Nanoemulsions: Increasing Possibilities in Oral Drug Delivery
R. G. Maskare*, N. H. Indurwade, R. A. Deshmukh, P. V. Kuthe, P. M. Londhe,
M. T. Deshmukh
Manoharbhai Patel Institute of Pharmacy (B. Pharm), Kudwa, Gondia, Maharashtra. 441614.
*Corresponding Author E-mail: rinamaskare@yahoo.co.in
ABSTRACT:
The objective of our work was to maximize the therapeutic efficacy of Atrovastatin by developing oil in water (o/w) nanoemulsion to increase its poor solubility and consequently enhance the oral bioavailability. The aim of investigation was to rectify the first-pass metabolism of Atorvastatin, using preparation of nanoemulsion as a prominent delivery system. Olive oil, surfactant (tween 80, tween 40, tween 20) and co-surfctants including polyethylene glycol was selected after solubility studies. Then pseudo-ternary phase diagram with surfactant/ co- surfactant ratio of 1:1, 2:1, 3:1 and 4:1 were constructed and spontaneous emulsification method. Titration method was used to optimize the Atorvastatin loaded nanoemulsions. The prepared nanoemulsion was characterized by globule size, refractive index, partition coefficient, viscosity, drug entrapment efficiency, in vitro drug release studies. Optimised nanoemulsion obtained was composed of olive oil (8.5 %w/w), tween 80 (11.5 %w/w) and water (80 %w/w). The results obtained showed an average globule size. The Optimized formulation showed an improvement in solubility of Atorvastatin. The use of pseudo-ternary phase diagram allows obtaining an optimal nanoemulsion with perfect stability for three months and a better solubilization capacity of Atorvastatin.
KEYWORDS: Nanoemulsion, Atorvastatin, Surfactant, Co-surfactant, Gastrointestinal tract, Pseudo ternary phase diagram, In-vitro drug release, o/w and w/o nanoemulsion.
INTRODUCTION:
A nanoemulsion is a colloidal system consisting of lipid and aqueous phase which is stabilized by addition of surfactant which decreases the interfacial tension. The particle size ranges from 5-200nm. It is kinetically stable and has low viscosity. Thus, nanoemulsion is considered to be the thermodynamically stabilized dosage form. Three types of nanoemulsion are formed depending on the composition Oil in water (o/w), Water in oil (w/o), Bicontinuous nanoemulsion where microdomains of oil and water are interdispersed within the system.
The three main components of nanoemulsions are oil, water and surfactant/co-surfactant [1]. Nanoemulsions are intended to increase the rate of absorption; it can be taken orally, intravenously or topically. It increases the bioavailability and efficiently penetrates the drug moiety.
According to Biopharmaceutical classification system (BCS)-class II drugs show the low solubility and consequently low bioavailability. On the basis of BCS, different strategies can be attempted to increase the solubility of drug; either by increasing amount of dissolved drug i.e. in contact with the absorbing membrane or increasing the permeability of the membrane. The BCS class II drugs, having limited dissolution but no limited permeation. Hence, there is need to increase the amount of dissolved drug at the absorption site and it is proven to be effective in many studies. Various techniques have been developed to enhance the solubility of poorly-soluble drugs [2]. Lipid-based formulation approach has been attracted wide attention in the context of improving the oral bioavailability of low water-soluble drugs and delivering the drug at a target site. The most popular approach is the incorporation of the active lipophilic component into inert lipid vehicles such as oils, surfactant dispersions, self nano or micro emulsifying formulations, solid emulsions, and liposomes[3].
Atorvastatin (ATR) is a competitive inhibitor of HMG-CoA reductase which catalyses’ the reduction of 3-hydroxy-3 methylglutaryl-coenzyme A (HMG-CoA) to mevalonate, the rate limiting step in hepatic cholesterol biosynthesis. ATR is most efficient drug for reducing plasma cholesterol level in atherosclerosis. Inhibition of the enzyme decreases de novo cholesterol synthesis and increases expression of low-density lipoprotein receptors (LDL receptors) on hepatocytes. This increases LDL uptake by the hepatocytes and decreases the amount of LDL-cholesterol in the blood. ATR also reduces blood levels of triglycerides and slightly increases levels of HDL-cholesterol. ATR is rapidly absorbed after oral administration. The absolute bioavailability of atorvastatin is approximately 14% and the systemic availability of HMG-CoA reductase inhibitory activity is approximately 30%. The absorption of ATR is slower and it completes in course of time after oral administration [3]. ATR is drug of choice in atherosclerosis. In addition it can be used in different diseases such as hypercholesterolemia, coronary heart disease, stroke, unstable angina and revascularization. Atherosclerosis is a gradual process in which plaques (collections) of cholesterol are deposited in the walls of arteries. Cholesterol plaques cause hardening of the arterial walls and narrowing of the inner channel (lumen) of the arteries. Hence, those narrowed arteries are unable to deliver enough blood to peripheral organs. Nanoemulsion formulation is suitable for targeted drug delivery system act as a super solvent for both hydrophilic and lipophilic drugs and it increases the solubility of ATR[4]. The objective of the present study is to prepare nanoemulsion of Atorvastatin for oral delivery and to improve its solubility and bioavailability.
MATERIALS AND METHODS:
Materials
ATR was provided by Ind Swift Pvt. Ltd. Olive oil, tween 80 and polyoxyethylene glycol, methanol purchased from Merck. Dialysis bags are prepared by using cellophane paper. Deionized water for HPLC analysis was prepared by Milli-Q-purification system. All other chemicals were of analytical grade. Double distilled water was prepared freshly whenever required.
Solubility Studies:-
The most important aspect for the selection of oils for nanoemulsion is the solubility of poorly soluble drug in oils. An excess amount of drug was added in 2 ml of each oil separately in 5 ml capacity stoppered vials, and mixed using a vortex mixer. These vials were then kept at 25±1.0oC in an isothermal shaker (IKA® KS 400i, Germany) for 72 hours to reach equilibrium. The equilibrated vials were removed from shaker and centrifuged at 10000 rpm for 30 min using centrifuge (Remi, India). The supernatant was taken and filtered through a 0.45 µm membrane filter. The concentration of Atorvastatin was determined in different oils by using UV at detection wavelength of 246nm[5,6,7].
Pseudo Ternary Phase Diagram:-
On the basis of the solubility studies of drug olive oil was selected as the oil phase. Tween 20 and PEG were used as surfactants and co- surfactant respectively. Water was used as an aqueous phase for the construction of phase diagram. Oil, surfactants and co-surfactant (Smix) were grouped together for phase study. Surfactant and co-surfactant in group was mixed in weight ratio (1:1). For phase diagram oil and specific ratio was mixed thoroughly in different weight ratios from 1:1, 2:1 and 3:1 in different glass vials. Three different combinations of oil and Smix, 1:1, 2:1 and 3:1 were made so that maximum ratio was covered for the study to delineate the boundaries of phases precisely formed in the phase diagrams. Pseudo-ternary phase diagram were develop using aqueous titration method. Slow titration with aqueous phase. It was done to each weight ratio of oil and Smix and visual observation was carried out for transparent and easily flow able oil in water nanoemulsion. The physical state of nanoemulsion was marked on pseudo three component phase diagram with one axis representing aqueous phase. The other representing oil and the third representing a mixture of surfactants and co-surfactants at fixed weight[8,9].
Figure: 1- Ternary phase diagram for 1:1, 2:1 and 3:1 oil/Smix.
Preparation of standard curve:
The absorption maxima of drug were determine by UV-Spectrophotometer and was used for preparation of standard curve in pH 7.2 phosphate buffer solution. Various concentrations of 2,4,6,8, & 10 microgram per milliliter sample were prepared and analyzed for absorbance. The data was then treated statistically to obtain the value of Y=MX+C.
Preparation of nanoemulsion:
The drug loaded nanoemulsions were prepared by dissolving 10mg (single dose) of Atorvastatin in oil (10%, 15%, 20% and 25% v/v). Respective smix ratio was added to the oil, mixed using magnetic stirrer and followed by addition of aqueous phase to obtain o/w nanoemulsion[9].
Dispersibility tests:
One ml of each formulation was added to 500 ml of 0.1 N HCl in USP Dissolution apparatus Type II at 37 ± 0.5oC to assess its efficiency of self emulsification [10]. A standard stainless steel dissolution paddle rotating at 75 rpm provided gentle agitation[10]. The formulation was visually assessed using the following grading system:-
Grade A: Rapidly forming (within 1 min) nanoemulsion, having a clear appearance.
Grade B: Rapidly forming, slightly less clear emulsion.
Grade C: Fine milky emulsion that formed within 2 min.
Grade D: Dull white emulsion having slightly oily appearance that is slow to emulsify (longer than 2 min).
Grade E: Formulation, exhibiting either poor or minimal emulsification with large oil globules present on the surface. Among the formulations which passed the stability and also dispersibility tests in Grade A and B were selected for preparing drug loaded batches utilizing minimum concentration of smix for each percentage of oil.
Stability tests:
Centrifugation and freeze thaw cycling were used to assess the physical stability of the prepared Nanoemulsion. The formulations were centrifuged at 3500 rpm for 30min. Those formulations that did not show phase separation were subjected to freeze thaw studies. Three freeze thaw cycles between – 20oC and +25oC with storage at each temperature for not less than 24h was done for the formulations. Those formulations, which passed these thermodynamic stress tests, were further taken for the dispersibility tests[10].
Evaluation Parameters[11, 12, 13, 14 ].
Viscosity determination
The rheological property of the formulations was determined as such without dilution using Brookfield DV-II ultra+ viscometer (Brookfield Engineering Laboratories, Inc., Middleboro, MA) using spindle # CPE 40 at 25 ± 0.5şC. The software used for the calculations was Rheocalc V2.6. Experiments were performed in triplicate for each sample, and results were presented as mean ± S.D.
Electroconductivity studies:
Electroconductivity of the resultant system was measured by an electroconductometer (Conductivity meter 305, Systronic). For the conductivity measurements, the tested nanoemulsions were prepared with a 0.01 N aqueous solution of sodium chloride instead of doubled distilled water. The measurements were made in triplicate at 25±1oC.
Refractive index and percent transmittance:
The refractive index of the system was measured by an Abbe refractometer (Bausch and Lomboptical Company, NY) by placing a one drop of nanoemulsion on the slide in triplicate at 25oC. The percent transmittance of the system was measured at 650 nm using UV spectrophotometer (Shimadzu, Japan) keeping doubled distilled water as blank. The measurements were made in triplicate.
Drug content:
The dose of the drug is well below the saturation point; hence it is presumed that the amount of drug incorporated will be available for the release. Since surfactant and co surfactant (smix) are added, there are chances of precipitation of the drug. Hence, the drug content was calculated by UV visible spectrophotometer. The formulation was diluted to required concentration using methanol as solvent and the absorbance was measured at 246nm against a solvent blank. Encapsulation efficiency was expressed as a percentage of Atorvastatin found in the system to the theoretical quantity of the drug added. All the measurements were made in triplicate.
In vitro drug release:
The quantitative in vitro release test was performed in 500 ml of Phosphate buffer pH 6.8 using USP Dissolution apparatus Type II at 75 rpm and 37±0.5oC using dialysis bag technique. Dialysis membrane (MWCO 12000 g/mole) was soaked in double-distilled water for 12h before use for experiment. Two millilitre of nanoemulsion formulation (containing single dose 10mg of Atorvastatin) was placed in treated dialysis bag. Samples (5ml) were withdrawn at regular time intervals (0, 0.5, 1, 1.5, 2, 4, 6, 8 and 12 h) and an aliquot amount of phosphate buffer was replaced. The release of drug from nanoemulsion formulation was compared with the conventional tablet formulation (Atorvastatin 10 mg) and the suspension of pure drug. To prepare drug suspension, drug and methylcellulose mucilage (3% w/v) were ground in a mortar to obtain a 5mg/ml drug suspension; this suspension was ultrasonicated for 2 minutes. The samples were analyzed for the drug content using UV-Visible spectrophotometer (Shimadzu, Japan) at 246 nm.
RESULTS AND DISCUSSION:
Solubility Studies
Atorvastatin lipophilicity and vulnerability to enzymatic degradation restrict its oral bioavailability. Solubility studies were aimed at identifying a suitable oil phase for the development of Atorvastatin nanoemulsion to achieve optimum drug loading [6]. The higher solubility of the drug in the oil phase is important for the nanoemulsion to maintain the drug in the solubilised form. In the oil phase tested, the solubility of Atorvastatin was found to be highest in olive oil (60±2.69 mg/ml) as compared to other oils Thus, olive oil was selected as the oil phase for the development of the formulation.
Figure:2- Charts showing solubility of Atorvastatin in different lipid phase.
Analytical development method:
It involves UV analysis. When UV analysis of ATR is done at 246 nm a standard curve is obtained, plotted between concentration and absorbance. From the plot slope is obtained given in Fig (2).
Figure: 3- UV Spectrophotometer
Figure: 4- Standard curve for Atorvastatin.
Stability tests:
Nanoemulsion can be differentiated from ordinary emulsions due to their thermodynamically stability [7]. In order to avoid phase separation, creaming or cracking, stability tests like centrifugation and freeze thaw cycle were performed. Those formulations, which survived stability tests (Table 1), were taken for dispersibility test in order to estimate the efficiency of dispersibility.
Table No.1- Dispersibility tests of different formulations selected from phase diagrams at a difference of 5%v/v of oil.
|
Smix ratio |
Oil (%v/v) |
Smix (%v/v) |
Aqueous (%v/v) |
Dispersibility grade |
Inference |
|
1:1 |
10 15 20 |
10 12 30 |
80 73 50 |
A --- B |
PASS FAIL PASS |
|
2:1 |
10 15 20 25 |
15 30 32 36 |
75 55 48 39 |
--- B A --- |
FAIL PASS PASS FAIL |
|
3:1 |
10 15 20 25 |
8 24 32 36 |
82 61 48 39 |
--- A B B |
FAIL PASS PASS PASS |
Table No.2- Optimized formulations selected from phase diagram.
|
Batch Code |
Smix ratio |
Oil (%) |
Smix (%) |
Aqueous (%) |
Oil: Smix: ratio |
Dispersibility grade |
|
NE1 |
1:3 |
10 |
23 |
67 |
1:2:3 |
A |
|
NE2 |
3:1 |
15 |
24 |
61 |
1:1:6 |
B |
|
NE3 |
1:2 |
20 |
27 |
53 |
1:1:35 |
A |
|
NE4 |
1:3 |
25 |
32 |
43 |
1:1:28 |
A |
Table No.3- Evaluation parameter Mean (± S,D., n=3)
|
Batch code |
Percent transmittance |
Refrective Index |
Viscosity(cP) |
Conductivity (u S/cm) |
Drug Content (%) |
|
NE1 |
99.43±0.03 |
1.352±0.017 |
18.08±1.14 |
412.3±3.47 |
98.62±0.56 |
|
NE2 |
99.62±0.03 |
1.354±0.014 |
20.12±0.95 |
387.2±2.43 |
99.41±0.14 |
|
NE3 |
99.51±0.04 |
1.366±0.021 |
23.25±0.97 |
366.5±1.21 |
98.44±0.23 |
|
NE4 |
99.48±0.05 |
1.412±0.013 |
28.55±1.16 |
342.2±4.22 |
98.84±0.63 |
Dispersibility tests:
The formulations that passed the dispersibility test in 0.1N HCL in grade A and B (as specified in Table 1) were considered to pass the dispersibility test and were selected for further study.
Oil content was increased from 10% v/v to 25% v/v, an increase in the viscosity of the formulations was observed. Overall, the viscosity of the optimized formulations was low as expected for o/w nanoemulsion. Formulation NE1 had a significant (p < 0.05) difference in electroconductivity compared to other formulations (Table 3). The higher conductivity of NE1may is due to higher percent of conducting ions in the aqueous media. The RI of the selected formulations was determined using an Abbe type refractometer. It indicates the isotropic nature of the formulation and was found to be in the range of 1.352-1.412. The results (Table 3) indicate that RI values increased with increase in concentration of oil and corresponding decrease in aqueous content. NE 4 exhibited highest RI of 1.412 which was significant in comparison to other formulations (p < 0.05). The RI of the developed system was found to be similar to that of the water (1.334). The transmittance of the developed formulations was found greater that 99% (Table 3). Amongst the selected formulations, formulation NE2 had highest percentage of transmittance which was significant (p < 0.05) in comparison to other formulations. The observed transparency of the system is due to the fact that the maximum size of the droplets of dispersed phase is not larger than 1/4th of the wavelength of visible light. Thus, nanoemulsion scatter little light and therefore transparent or translucent.
Drug Content:-
Atorvastatin content in the nanoemulsion formulations was analyzed spectrophotometrically at 246 nm, against solvent blank. Drug content of the optimized formulations was found in range of 98.84-99.41%. The drug content varied for up to 0.57% between formulations NE1 to NE4 (Table 3). However, there was no significant (p > 0.05) difference in drug content among various formulations.
Figure 5:-In vitro release profile of Atorvastatin from different optimized nanoemulsion formulations (NE1 to NE4).
CONCLUSION:
In this investigation, lipid nanoemulsion containing Atorvastatin was successfully optimized based on physicochemical parameters, in vitro and in vivo performance. The absorption of Atorvastatin from nanoemulsion resulted in increase in relative bioavailability. The dose of Atorvastatin nanoemulsion needs to be corrected in accordance with increased bioavailability; to minimize its dose related adverse effects. Results from stability studies indicate stability of optimized formulation, as there was no significant change is observed in physical parameters. Our studies demonstrate that the lipid nanoemulsion formulation is the promising strategy for the formulation of lipophilic compounds with low oral bioavailability.
ACKNOWLEDGEMENT:
We are highly thankful to the Principal, Dr. N. H. Indurwade and the Department of Pharmacy, Manoharbhai Patel Institute of B. Pharmacy Kudwa, Gondia (M.S.) for providing various facilities like library facility during literature survey, laboratory and chemicals/ reagent facilities during experiments.
CONFLICT OF INTEREST:
The authors declare no conflict of interest.
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Received on 25.11.2020 Modified on 11.12.2020
Accepted on 29.12.2020 ©Asian Pharma Press All Right Reserved
Asian J. Pharm. Tech. 2021; 11(1):53-58.
DOI: 10.5958/2231-5713.2021.00009.X