analyzer (PSS Inc., Santa Barbara, CA). PI, which is a measure of homogeneity and

Table 2. The 12 runs of the PlackettBurman screening design and the observed responses for each run.

Run

Factors (independent variable) Observed responsesX

1X

2X

3X

4X

5X

6X

7X

8Y

1Y

2Y

3Y

4

1 25 25 1.2 2.0 0 30 10 70 166 0.227 100 422 25 5 2.4 0.5 0 30 20 70 209 0.161 100 423 5 5 1.2 2.0 0.5 70 10 70 519 0.505 47 314 25 5 2.4 2.0 0 70 10 25 373 0.376 84 415 5 5 1.2 0.5 0 30 10 25 324 0.317 100 306 5 5 2.4 2.0 0.5 30 20 70 299 0.219 100 307 25 25 2.4 0.5 0.5 70 10 70 290 0.236 91 538 25 5 1.2 0.5 0.5 70 20 25 342 0.337 85 489 5 25 2.4 0.5 0.5 30 10 25 274 0.224 100 32

10 5 25 2.4 2.0 0 70 20 25 372 0.409 62 3311 25 25 1.2 2.0 0.5 30 20 25 147 0.115 100 5112 5 25 1.2 0.5 0 70 20 70 431 0.345 69 35

Table 1. List of independent factors and dependent responses of the PlackettBurman screening design. Also shown are the low and high levels of each independent factor, which were coded as 1 and 1 in ANOVA, respectively.

Low level (1)

High level (1)

Independent factors X

1: Homogenization pressure (psi) 1000 5 25

X2: Number of cycles 5 25

X3: Lipoid E80 S (% w/w) 1.2 2.4

X4: Tween 80 (% w/w) 0.5 2

X5: Cholesterol (% w/w) 0 0.5

X6: % Vitamin E in the oil phase (% w/w) 30 70

X7: % Oil phase in the emulsion (% w/w) 10 20

X8: Temperature during high shear

homogenization (C)25 70

Dependent factors (responses) Y

1: Particle size (nm)

Y2: Polydispersity index (PI)

Y3: % Vitamin E remaining emulsified after 48 h of storage

Y4: The temperature of the emulsion at the end of the

homogenization run (C)

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width of the size distribution, ranges from 0 (indicating a monodisperse system) to 0.5 (indicating a relatively broad distribution23). When needed, samples were diluted with filtered DI water. Analyses were performed in triplicates unless otherwise specified. Short-term stability was assessed by measuring the size and concentration of vitamin E remaining emulsified after storage at ambient conditions for 48 h. The percentage of vitamin E emulsified was determined by first removing the separated oils from the surface of the emulsion. The content of vitamin E remaining emulsified was then determined by analyzing a sample collected from the bulk of the emulsion spectrophotometrically at 295 nm (Cary 50 probe-UV spectrophotometer, Varian Inc., Cary, NC) after dilution with appropriate amount of methanol.

Results and discussion

Viscosity measurementThe viscosity of the oil phase plays an essential role in determining the physical properties of the emulsion24. It is known that the viscosity of the oil phase is important for the break-up of emulsion droplets during the emul-sification and homogenization process. The higher the viscosity the longer it takes to deform a droplet, where deformation time (

def) can be estimated from the ratio of

oil viscosity () to the external stress acting on the droplet (25):

ref=

This effect was seen in preliminary studies during which it was observed that emulsifying vitamin E into an aqueous surfactant solution was difficult. Due to its high viscosity, vitamin E would form a paste or a crude dispersion that would quickly phase separate. To aid in vitamin E emulsification, binary blends with MCT were used as the oil phase to lower the viscosity of vitamin E. While soybean oil (LCT) is commonly used in commer-cial emulsions, MCT was used in this study for its meta-bolic advantages. MCT has a faster rate of hydrolysis by lipoprotein lipase leading to more rapid clearance when administered26,27. Furthermore, MCT/LCT (1:1, wt/wt) emulsions are used in commercial products in Europe, Asia, and South America, in order to avoid linoleic acid overload, a condition caused by excessive quantity (5254%) of linoleic acid (C18:2 -6), an -6 polyunsatu-rated fatty acid in soybean oil28,29. Linoleic acid, in large amounts, may have deleterious effects because of its conversion to arachidonic acid (C20:4 -6) and may lead to proinflammatory eicosanoids via the cyclooxygenase and lipoxygenase pathways29.

To demonstrate the impact of MCT, the viscosity of the binary blends was measured at different vitamin E/MCT ratios (Figure 1). The viscosity of the blends decreased from a high of 3700 mPa.s for vitamin E alone to a low of 27 mPa.s, which is the viscosity of pure MCT. At the lower

end, the viscosity reached a plateau at an approximate ratio of 30/70 for vitamin E/ MCT blends. Therefore, two levels of vitamin E loading in the emulsion were evalu-ated in this study representing a high viscosity system at a 70/30 ratio and a low viscosity system at a 30/70 ratio of vitamin E to MCT. The effect of viscosity on emulsion processing and stability is further discussed in the subse-quent sections.

Experimental designA 12-run PlackettBurman screening design was used in this study to estimate the main effects of eight indepen-dent variables. These variables included process param-eters [homogenization pressure, number of cycles, and Temperature during high-shear homogenization] and composition parameters [concentration of primary and secondary emulsifier, % oil loading, and % vitamin E in the oil phase]. While the PlackettBurman design does not consider the interaction terms between the indepen-dent variables, it greatly reduces the number of experi-ments that are required to evaluate their main effects19.

The upper and lower level of each independent variable [X

1X

8] is listed in Table 1. These levels were

identified in preliminary studies. In addition, the list of dependent variables [Y

1Y

4] that were evaluated in this

study is given in Table 1. The exact level of independent variables [X

1X

8] and the observed responses [Y

1Y

4] for

each of the 12 runs are given in Table 2.The results from Table 2 were used to generate polyno-

mial equations for each response (Table 3). Polynomial equations were vital to understand the relationship between the independent and dependent variables. The magnitude and direction of the factor coefficient in each equation (as given in subsequent sections) was used to explain the nature of factor effect [X

1X

8] on the responses

[Y1Y

4]. Factors with coefficients of greater magnitude

Figure 1. Viscosity profile of the binary blends of vitamin E and MCT as a function of % (w/w) MCT in the blend.

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show a high effect on the response. The regression coefficient obtained for Y

1, Y

2, Y

3, and Y

4 was 0.98, 0.96,

0.87, and 0.96, which indicates that the model as fitted explains 98, 96, 87, and 96% of the variability around the mean, respectively.

By using ANOVA, it was possible to calculate the significance of the ratio of mean square variation due to regression coefficient and residual error. The ANOVA of the model parameters for the response Y

1 [particle size] is

given in Table 4. Three factors [X1, X

2, and X

6] had p values

less than 0.1, indicating that they had a significant effect on particle size whereas the remaining factors [X

3X

5,

X7, and X

8] had no significant effect on the response.

Similar analysis was done for the responses Y2 [PI], Y

3 [%

vitamin E that remained emulsified after 48 h of storage] and Y

4 [the temperature of the emulsion at the end of

the homogenization run]. The ANOVA of the model parameters for the response Y

2 is given in Table 4. Three

factors, homogenization pressure (X1), number of cycles

(X2), and % vitamin E in oil phase (X

6) had a significant (p

value < 0.10) effect on this response. For Y3, however, X

6

was the only factor that had a significant effect (p value < 0.10) on this response. For the response Y

4, two factors

X1 and X

2 had a significant effect on the temperature of

the emulsion at the end of the homogenization run. The statistical significance of the effect of each independent variable on the responses was estimated by ANOVA, which was visually presented by Pareto Charts, for each response (Figure 2). An in-depth discussion on the effect of each independent variable on the responses is given in the subsequent sections.

Effect of homogenizing pressure (X1) and

number of homogenization cycles (X2)

Despite the common use of high-pressure homogenizers, few studies dealt with the effect of homogenization pres-sures within a broad range on emulsion properties30,31. Therefore, in this study homogenization pressure at two levels, low (5000 psi) and high (25,000 psi) was evaluated for its effect on the physical stability of vitamin E emulsions. From the statistical analysis of the results (Table 4 and Figures 2A and 2B), it could be deduced that an increase in homogenization pressure led to a significant reduction in particle size (Y

1) and PI (Y

2). This was in line with previously

reported data where it was demonstrated that the average

diameter of fat globules decreases with emulsification pres-sure32. A positive correlation was also observed between the temperature of the emulsions at the end of the homog-enization run and the applied pressure and the number of cycles (Figure 3). Homogenization pressure, however, had an insignificant effect on the % vitamin E that remained emulsified into the aqueous phase of the emulsion after 48 h of storage. Similar observations were observed when the effect of the number of homogenization cycles on the quality of the emulsions was assessed. The estimation of the optimum number of cycles during homogeniza-tion was the focus of many studies24,30,31,33. Muller et al. for example, found that the optimum number of cycles for cas-tor oil emulsions without causing any over processing was eight. Processing of vitamin E, which has higher polarity and viscosity than castor oil is, however more challenging. Therefore, a wider range from 5 to 25 cycles was screened in this study. As expected, increasing the number of cycles improved the physical properties of the emul-sion. It significantly reduced particle size and marginally reduced PI. Furthermore, no over processing or instabil-ity was observed when the emulsions were processed through 25 cycles. While 25 cycles may be excessive, in certain applications such as the case with viscous lipids, it may be essential. The fact that stable emulsions were obtained is a strong indication that running emulsions through a large number of cycles is acceptable. On the other hand, running the emulsion though only five cycles was insufficient to produce stable emulsions as observed by the separation of vitamin E after 48 h of storage. An interaction effect is expected between homogenization pressure and the number of cycles. The PlackettBurman design, however, only evaluates the linear effect of each factor independently of their potential interaction with other factors. Such complex correlation is the subject of future studies. Visually, the effect of pressure and homog-enization cycles on practice size and polydispersity index is illustrated by the linear 3D surface plot given in Figures 4A and 5A, respectively.

Effect of surfactants and co-surfactant concentrations (X

3, X

4, and X

5)

Surfactants are considered the backbone of any emulsion. Therefore, studying the effect of surfactants on the process of developing lipid emulsions is essential34,35. Biocompatible

Table 3. (A) Regression equations of the fitted models for the responses (Y1Y4) and (B) adjusted regression equations for Y1Y4 showing factors with significant effect on the response at = 0.1.(A) Regression equations of the fitted modelsY

1 = 312.17 57.67 X

1 32.17 X

2 9.33 X

3 + 0.50 X

4 0.33 X

5 + 75.67 X

6 12.17 X

7 + 6.83 X

8

Y2 = 0.2893 0.0473 X

1 0.0299 X

2 0.0184 X

3 + 0.0193 X

4 0.0166 X

5 + 0.0788 X

6 0.0249 X

7 0.0071 X

8

Y3= 86.5 + 6.83 X

1 + 0.5 X

2 + 3.0 X

3 4.33 X

4 + 0.667 X

5 13.5 X

6 0.5 X

7 2.0 X

8

Y4 = 39.0 + 7.167 X

1 + 2.0 X

2 0.5 X

3 1.0 X

4 + 1.833 X

5 + 1.167 X

6 + 0.833 X

7 0.167 X

8

Adjusted regression equations at = 0.1 [showing factors with significant effect on the responses (p < 0.1)]Y

1 = 312.17 57.67 X

1 32.17 X

2 + 75.67 X

6

Y2 = 0.2893 0.0473 X

1 0.0299 X

2 + 0.0788 X

6

Y3= 86.5 13.5 X

6

Y4 = 39.0 + 7.167 X

1 + 2.0 X

2

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phospholipids are the primary emulsifiers in commercial lipid emulsions5. Stable vitamin E emulsions, however, could not be prepared when phospholipids were the only emulsifiers used8 Poor stability of vitamin E emulsions could be attributed to its polarity and viscosity. Vitamin E is more polar than LCT and MCT because of the hydroxyl group on the aromatic chromanol ring. Such polarity may result in higher solubility of phospholipids in vitamin E with the result that the emulsifier becomes less available at the vitamin E/water interface. It was reported that combining phospholipids with other surfactants is essential to improve the stability of vitamin E emulsions as compared with the stability of emulsions when prepared using only a single emulsifier9. This phenomenon was observed in our preliminary studies, where stable emulsions with high vitamin E loading could not be formed using Lipoid E80S alone. Therefore, Tween 80, a hydrophilic emulsifier with a high Hydrophilic-lipophilic balance (HLB) value was co-admixed with Lipoid E80S to form the primary emulsifier in this study.

The concentration of Tween 80 in the emulsion ranged from 0.52%, whereas Lipoid E80S was evalu-ated within a concentration range from 1.2 to 2.4%. From the Pareto chart (Figure 2) and Table 4, however, it could be readily concluded that both factors had no significant effect on any of the responses. This indicates that vitamin E emulsions 1020% oil phase could be readily manu-factured irrespective of the concentration of the emulsi-fiers used, as long as the minimum amount of primary emulsifiers was added to the system. This minimum was 1.2% for Lipoid E80S, which is exactly the same concen-tration used in commercial 20% soybean oil lipid emul-sions. A minimum of 0.5% Tween 80 was also essential for the reasons discussed above. Higher concentrations of Lipoid E80S and Tween 80 had no added advan-tage. In addition, cholesterol had no significant effect on the quality of the emulsions, which might be attributed to the presence of Tween 80, a stronger emulsifier, in the phospholipids layer. Cholesterol is commonly used to stabilize phospholipids bilayers in liposomes36. It was also used in the preparation of several emulsions3739. Therefore, it was of interest to observe whether the pres-ence or absence of cholesterol had any impact on the formation and quality of vitamin E emulsions. It was hypothesized that the hydroxyl group in cholesterol would stabilize the chromanol ring of vitamin E at the water interface. Such effect, however, was insignificant (Table 4). Nonetheless, it may be worth investigating the effect of cholesterol at higher concentrations on the stability of lipid emulsions, which is beyond the scope of this study.

Effect of vitamin E to MCT ratio (X6)

This was by far the most significant factor with a profound impact on the quality of the emulsions. Increasing the ratio of vitamin E in the oil phase from 30/70 to 70/30 significantly increased particle size and PI (Figure 2A Ta

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and 2B). It also had a negative impact on the stability of the emulsions as measured by the % vitamin E remaining emulsified after 48 h of storage (Figure 2C). At higher vitamin E ratio, the emulsions broke and a significant portion of vitamin E separated into a visible oily layer on the surface of the emulsion. The negative impact of high vitamin E to MCT ratio (Figure 6) could be attributed to its high viscosity and polarity as discussed above. In general, oil phases with higher viscosities require higher homogenization pressures to achieve smaller particle

size distributions, otherwise coarse and unstable large oil droplets coalesce resulting in a destabilization effect34. Furthermore, because of the higher polarity of vitamin E as compared with MCT, less surfactant will be available at the vitamin E/water interface. It is worth noting that the negative effects of vitamin E were not observed when the emulsions were loaded with 20% oil phase containing vitamin E and MCT at a ratio of 30/70 (Figures 4B and 5B), which is equivalent to 6% vitamin E of the total emulsion. On the other hand, emulsions loaded with 10% oil phase with vitamin E to MCT ratio of 70/30 (equivalent to 7% vitamin E of the total emulsion) were unstable, even in the presence of excess phospholipids. Therefore, it could be concluded that emulsifying vitamin E alone in the absence of secondary low viscosity oil is challenging. Addition of MCT lowered the viscosity of the blend and provided a hydrophobic core to stabilize the oil/water interface. Furthermore, within the range of parameters that were evaluated in this study, only homogenization pressure and number of homogenization cycle seemed to aid in the emulsification of emulsions with high vitamin E to MCT ratio in the oil phase. This could be seen from their significant effect on emulsion stability. The optimum manufacturing conditions and the interaction effect between pressure and number of cycles, however, warrants further investigation. Increasing the concentration of primary and secondary surfactants had insignificant effect on the stability of the emulsions. This indicates that simply increasing the concentration of these emulsifiers is not expected to improve the quality of the emulsion. Rather, factors other than those evaluated

Figure 2. Standardized Pareto charts showing the significance ( = 0.1) of each independent variable (X18

) on the responses (A) Y1 [particle

size], (B) Y2 [polydispersity index], (C) Y

3 [% vitamin E that remained emulsified after 48 h of storage] and (D) Y

4 [emulsion temperature].

White bars indicate a positive effect whereas black bars indicate a negative effect on the response. Bars that extend beyond the vertical line indicate a significant effect (p < 0.1) of the factor on the response.

Figure 3. 2D contour plot showing the effects of homogenization pressure (psi) (X

1), and number of cycles (X

2) on the response Y

4 [the

temperature of the emulsion at the end of the homogenization run].

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in this study, such as the presence of a ternary emulsifier or a different primary emulsifier altogether may be needed to stabilize the emulsions with high vitamin E to MCT ratio in the oil phase.

Effect of % oil loading (X7)

Loading emulsions with high concentrations of the oil phase has its advantages. It provides a reservoir to solubilize lipophilic drugs and delivers more energy when used in nutritional applications. High oil concentrations, however, often lead to an increase in particle size and viscosity of the system30 which in turn may compromise emulsion stability. The increase in particle size may result from an impoverishment of the surfactant at the interface and an increase in the surface tension of the dispersed oil phase30. In this study, however, no significant differences between the 10 and 20% emulsions was observed. In essence, loading an emulsion with 10 or 20% oil phase while keeping

the amount of the other constituents constant resulted in insignificant change in size, PI, or emulsion stability. This should be of no surprise as several comparable and stable emulsions with 20% oil load are commercially available. This further confirms an earlier observation that using 1.2% Lipoid E80 S is sufficient to stabilize these emulsions even when the oil phase was partially substituted with vitamin E. In fact, several studies showed that 10% fat emulsions contain phospholipids in amounts that usually exceed what is needed to stabilize the lipid droplets26. Excess phospholipids (PL) in emulsions with low oil loading form vesicular PL-rich or TG (triglyceride)-free particles that can induce plasma lipid accumulation in children and adults26. Hyperphospholipidemia has been reported in studies of animals, infants, and adults receiving regular 10% MCT/LCT fat emulsions with a PL: TG ratio of 0.1226 Therefore, it is essential from a clinical perspective to use the minimum concentration of phospholipids that is sufficient to produce stable parenteral emulsions. While

Figure 4. 3D response surface plots showing (A) the effect of homogenization pressure (psi) (X


2) on

the response Y1 [particle size of the emulsions] and (B) the effect of

oil loading in the emulsion and the percentage of vitamin E in the oil phase on the same response (Y

1).

Figure 5. 3D response surface plots showing (A) the effect of homogenization pressure (psi) (X


2) on

the response Y2 [polydispersity index (PI) of the emulsion] and

(B) the effect of oil loading in the emulsion and the percentage of vitamin E in the oil phase on the same response (Y

2).

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the least concentration of Lipoid E80S could not be identified from the current PlackettBurman design, it clearly demonstrated that higher concentrations are not necessary beyond what is used in commercial products.

Effect of temperature during high-shear homogenization (X

8)

In the literature, different temperatures were reported for the primary high-shear homogenization step when

preparing the crude emulsions using Lipoid E80 as the primary emulsifier. It ranged from ambient conditions, where no heat was applied, to 5070C9,30,40. Some stud-ies considered this a critical factor during the homog-enization process. While temperatures above the phase transition temperature of phospholipids is important to fluidize the bilayers and aid in liposome formation41,42, such requirement may not be critical during emulsion formation by the high-pressure homogenization pro-cess. Furthermore, it is well known that the degradation of heat-labile drugs, including vitamin E, and lipids is time and temperature dependent. Nonetheless, it could be argued that temperatures may lower the interfacial tension and viscosity of the oil phase25 thereby facili-tating emulsion formation. To investigate the effect of temperature, formulations were prepared at either low or high temperatures during the high-shear homogeni-zation step. For low temperature, the formulations were prepared at ambient conditions whereas for high tem-perature, formulations were prepared at 70C, which was frequently cited in the literature. The results of this study (Table 4 and Figure 2) showed that temperature had no significant effect on the physical properties and stability of the emulsions. High temperature did not improve the properties of the emulsion. Therefore, it was concluded that emulsions could be readily prepared at room tem-perature. Instead, a more critical parameter may be the operational temperature at the end of the high-pressure homogenization step. Increasing pressure and number of cycles during homogenization was found to raise the temperature of the developed emulsion (Figure 3). The average temperature of the emulsions at exit ranged from 30C when the emulsions were prepared using five cycles at 5000 psi to 53C for the emulsion made using 25 cycles at 25,000 psi. Evaluating the effect of tempera-ture during high-pressure homogenization, however, was beyond the scope of this study. Nonetheless, it could be readily seen that no correlation existed when the exit temperature from each run was plotted against mea-sured particle size and the stability of the emulsions after 48 h of storage (Figure 7). Stable submicron emulsions were successfully prepared at low temperatures. This may indicate that temperature has little or no effect on the quality of vitamin E/MCT emulsions when prepared using the parameters outlined in this study. To accurately estimate the effect of temperature during high-pressure homogenization, this parameter will be evaluated as an independent variable in future studies by maintaining the temperature constant during homogenization with the aid of heat exchanger.

Conclusions

Parenteral lipid emulsions could be readily prepared by the high-shear homogenization process. However, preparing emulsions for highly viscous or polar oils, such as the case with vitamin E, is challenging. There is a need for emulsions with high vitamin E loading to serve as versatile carriers

Figure 7. Observed particle size (nm) and stability of the emulsions for each of the 12 PlackettBurman runs as a function of the measured temperature of the same emulsions at the end of the high-pressure homogenizing cycles.

Figure 6. Linear plot of the fitted model showing the negative effect of the percentage of vitamin E loaded in the oil phase (fraction of the Vit E/MCT blend, X

6) on the stability of the emulsions, expressed as

the percentage of vitamin E that remained emulsified in water after 48 h of storage at ambient conditions (Y

2).

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for co-administered drugs. To circumvent the challenges associated with emulsifying vitamin E, high viscosity could be addressed by emulsifying blends of viscous vitamin E with low viscosity oil, such as MCT. This may also help lower the polarity of the oil phase and thereby increasing the ratio of emulsifiers at the oil/water interface. This approach was confirmed in this study where the ratio of vitamin E to MCT was found to play the most significant effect on the quality and stability of the emulsions. Increasing the concentration of phospholipids or adjusting homogenization temperature did not improve the quality of the emulsions; rather a more complex emulsifying system may be needed. Other factors, such as homogenization pressure and number of homogenization cycles was also shown to improve the quality of the emulsions, though optimum condition may require further optimization. Overall, stable submicron emulsions with high vitamin E loading could be prepared at ambient temperature using 1.2% phospholipids and 0.5% Tween 80 (0.5%) at low vitamin E to MCT ratios.

Declaration of interest

This study was supported by grant from First Tech International Ltd (Wanchai, Hong Kong). The authors alone are responsible for the content and writing of this article.

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Documents

Alayoubi - Vitamin E Fortified Parenteral Lipid Emulsions