Self-Nanoemulsification of Healthy Oils to Enhance the Solubility of Lipophilic Drugs

Özet

Oils used for drug delivery applications can disrupt the lipid profile of patients, which is undesirable in cardiovascular diseases. Omega-3 fatty acids-rich oils are a healthy alternative to conventional oils and have enormous potential for self-emulsified drug delivery systems.

Abstract

The low aqueous solubility of many drugs reduces their bioavailability in the blood. Oils have been used for centuries to enhance the solubility of drugs; however, they can disturb the lipid profile of the patients. In this study, self-nanoemulsifying drug delivery systems of omega-3 fatty acids-rich oils are prepared and optimized for the delivery of lipophilic drugs. Rosuvastatin, a potent hypolipidemic drug, was used as a model lipophilic drug. Fish oil showed more than 7-fold higher solubility of rosuvastatin than other oils and therefore it was selected for the development of self-nanoemulsifying drug delivery systems (SNEDDS). Different combinations of surfactants and co-surfactants were screened and a surfactant mixture of Tween 80 (surfactant) and Capryol PGMC (cosurfactant) were selected for compatibility with fish oil and rosuvastatin. A pseudoternary phase diagram of oil, surfactant, and co-surfactant was designed to identify the emulsion region. The pseudoternary phase diagram predicted a 1:3 oil and surfactant mixture as the most stable ratio for the emulsion system. Then, a response-surface methodology (Box-Behnken design) was applied to calculate the optimal composition. After 17 runs, fish oil, Tween 80, and Capryol PGMC in proportions of 0.399, 0.67, and 0.17, respectively, were selected as the optimized formulation. The self-nanoemulsifying drug delivery systems showed excellent emulsification potential, robustness, stability, and drug release characteristics. In the drug release studies, SNEDDS released 100% of the payload in around 6 h whereas, release of the plain drug was less than 70% even after 12 h. Therefore, omega-3 fatty acids-rich healthy lipids have enormous potential to enhance the solubility of lipophilic drugs whereas, self-emulsification can be used as a simple and feasible approach to exploit this potential.

Introduction

Lipids have been used for centuries to increase gastrointestinal absorption of water insoluble components of food and medicines¹. Emulsions are the formulations used most widely for oral, intravenous (nutritional supplementation), and topical use². A variety of lipids (fats and oils) are used in the manufacturing of pharmaceutical emulsions and lipid-based self-nanoemulsifying drug delivery systems (SNEDDS). Self-emulsification techniques are widely adopted in pharmaceutical sciences for transmucosal drug delivery. Unlike emulsions, SNEDDS consist of an oil and a surfactant mixture that self-emulsifies in an aqueous medium of the stomach to form emulsion droplets³. They can load lipophilic drugs in the oil phase and prevent them from degrading in the stomach environment⁴. SNEDDS have been shown to effectively enhance the bioavailable fraction of lipophilic drugs (four to six folds) by enhancing solubility and permeability^5,6. The absence of an aqueous phase in SNEDDS offers significant advantages in terms of ease of manufacture and stability as compared to emulsions that are metastable dispersions prone to chemical degradation⁷. Many lipid excipient combinations are commercially available due to their desirable characteristics^8,9.

Cardiovascular disorders are a leading cause of mortality worldwide¹⁰ and, hyperlipidemia causes the blood vessels to obstruct blood flow due to thickening of the blood vessels¹¹. Increased dietary lipid uptake and a sedentary lifestyle are the major risk factors for development of hyperlipidemia. In addition to this, lipids have also been shown to directly damage the myocardium of the heart leading to non-ischemic heart failure¹². Rosuvastatin is a potent hypolipidemic drug that belongs to the statin class and inhibits cholesterol synthesis leading to the lowering of lipid levels for the treatment of hyperlipidemia/dyslipidemia¹³. Rosuvastatin is a biopharmaceutical classification system (BCS) class II with poor aqueous solubility (0.01796 mg/mL)¹⁴. Recent advances in pharmaceutical research have recognized that lipids used in drug delivery can disturb the lipid profile of patients. The role of emulsions to increase low- and high-density lipoproteins and free cholesterol was demonstrated in the late twentieth century¹⁵. In addition to this, lipid-based drug delivery systems have shown to increase triglycerides¹⁶ and other lipid metabolites in the blood¹⁷. Therefore, there is a dire need to develop pharmaceutical formulations of oils that are unable to disturb the lipid profile of cardiovascular and hyperlipidemic patients.

Fish oil is a rich source of omega-3 fatty acids such as eicosapentaenoic acid and docosahexaenoic acid. Fish oil has shown many health effects with substantial evidence of its beneficial role in cardiovascular and nervous systems¹⁸. The aim of the study was to utilize fish oil as an alternative to the conventional oils to formulate SNEDDS for the delivery of a lipophilic drug, rosuvastatin. No previous study has employed fish oil as a carrier to formulate drug delivery systems. Appropriate formulation and processing parameters were selected, and optimization was performed using design expert software.

Protocol

1. Screening of the oils, surfactants, and cosurfactants

1. Screening of the oils, surfactants, and co-surfactants for drug solubility
   1. Mix 100 mg of rosuvastatin separately in 1 mL of different omega-3 fatty acids-rich oils (fish oil, olive oil, sesame oil, and linseed oil) and 1 mL of surfactants and cosurfactants (Tween 80, Capryol PGMC, PEG 400, and ethanol) by vortexing for 5 min at a fixed-speed of 2,500 rpm. Then, place in a shaking water bath for 48 h at 50 °C.
   2. After shaking, let the mixture settle for at least 6 h at room temperature so that the undissolved drug is precipitated. Take 0.1 mL of supernatant by using a micropipette and dilute up to 1 mL with methanol.
   3. Analyze by using a UV-visible spectrophotometer at 242 nm and calculate the concentration by adding the absorption to the straight-line equation of the calibration curve^19,20.
      NOTE: Establish the calibration curve by preparing a stock solution of 100 µg/mL and making serial dilutions up to 0.5 µg/mL. Absorbance is taken for all dilutions and a graph is plotted between absorbance and concentration in a spreadsheet. The straight-line equation (y = mx + b) of the graph is rearranged so that value of absorbance (y) can be used to calculate the unknown concentration (x)^19,20.
2. Screening of the surfactant and co-surfactants for miscibility with oil
   1. Mix the surfactant and the co-surfactant (Smix) in a 3:1 ratio. Add Smix and oil in different ratios; mix and heat up to 50 °C to ensure homogenization.
   2. Take 0.1 mL from each mixture and dilute with 25 mL of distilled water in a glass test tube.
   3. Invert the test tube; the number of inversions at which an emulsion is formed represents the emulsification efficacy and ease of emulsification. Measure the transparency (T%) by measuring the emulsion at 650 nm with a UV-visible spectrophotometer using distilled water as a blank²¹.

2. Construction of the pseudo ternary phase diagram

1. Mix the surfactants and the co-surfactants in various volume ratios (1:0, 1:1, 1:2, 1:3,1:4, and 1:5) to form surfactant mixtures (Smix). Add oil to the Smix in separate vials at various volume ratios of 1:1,1:2,1:3,1:4,1:5,1:6,1:7, 1:8, 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1, and mix by vortexing.
2. Add oil and Smix mixtures in a 10 mL glass vial and heat up to 50 °C with constant stirring at 300 rpm for optimal mixing.
3. Cool the mixture to 37 °C and transfer 1 mL from it to a 250 mL water beaker and add dropwise the preheated distilled water (37 °C) under gentle stirring at 50 rpm. Examine the dispersion visually and the mixture that forms a bluish transparent nanoemulsion is regarded as SNEDDS²¹.
4. Construct the pseudoternary plot at different Smix ratios (Supplementary Table 1) through software (e.g., Triplot).

3. Optimization via software using a response surface methodology (RSM)

1. Select three independent variables as oil (A), surfactant (B) and co-surfactant, and run the software for optimization and screening. Then, select the flexible design by choosing 'NO' to a hard-to-change selection.
   1. Observe the effect of these factors critically on dependent variables such as particle size (Y1, nm), zeta potential (Y2, mV), emulsification time (Y3, s) as well as entrapment efficiency (Y4, %) as mentioned in Table 2.
      NOTE: The runs were increased until the warning signs disappeared and the software itself selected the Box-Benhken design for optimization.
2. Select the higher and lower values as -1 identifying the lowest variable value, whereas +1 depicts the highest value. The mid-value depicted the middle value. Box-Benhken design suggested a total of 17 runs with five center points as a measure to reduce the error.
3. Record the responses to individual runs and fit to linear, 2F1, and quadratic models to ensure the best-fitted model. Generate polynomial equations and utilize to make the inference on the basis of the magnitude of the coefficient corresponding numerical signs.
4. Display data of polynomial regression as 3-D plots. Evaluate the best fitted data model by comparing adjusted R² and predicted R² value ²².
   NOTE: The required selection criteria of the formulation was based on grounds of maximum entrapment efficiency, less particle size, higher zeta potential, and minimum emulsification time frame.
   Y = β₀ + β₁A + β₂B + β₃C + β₁₂AB + β₁₃AC + β₂₃BC + β₁₁A₂ + β₂₂B₂ + β₃₃C₂ Eq. (1)
   Here, β₀ appeared as intercept, Y as a response, ad β₁, β₂, and β₃ as linear coefficients. Β₁₁, β₂₂, and β₃₃ as quadratic terms and squared coefficients while β₁₂, β₁₃, and β₂₃ as interaction coefficients. A, B, and C employed as independent variables that were selected from results of preliminary study.

4. Characterization

1. Thermodynamic stability studies
   1. Store the diluted SNEDDS at 4 °C in a refrigerator, and then transfer it to a 50 °C incubator . Perform six such cycles, each cycle being equal to or greater than 48 h.
   2. Examine the formulation for phase separation²³.
2. Centrifugation test
   1. Centrifuge the diluted SNEDDS for 30 min at 3,500 rpm at -4 °C.
   2. Examine the SNEDDS at room temperature for phase separation or sedimentation of drug²⁴.
3. Dispersibility test for self-emulsification efficacy
   1. Add 1 mL of the formulation dropwise to 500 mL of double distilled water maintained at 37 °C and 50 rpm. Note the time in which a clear homogenous emulsion is formed by visual inspection.
   2. Perform a visual assessment according to the following grading system²⁵.
      Grade 1: a clear bluish emulsion with emulsification time <1 min
      Grade 2: a bluish-white emulsion with emulsification time <1 min
      Grade 3: milky appearance with emulsification time <2 min
      Grade 4: gray, white appearance oil on top with emulsification time >2 min
      Grade 5: emulsification failure with larger oil droplet on top.
      NOTE: The time required for emulsification or rate of emulsification is vital to emulsification efficacy. The test is performed in a USP type II dissolution apparatus.
4. Robustness to dilutions
   1. Dilute the optimized SNEDDS formulation 50, 100, and 1,000 times with different carriers such as distilled water, 0.1 N HCl (pH 1.0) to mimic gastric pH and phosphate buffer (pH 6.8) to mimic intestinal pH.
   2. Thoroughly mix the diluted mixtures and set them aside for 12 h.
   3. Visually examine the formulations for drug precipitation, phase separation, and any other stability issue^26,27.

5. In vitro dissolution studies

1. Perform the release of drug from SNEDDS and pure drug suspension by using a water shaking bath maintained at 37 °C and 50 rpm.
2. Soak the dialysis membranes in the respective media solution for 24 h before the drug dissolution assay to attain good integrity and activation. Fill the drug suspension (in water) and SNEDDS equivalent to 10 mg of rosuvastatin in the dialysis membrane; tie and place in separate beakers (50 mL).
3. Take 1 mL of the sample at specific intervals and replenish the beaker with fresh media (1 mL) after each sample.
4. Filter the drawn samples and analyze by using a UV-visible spectrophotometer at 242 nm^21,28,29,30.

Representative Results

Herein, nanoformulation of omega-3 fatty acids-rich fish oil are prepared and optimized by self-emulsification with different surfactants and co-surfactants. Figure 1 shows the solubility of rosuvastatin in different oils, surfactants, and co-surfactants. Based on solubility, fish oil was selected as the oil, Tween 80 as the surfactant, and Capryol PGMC as the co-surfactant in the following studies. Table 1 shows the screening of Smix at different ratios to emulsify fish oil. Figure 2 presents pseudoternary phase diagrams between different ratios of oil, surfactant, and co-surfactant. The highest nanoemulsion region was observed with Smix 1:3. Table 2 represents factors and responses with their level of flexibility, high and low limits. Figure 3 represents the effect of surfactant and oil concentration on different parameters of SNEDDS. Analysis and optimization parameters of computer-aided optimization are given in supplementary data. Table 3 represents data of the stability and dispersibility studies after SNEDDS are diluted with water. Table 4 represents data for robustness to dilutions test of SNEDDS in distilled water and medias representing stomach and intestinal conditions.

Figure 1: Screening of different excipients for solubility of rosuvastatin presented as (A) solubility of rosuvastatin in different oils, and (B) solubility of rosuvastatin in different surfactants and co-surfactants. Please click here to view a larger version of this figure.

Figure 2: Pseudoternary phase diagrams constructed at different ratios of fish oil and Smix (Tween:Capryol PGMC) such as (A) 1:1, (B) 1:2, (C) 1:3, (D) 1:4, and (E) 1:5 by Triplot. Please click here to view a larger version of this figure.

Figure 3: 3D plots depicting the role of independent variables on zeta size, potential, emulsification time and entrapment efficiency. Please click here to view a larger version of this figure.

Figure 4: Cumulative drug release (%) graphs of rosuvastatin from aqueous suspension and SNEDDS. Please click here to view a larger version of this figure.

Serial No.	S-mix (mL)			Percentage
	Tween-80	Capryol PGMC	Oil (mL)	Transmittance	Sonuçlar
1	0.675	0.225	0.1	99%	+++++
2	0.6	0.2	0.2	98.10%	+++++
3	0.525	0.175	0.3	96.40%	++++
4	0.45	0.15	0.4	95.30%	++++
5	0.375	0.125	0.5	92.70%	+++
6	0.3	0.1	0.6	85.98%	++
7	0.225	0.075	0.7	81.55%	++
8	0.15	0.05	0.8	78.76%	–
9	0.075	0.025	0.9	70.25%	–

Table 1: Screening of surfactant to co-surfactant mixture to emulsify fish oil.

Run	A: Oil	B: Surfactant	C: Co-surfactant	Zeta Size	Zeta Potential	Emulsification time	Entrapment Efficiency
	mL	mL	mL	nm	mv	s	%
1	0.25	0.5625	0.1875	125	18.22	166	78
2	0.1	0.675	0.1875	107	19.13	128	84
3	0.25	0.5625	0.1875	124	18.27	172	77
4	0.25	0.675	0.225	78	23.5	82	98
5	0.4	0.675	0.1875	104	22.56	138	93
6	0.25	0.5625	0.1875	124	18.27	172	76
7	0.25	0.45	0.15	196	13.21	292	67
8	0.1	0.5625	0.15	146	14.79	227	66
9	0.1	0.5625	0.225	116	18.23	122	83
10	0.1	0.45	0.1875	173	13.45	237	65
11	0.4	0.5625	0.15	142	17.56	222	94
12	0.25	0.5625	0.1875	126	18.38	173	77
13	0.4	0.5625	0.225	117	21.45	126	99
14	0.4	0.45	0.1875	197	29.7	120	90
15	0.25	0.675	0.15	122	19.67	183	84
16	0.25	0.45	0.225	169	17.28	181	83
17	0.25	0.5625	0.1875	121	18.19	172	76

Table 2: Composition and different characteristics of the seventeen SNEDDS formulation.

Test	Experimental Conditions	Visual Inspection	Sonuçlar
Heating-Cooling cycle	4°C for 48 hours	• No phase separation	Stable
		• No pallet formation
	50 °C for 48 hours	• No phase separation	Stable
		• No pallet formation
Centrifugation test	3,500 rpm for 30 minutes	• No phase separation	Stable
		• No pallet formation
Dispersibility test	Dropwise addition to 500 mL of double distilled water; maintain at 37 °C and 50 rpm stirring.	• Clear, bluish (Grade 1) emulsion forms within 60 seconds	Stable

Table 3: Stability study data of SNEDDS after heating-cooling cycles, centrifugation and dilution with distilled water.

Dilution	Water pH 7	0.1 N HCl	Phosphate Buffer (pH 6.8)
		(pH 1.2)
50 folds	+++	++	+++
100 folds	+++	+++	++++
1000 folds	+++++	++++	+++++

Table 4: Robustness to dilution test of SNEDDS in different medias relevant to gastrointestinal conditions.

Supplementary File: Please click to download this file.

Discussion

This study was designed to explore the potential of omega-3 fatty acids-rich oil, such as fish oil, sesame oil, olive oil, and linseed oil to act as drug carriers. Self-nanoemulsification was selected as a preferred technique for fabricating the delivery system that lacks water, making it more stable than classical emulsion systems³². Omega-3 fatty acids-rich oils are known for their beneficial health effects¹⁸. They have been used as a supplement for various diseases such as hyperlipidemia-associated cardiovascular diseases³³. Omega-3 fatty acids are routinely added to the nutritional emulsions and many researchers have incorporated small quantities of omega-3 fatty acids-rich oils to pharmaceutical emulsions and SNEDDS for their bioactivity^34,35,36. However, omega-3 fatty acids have been employed in a very low fraction compared to the conventional oils as part of the emulsification systems.

Excipients used in the pharmaceutical dosage forms need to be efficient, inert, and economical. Therefore, the first step in the formulation of SNEDDS is the selection of an oil and a Smix that can load the maximum amount of the drug³⁷. In this study, the highest solubility was exhibited by the fish oil, hence, it was selected as the carrier oil. Fish oil is a rich source of omega-3 fatty acids (i.e., 1 mL of fish oil contains 180 mg of eicosapentaenoic acid and 120 mg of docosahexaenoic acid³⁸). Tween-80 was selected as the surfactant and capryol PGMC as the cosurfactant due to the highest solubility of rosuvastatin. Next, pseudoternary phase diagrams were established in which Smix of different compositions were titrated against fish oil. These plots are typically used to study the phase behavior of three components (oil, surface active agent, and co-surface-active agent). Pseudoternary phase diagrams are commonly used to design pharmaceutical formulations by changing the ratio of its components. Figure 2 shows the area under the curve (colored) where Smix of different co-surfactant to surfactant ratios (1:1 to 1:5) were used. The area under the curve represents the emulsification region; a broader emulsification region indicates better emulsification efficiency^21,39. The results showed that fish oil with Smix at a 1:3 ratio can effectively self-emulsify to form nanoemulsions.

Computer-aided optimization is revolutionizing the pharmaceutical landscape and provides an opportunity to comprehensively understand the formulation parameters⁴⁰. In this study, design software was employed for the possible screening and optimization of three factors at three levels.

The results depicted a strong correlation between independent variables (i.e., oil, surfactant, and co-surfactant). The combination of factors (i.e., AB, AC, and BC) showed nonlinear trends for the zeta size, whereas the zeta potential had a linear relation with all the factors. The effect of the variables on particle size is given by the following polynomial equation (Eq. 2):

Zeta size = 124 + 2.25 A + 40.50 B – 15.75 C – 6.75 AB + 1.25 AC – 4.25 BC + 5.12 A² +16.12 B² + 1.13 C² (Eq. 2)

The mean result of the change of one variable at a specific time from its lowest level to its highest level is represented by the effect of "A", "B" and "C". Results from the above equation clearly showed that oil (A; p 0.172, F-value of 105.88) has a minimal effect compared to the co-surfactant (C; p < 0.0001, F-value of 113.40) and the surfactant (B; p < 0.0001, F-value of 749.83). The oil concentration had non-significant effect on particle size while the surfactant and co-surfactant had a significant effect on reducing the size of nanoparticles. Both significant and non-significant results for the individual and combination factors are represented in Supplementary Table 3.

3D plots also depicted the same trend (i.e., as the concentration of surfactant and co-surfactant was decreased from 0.67 mL and 0.20 mL, the particle size increased). Whereas, by increasing the concentration of oil and decreasing the surfactant, the particle size tends to increase. The combinational impact of the oil and the co-surfactant depicted that increase in the co-surfactant concentration and the oil tends to decrease the particle size. All the results were depicted in Figure 3. AB, AC, and BC represent effects on dependent variable when the two factors were simultaneously changed while, A2, B2, and C2 represent exponential terms for the dependent variable discussed.

When the surfactant concentration was kept constant, oil tends to decrease the emulsification efficacy of the surfactant, which further results in agglomeration of the particles and resulted in increasing particle size. As the surfactant concentration was increased the interfacial tension between the lipoidal and aqueous phase also decreased, which ultimately resulted in decreased particle size⁴¹.

The polynomial equation for the zeta potential variable is given as Eq. 3:

Zeta potential = 18.93 + 3.21 A + 1.40 B + 1.90 C (Eq. 3)

From the equation, it is clear that oil (A; p 0.0098, F-value of 2.12) has a predominant effect in relation to surfactant (B; p 0.209, F-value of 1.75) and co-surfactant (C; p 0.096, F-value of 3.22). The surfactant and co-surfactant have a non-significant effect while the oil concentration has a significant effect on the zeta potential. The negatively charged oil concentration increases the zeta potential linearly. At lower oil and surfactant concentrations, the potential was lower but, it reached a plateau at 0.20 mL and 0.56 mL of oil and surfactant, respectively. This trend remained constant up to 0.29 mL and 0.56 mL of oil and surfactant, and then started to rise after further increase in both levels.

The polynomial equation for the emulsification time is given as Eq. 4:

Emulsification time = 171.35 + 13.50 A – 37.38 B – 51.63 C (Eq. 4)

The above relation depicts that the oil concentration (A; p 0.1691, F-value of 2.12) has the least effect in relation to the surfactant concentration (B; p 0.0014, F-value of 16.25) and co-surfactant concentration (C; p < 0.0001, F-value of 31.01). The surfactant and co-surfactant concentrations have a significant impact while the oil concentration had a non-significant impact on emulsification time. This is because surfactants and co-surfactants ensure emulsification by decreasing surface tension between two phases. The entrapment of different formulations was in the range of 65% to 99%. The higher entrapment is desirable for the optimized formulation for the maximal effect at the target site. The relation depicting the variable effects on the entrapment efficiency is given below (Eq. 5):

Entrapment Efficiency = 76.80 + 9.75 A + 6.75 B + 6.5 C – 4 AB – 3 AC – 0.5 BC +4.35 A² + 1.85 B² + 4.35 C² (Eq. 5)

From the above equation, it can be deduced that all the independent variables had a positive impact on increasing the entrapment efficiency whereas, all the combinational factors had a negative impact on the drug entrapment. The R² and adjusted R² values were 0.99 and 0.98, respectively, which were close to each other in the case of zeta size depicting model significance and low noise. A similar trend was shown by emulsification time and entrapment efficiency. These values are presented in Table 4. Adequate precision gives a signal-to-noise ratio that is greater than 4. All the adequate precision values for the independent variables were greater than 4 (i.e., 35.53%, 7.02%, 13.99%, and 20.89% for zeta size, potential, emulsification time, and entrapment efficiency, respectively).

High entrapment and high value of zeta potential were the main objectives in this study. The design software predicted 0.399, 0.67, and 0.17 for oil, surfactant, and co-surfactant proportions, respectively as independent variables. At this optimal condition, the responses were 104.56 nm, 23.11 mV, and 132.2 s, 95.04% for particle size, zeta-potential, emulsification time, and entrapment efficiency, respectively.

After formulation ad optimization, SNEDDS were characterized by different techniques. The robustness to dilution test determines the stability of SNEDDS to the different levels of dilutions and pH conditions, which may be encountered in the physiological conditions⁶. Fish oil SNEDDS were stable in both gastric and intestinal pH when diluted up to 1,000 times. Dissolution studies were employed to demonstrate the release of rosuvastatin from drug suspension and SNEDDS. Figure 4 shows that fish oil SNEDDS are an effective delivery system of rosuvastatin with complete drug release in about 400 min. Rosuvastatin release from suspension remained incomplete even after 800 min. A few researchers have designed bioactive SNEDDS containing rosuvastatin. For example, garlic acid was added to rosuvastatin SNEDDS for the treatment of hypertriglyceridemia⁴². Although results showed a significant decrease in triglyceride levels, yet the mechanism of synergistic action of rosuvastatin and garlic oil is unknown. On the other hand, fish oil or omega-3 fatty acids are widely reported for their health effects. Therefore, fish oil has the potential to be used as a bioactive carrier of lipophilic drugs, which may synergize the treatment of various diseases, such as cardiovascular, nervous, and inflammatory disorders.

The study has successfully demonstrated that fish oil can be used as a healthy alternative to the conventional oils. Optimization of the SNEDDS formulations was performed by the construction of the pseudoternary phase diagram and response surface methodology. A pseudoternary phase diagram construction is a laborious process and requires extensive resources for multiple trials, which remains a limitation of this study. However, this method was adopted because fish oil was not used as a carrier previously and careful optimization of the formulations was deemed necessary. Furthermore, this study builds the rationale for using fish oil as a healthy carrier of lipophilic drugs based on the finding of in vitro studies. In the future, studies on healthy animals for bioavailability assessment and hyperlipidemia animal models for synergistic activity evaluation may be performed as part of pre-clinical studies.

Materials

Ammonium acetate	Sigma-Aldrich, Germany	A1542	Analytical grade
Capryol PGMC	Gattefossé, France	RT9P9S09QI	Analytical grade
Design Expert Software	StatEase, United States	Version 12.0.3.0	Analytical software (freely available for subscription)
Dialysis tubing (12,000 Daltons MWCO)	Visking, UK	12000.02.30	Pure regenerated natural cellulose membranes with 12,000 Daltons MWCO
Dissolution apparatus	Memmert, Germany	SV 1422	USP type II dissolution apparatus
Ethanol	Honeywell, Germany	24194	Analytical grade
Fish oil	Wilshire Labs Pvt(Ltd), Pakistan	not applicable	Received as gift sample.
Hydrochloric acid	BDH Laboratories Ltd, UK	BDH3036-54L	Analytical grade
Methanol	Honeywell, Germany	34966	Analytical grade
Refrigerator (Pharmaceutical)	Panasonic, Pakistan	MPR-161 DH-PE	Refrigerator for storage at 4 °C
Rosuvastatin calcium	Searle Pharmaceuticals Pvt(Ltd) Pakistan	not applicable	Received as gift sample.
Sodium Hydroxide	Honeywell, Germany	38215	Analytical grade
Span 80	BDH Laboratories Ltd, UK	MFCD00082107	Analytical grade
Triplot Software	MS Excel spreadsheet developed by Tod Thompson	Triplot Ver. 4.1.2	Analytical software (freely available)
Tween-80	Sigma-Aldrich, Germany	P1754-500ML	Analytical grade
UV-Vis spectrophotometer	Dynamica, UK	Halo DB-20	Double beam spectrophotometer
Water Bath	Memmert, Germany	WNB 7	Water batch for heating up to 70 °C