Ece 6

Indo Global Journal of Pharmaceutical Sciences, 2020; 10(1): 1-11
INDO GLOBAL JOURNAL OF

PHARMACEUTICAL SCIENCES
ISSN 2249- 1023
Effect of Surfactants and Co-surfactants on Phase Behaviour and

Physicochemical Properties of Self-nanoemulsifying Drug Delivery
System Loaded with Plumbagin
Kamble Pavan Ram, Shaikh Karimunnisa Sameer *
Progressive Education Society’s Modern College of Pharmacy, Yamunanagar, Nigdi, Pune 411044, Maharashtra, India
Address for Correspondence: Shaikh Karimunnisa Sameer, [email protected]
Received:
19.02.2019 ABSTRACT: Purpose: Plumbagin has wide pharmacological actions but limited solubility and low oral
Accepted: bioavailability. Self-nanoemulsifying drug delivery system (SNEDDS) has potential to improve solubility and
27.05.2019 dissolution rate. Selection of surfactant and co-surfactant is critical for a successful SNEDDS. Aim of present
work was to investigate effect of different surfactants and co-surfactants on phase behaviour and
Keywords physicochemical properties of SNEDDS loaded with plumbagin. Methods: Solubility of plumbagin in various
Plumbagin; oils, surfactants and co-surfactants was estimated. Out of those, Capmul®MCM (oil), Tween®20 and Tween®
Surfactants; 80 (surfactants), polyethylene glycol 400 and propylene glycol (co-surfactants) in varying concentrations and
Solubility; combinations of surfactant: co-surfactant (Smix) were employed to construct pseudo-ternary phase diagrams.
Ternary. Thermodynamic stability, dispersibility, robustness to dilution, self-emulsification time, % transmittance and
globule size tested. Results: Capmul®MCM, Tween®20, Tween® 80, polyethylene glycol (PEG) 400 and
propylene glycol (PG) demonstrated the highest solubilisation and emulsification ability and studied further
using ternary phase diagrams. Tween®20 showed larger self-emulsification region compared to Tween® 80 in
combination with all co-surfactants studied. Tween® 80 could not form any nano-emulsion with PEG 400.
Propylene glycol gave clear isotropic regions with 30% of oil. With Tween 20, it could emulsify up to 40% of
oil. SNEDDSs prepared using Tween®20 with each of the co-surfactants passed dispersibility test. SNEDDSs
containing Tween®20 and propylene glycol displayed smaller globule size, less self-emulsification time, high
transmittance and than those prepared with Tween®20 and PEG 400. Conclusion: Tween®20 and propylene
glycol are suitable surfactant and co-surfactant for plumbagin loaded SNEDDS. © 2019 iGlobal Research and
Publishing Foundation. All rights reserved.
Cite this article as: Kamble, P.R.; Shaikh, K.S. Effect of surfactants and co-surfactants on phase behaviour
and physicochemical properties of self-nanoemulsifying drug delivery system loaded with plumbagin. Indo
Global J. Pharm. Sci., 2020; 10(1): 1-11. DOI: http://doi.org/10.35652/IGJPS.2020.10101 .
INTRODUCTION
Most of the new drug candidates and many existing drug considerable attention has been focused on lipid based
molecules show poor aqueous solubility, which leads to poor formulations to improve the oral bioavailability of poorly
oral bioavailability, high intra-and inter-subject variability and water-soluble and lipophilic drugs. In fact, the most popular
lack of dose proportionality [1]. The greatest challenge is to strategy is the incorporation of the drug molecule into inert
present the poorly water-soluble drugs into orally administered lipid vehicles such as oils and surfactant dispersions, self-
medications with sufficient bioavailability. To increase the emulsifying formulations, emulsions, and liposomes with
oral bioavailability of poorly water soluble drugs, various particular emphasis on self-nanoemulsifying drug delivery
formulation strategies have been adopted including the use of systems (SNEDDS) [3]. Self nanoemulsifying drug delivery
cyclodextrins, nanoparticles, solid dispersions, permeation systems (SNEDDS) are isotropic and thermodynamically
enhancers and lipid-based formulations [2]. In recent years, stable mixtures of oil, surfactant, co-surfactant and drug that
1
have a novel property of forming fine oil-in-water (o/w) ultrasonication. Chellampillai et al. [10] prepared surface
nanoemulsion in the nanometric range (10-100 nm) when modified PLB crystals by cold recrystallization technique.
introduced into the aqueous phase under gentle agitation [4]. Rajalakshmi et al. [13] prepared different PLB crystals using
Upon administration, the isotropic mixture will come in anti-solvent precipitation, melt solidification, melt quenching,
contact with the aqueous phase of gastrointestinal tracts and sonocrystallization and melt sonocrystallization processes.
form an oil-in-water nanoemulsion with the aid of Sunil Kumar et al. [14] prepared PLB loaded long circulating
gastrointestinal motility. This spontaneous formation of PEGylated liposomes. Mandala et al. [15] prepared chitosan
nanoemulsion in the gastrointestinal tract presents the drug in based PLB microsphere.
a solubilized form, in small droplets of oil, all over its transit
through the GIT [5]. The nano-sized droplets provide a large Poor water solubility and consequent restricted absorption is a
interfacial surface area for drug release and absorption. Apart major limitation with many drugs despite their good efficacy.
from solubilization, the presence of oily phase in the SNEDDS provides an opportunity for the improvement of the
formulation helps improve bioavailability by affecting the in vitro and in vivo performance of poorly water soluble drugs
drug absorption [6]. Selection of a suitable self- and thus serves as an ideal carrier for the delivery of drugs
nanoemulsifying formulation depends mainly upon the belonging to Biopharmaceutics Classification System (BCS)
assessment of drug solubility in various components, the area classes II and IV [32]. Thus, SNEDDS is an important viable
of the self-nanoemulsifying region obtained in the phase option for PLB oral delivery. The performance of a SNEDDS
diagram, and the droplet size of the resultant emulsion depend sound selection of its component system, most
following self-emulsification [7]. Finally, SNEDDS offer the importantly, the surfactant and co-surfactant system.
opportunity to deliver poorly water soluble drugs to the The present study reports the effect of different surfactants and
gastrointestinal tract in a dissolved state which leads to co-surfactants on the phase behaviour and properties of
avoiding the dissolution step (which can limit the absorption SNEDDS by use of ternary phase diagrams. This study helped
rate of lipophilic drugs), reduction in inter-and intra-subject in selection of product variables for optimization of PLB
variability, reduction of food effect and ease of manufacturing SNEDDS.
and scale-up [8]. Plumbago zeylanica Linn (family
Plumbaginaceae), is an erect semi-climbing shrub and grows MATERIALS AND METHODS
throughout Asia, Africa and Australia. Traditionally, the aerial
Materials
part of plant has long been used as medicine to treat rheumatic
Plumbagin (PLB) was purchased from PEE ESS Aromatics
pain, scabies, skin diseases, dysmenorrhea, injury by bumping,
Chennai, India. Capmul® MCM EP was a kind gift from
wounds and even cancer. The roots of the plant and its
IMCD Ltd, Mumbai. Oleic acid was obtained from Research
constituents have been reported to possess potential
Lab Fine Chem. Sesame oil, Tween® 20, Tween® 80, Span®
antiatherogenic, cardiotonic, hepatoprotective,
80, PEG 200, PEG 400 were purchased from S.D. Fine Chem.
neuroprotective, antimicrobial and anti-ulcer activity.
Ltd., Mumbai, India. Cremophor® RH 40 were obtained from
Plumbagin (5-hydroxy-2-methylnaphthalene 1, 4-dione) is a
BASF, Germany. Labrafac® was a kind gift sample from
yellow crystalline naphthoquinone abundantly present in the
Gattefosse Canada Inc. Castor oil was purchased from
roots of P. zeylanica. Plumbagin (PLB) has been explored for
Himedia Pvt, Ltd. Propylene glycol (PG) (99.5%) was bought
its anti-cancer, anti-inflammatory, anti-bacterial, anti-fungal,
from BDH Laboratory, UK. Glycerol and Span®80 were
CNS stimulant and anti-ulcer activity [9]. Despite the great
obtained from Loba Chemie.
therapeutic interest, PLB showed low oral bioavailability
(39%) due to its high lipophilicity (log P = 3.04) and poor
Methods
aqueous solubility (79.3 ± 1.7 μg/ml); factors considered as
Solubility study
major challenges in developing formulations for clinical
The solubility of PLB in various oils (Capmul® MCM , oleic
efficacy [10]. Besides, the higher and frequent dose often
acid, arachis oil, cinnamon oil, olive oil, sesame oil, castor oil,
causes severe side effects including diarrhea, skin rashes,
Labrafac®PG), surfactants (Tween® 20, Tween® 80,
increase in white blood cells and neutrophil count [9]. To
Cremophor® RH 40, Span® 80) and co-surfactants (PG, PEG
overcome this problem a number of attempts have been made
200, glycerol, PEG 400 was determined. An excess amount of
to enhance the solubility and, therefore, bioavailability of
PLB was added to 2 ml of each component in screw-capped
PLB. Singh and Udupa [11] prepared inclusion complex of
glass vials and mixed continuously for 2 min using magnetic
PLB with beta-cyclodextrin employing neutralization method.
stirrer (REMI 1 MLH). The mixtures were then shaken (100
Sheik et al. [12] developed nanoplumbagin encapsulated with
rpm) for 72 h at 25 ± 0.5°C in an orbital shaker (REMI-BL)
cetyl trimethylammonium bromide or beta-cyclodextrin using
followed by equilibrium for 24 h [16]. The equilibrated
2
samples were removed and centrifuged (REMI C-24 BL) at ternary phase diagram [21]. Self-nanoemulsifying systems
5000 rpm for 30 min. The supernatant solution was taken and form fine o/w emulsions when introduced into aqueous media
filtered through a Millipore membrane filter (0.45 μm) and with gentle agitation. Surfactant and co-surfactant get
then suitably diluted with methanol water system (50:50). The preferentially adsorbed at the interface, reduce the tension, and
concentration of PLB was determined spectrophotometrically provide mechanical barrier to prevent the globules from
using UV-Visible spectrophotometer (JASCO V-630) at 268 coalescence. The decrease in free energy required for
nm using methanol water system (50:50) as a blank. The emulsion formation consequently improves the
experiment was repeated in triplicates. thermodynamic stability [22]. On the basis of the solubility
studies and preliminary screening of surfactants, Capmul®
Preliminary screening of surfactants MCM was selected as the oil phase, Tween® 20 and Tween®
Different surfactants for the per-oral use were screened for 80 as the surfactants and PEG 400 and PG as the co-
emulsification ability according to the method described by surfactants. Double distilled water was used as the aqueous
Date and Nagarsenker [17]. Briefly, 300 mg of each selected phase for construction of phase diagrams. Pseudo-ternary
surfactant was added to 300 mg of the chosen oily phase phase diagrams of mixed surfactant and co-surfactant (Smix),
(Capmul® MCM). The mixtures were gently heated at (45°C - oil and water but without drug incorporation were plotted, and
60°C) for homogenization of the components. 50 mg of each each of them represents a side of the triangle. For any mixture,
mixture was then diluted with double distilled water to 50 ml the total of surfactant, co-surfactant and oil concentrations
in a stoppered volumetric flask to yield fine emulsion. Ease of always added to 100% [23]. Surfactant and co-surfactant were
emulsification was judged by the number of flask inversions mixed in three ratios, namely; 1:1, 2:1 and 3:1 (Smix). For
required to yield homogenous emulsion. The resulting each phase diagram, oil and specific Smix ratio was mixed
emulsions were allowed to stand for 2 h and their % thoroughly in nine different ratios from 1:9 to 9:1 in different
transmittance was evaluated spectrophotometrically at 638 nm glass vials. The nine different combinations of oil and Smix;
by UV spectrophotometer (JASCO V-630 ) using double 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1 were made so that
distilled water as a blank. Emulsions were furthermore maximum ratios were covered for the study to delineate the
observed visually for any turbidity or phase separation [18, boundaries of phase precisely formed in the phase diagrams.
19]. Pseudo-ternary phase diagrams were developed using the
aqueous titration method. Slow titration with aqueous phase
Preliminary screening of co-surfactants was done to each weight ratio of oil and Smix. The total water
The selected oily phase and surfactant were used for further consumed was noted during titration of oil–Smix ratio and
screening of the different co-surfactants (PEG 400, PEG 200, observations were made for phase clarity [22]. The formation
PG, Glycerol) for their emulsification ability. Mixtures of 100 of the nanoemulsion was visually observed as
mg of co-surfactant, 200 mg surfactant, and 300 mg oil clear/transparent and easily flowable/dispersible with low
mixture were prepared and this mixture was homogenized viscosity o/w nanoemulsion and marked on the pseudo-ternary
with the aid of the gentle heat (45°C-60 ºC). The isotropic phase diagram [24]. The point from clear to turbid and turbid
mixture, 50 mg, was accurately weighed and diluted to 50 ml to clear was considered as the end of titration. The amount of
with double distilled water to yield fine emulsion. The ease of each component (oil, Smix and water) at this point was
formation of emulsions was noted by noting the number of recorded and presented in a pseudo-ternary phase diagram
flask inversions required to give uniform emulsion. The [25].
resulting emulsions were observed visually for the relative
turbidity. The emulsions were allowed to stand for 2 h and Formulation of PLB loaded SNEDDS
their transmittance was measured at 638 nm by UV Once the self-emulsifying region was identified, the desired
spectrophotometer (JASCO V-630 ) using double distilled component ratio of SNEDDS were selected (Table 1). PLB
water as blank. As the ratio of co-surfactants to surfactant/s is loaded SNEDDS were prepared by the method reported by
the same, the turbidity of resulting nanoemulsions will help in Gupta et al [26]. The calculated amount of surfactant (Tween®
assessing the relative efficacy of the co-surfactants to improve 20) and co-surfactant (PG) were mixed in a vial on magnetic
the nanoemulsification ability of surfactants [20]. stirrer (Mix 1). The weighed amount of drug (PLB) was
dissolved in the calculated amount of oil (Capmul® MCM) in
Construction of pseudo-ternary phase diagrams a separate beaker (Mix 2). The oil phase (Mix 2) was added
The optimum concentrations or concentration ranges of oil, drop wise to the surfactant-cosurfactant mix (Mix 1) and
surfactant and co-surfactant necessary to promote self- stirring was continued for 1 h to obtain PLB loaded liquid
emulsification are determined by construction of a pseudo SNEDDS.
3
Table 1: Composition of prepared PLB-SNEDDS formulations by using Capmul® MCM as an oil Tween® 20 and Tween® 80 as
surfactants and PG and PEG 400 as co-surfactants.
Formulation Oil Smix
code (%) Ratio Surfactant (%) Cosurfactant (%)
Tween® 20 Tween®80 PG PEG 400

1 10 01:01 45 - 45 -
2 15 01:01 42.5 - 42.5 -
3 20 01:01 40 - 40 -
4 25 01:01 37.5 - 37.5 -
5 30 01:01 35 - 35 -
6 10 02:01 60 - 30 -
7 15 02:01 56.66 - 28.33 -
8 20 02:01 53.33 - 26.66 -
9 25 02:01 50 - 25 -
10 30 02:01 46.66 - 23.33 -
11 10 03:01 67.5 - 22.5 -
12 15 03:01 63.75 - 21.25 -
13 20 03:01 60 - 20 -
14 25 03:01 56.25 - 18.75 -
15 30 03:01 52.5 - 17.5 -
16 10 02:01 60 - - 30
17 15 02:01 56.66 - - 28.33
18 10 03:01 67.5 - - 22.5
19 15 03:01 63.75 - - 21.25
20 10 01:01 - 45 45 -
21 15 01:01 - 42.5 42.5 -
22 20 01:01 - 40 40 -
23 10 02:01 - 60 30 -
24 15 02:01 - 56.66 28.33 -
25 20 02:01 - 53.33 26.66 -
26 10 03:01 - 67.5 22.5 -
27 15 03:01 - 63.75 21.25 -
28 20 03:01 - 60 20 -
Thermodynamic stability studies The formulations that did not show any signs of instability
The objective of these tests was to evaluate the stability and were chosen for heating–cooling cycle.
phase integrity of PLB loaded SNEDDS under different
conditions of temperature variation and centrifugal force [25]. Heating and cooling cycle [27]
Three different tests were carried out in this study. Heating cooling cycle so performed involved three cycles
between 4°C and 45°C with storage at each temperature for
Centrifugation study [27] not less than 48 h. The formulations that passed at these
Formulations were centrifuged (REMI C-24 BL) at 5000 rpm temperatures, without undergoing any creaming, cracking,
for 30 min and were then checked visually for instability such coalescence, phase separation or phase inversion, were chosen
as phase separation, creaming, cracking or drug precipitation. for freeze thaw stress test.
4
Freeze thaw cycle (accelerated aging) [27] % Transmittance
Freeze thaw cycle involved three freeze thaw cycles at The PLB-loaded SNEDDS were reconstituted with distilled
temperatures between -20°C and +25 °C with storage at each water and the resulting nanoemulsion was observed visually
temperature for not less than 48 h. The formulations were then for any turbidity. Thereafter, its % transmittance was
visually observed for phase separation. Only formulations that measured at 638.2 nm using UV visible spectrophotometer
were stable to phase separation were selected for dispersibility (JASCO V-630) against distilled water as the blank. The
study. studies were conducted after 50, 100, 250 and 1000 times
dilution.
Dispersibility study
The dispersibility studies were carried study the self- Self-emulsification time
emulsification efficiency and self-emulsification time [22]. A quantity of 1ml of each formulation was added to 900 ml of
Self emulsification time is the time required by the distilled water under continuous stirring (50 rpm) using USP
preconcentrate to form a homogeneous mixture upon dilution, 31 dissolution test apparatus II at 37 ± 0.5ºC. The time
when disappearance of SNEDDS is observed visually [27]. required to disperse the system completely and uniformly was
Briefly, 0.5 ml of each formulation was added drop wise to determined, and the emulsification time was recorded in
250 ml of double distilled water in a glass beaker with gentle second [31].
agitation by placing it on a hotplate magnetic stirrer (REMI 1
MLH) at 100 rpm with temperature adjusted to 37 °C ± 0.5°C RESULTS & DISCUSSION
[22]. The time required for the disappearance of the SNEDDS Solubility study
was recorded [30]. The efficiency of self-emulsification of Solubility of drug in excipients plays an important role in
SNEDDS was visually assessed using the five grading system determining stability of formulation, as many formulations
[28, 29]. undergo precipitation before undergoing in situ solubilization
[16]. Formulations of self-emulsifying property essentially
Grade A: refers to rapidly forming (within 1 min) contain combination of oils, surfactants, and co-surfactants.
nanoemulsion, having a clear or bluish appearance. This mixture should be clear, isotropic, monophasic liquid at
Grade B: is rapidly forming (within 2 min), slightly less clear room temperature [33] and should have good solubilizing
nanoemulsion, having a bluish white appearance. capacity to incorporate dose of drug in minimum volume of
Grade C: is fine milky emulsion that was formed within 2 the mixture [34]. The solubility of plumbagin in various oils,
min. surfactants and co-surfactants is presented in Table 2. Among
Grade D: is dull, grayish white emulsion having slightly oily various vehicles screened, Capmul® MCM was selected as the
appearance that is slow to emulsify (longer than 2 min). oil phase as it demonstrated the highest solubilization capacity
Grade E: is a formulation, exhibiting either poor or minimal (128.10 ± 2.64 mg/ml). Similarly, Tween® 20 (34.10±1.50
emulsification with large oil globules present on the surface mg/ml) and propylene glycol (15.79 ±1.41mg/ml) showed
(longer than 3 min). Formulations those of grade A or B were highest solubilization capacity. Previous reports demonstrated
selected for further optimization. that medium chain monoglyceride like Capmul® MCM shows
good solvent capacity for hydrophobic drugs and also promote
Robustness to dilution water penetration and self-dispersibility of lipid formulations
Robustness of selected plumbagin SNEDDS formulations to upon hydration. Additionally, Capmul ® MCM is likely to
dilution was studied by diluting it 50, 100 and 1000 times with increase the interfacial fluidity of surfactant boundaries in the
various diluents i.e. double distilled water, 0.1 N HCl and micelles because the entrapment of Capmul® MCM in high
phosphate buffer pH 7.4. The diluted samples were stored for HLB surfactant enhances the emulsification process upon
24 h and observed for any signs of physical changes i.e. phase dilution with an aqueous medium [34, 35].
separation or drug precipitation [27, 18].
Screening of surfactant and co-surfactant for their
Globule size emulsification ability
The mean droplet size of the emulsion globules were Emulsification process and efficiency are controlled by
determined using nanoparticle size analyzer (Horiba SZ-100, multiple variables including lipid-surfactant affinity,
Kyoto, Japan). For droplet size analysis the dispersed hydrophilic-lipophilic balance (HLB) value of surfactant and
formulation was measured after 100 dilutions with double visco elasticiy of the emulsion base [36]. HLB value of
distilled water. Light scattering was monitored at 25°C at 90° surfactant affects the spontaneity of emulsification and
angle and Z average measured [30]. emulsion droplet size. Surfactants with HLB >10 are suitable
5
for formation of o/w nanoemulsions [20]. Non-ionic Table 3. Emulsification ability of selected surfactants using
surfactants are often considered for pharmaceutical Capmul® MCM as an oily phase and emulsification ability
applications and nanoemulsion formulations since these are of selected co-surfactants using Capmul® MCM and
less toxic [18], less affected by pH and ionic strength changes Tween® 20 as an oily phase and surfactant respectively.
[37] and typically have lower critical micelle concentration Sr. No of
Surfactant %Transmittance
compared to their ionic counterparts [36,38]. They are usually No. inversions
accepted for oral ingestion [39]. The selected surfactants were Cremophor®
1 7.33±0.57 81.55±1.32
compared for their emulsification efficiencies using Capmul ® RH 40,
MCM as the oily phases. It has been reported that well 2 Labrasol® 10.00±1.00 57.16±0.44
formulated SNEDDS is dispersed within seconds under gentle 3 Tween® 20 3.33±1.52 97.44±0.64
stirring conditions [39]. Transmittance values as well as 4 Tween® 80 5.33±0.577 92.88±2.60
number of flask inversions of different mixtures are given in
5 Span® 80 17.00±1.00 46.33±02.10
Table 3. The results inferred that highest % transmittance, i.e.
Sr. No of
highest emulsification efficiency, is demonstrated by Tween® No.
Co-surfactant
inversions
Transmittance
20 (HLB 16.7) Tween® 80 and (HLB 15.0). The observed
1 PEG 400 6.00±1.00 95.7±2.19
difference in their emulsifying ability could be attributed to
2 PEG 200 11.66±1.04 87.6±1.70
the HLB values of the examined surfactants, where Tween ® 20
possessed more HLB value than Tween® 80 in addition to the Propylene
3 3.00 ±1.00 99.16±1.04
glycol
difference in their structure and chain length [17, 19]. Higher
HLB surfactants lead to the formation of more stable 4 Glycerol 19.33±1.52 55.15±1.63
nanoemulsion upon exposure to water [20]. Tween® 20 and Data expressed as mean ± SD (n = 3).
Tween® 80 showed highest solubility as well as emulsification
efficiency (yielded clear nanoemulsions requiring shorter time Construction of pseudo-ternary phase diagrams
for nanoemulsification). Among various vehicles screened, Ternary phase diagrams were constructed in the absence of
Capmul® MCM was selected as the oil phase showing the PLB to identify the self-nanoemulsifying region and to select a
highest solubilization capacity. Tween® 20 and Tween® 80 suitable concentration of oil, surfactant and co-surfactant for
used as surfactant and PG and PEG 400 co-surfactant the development of liquid SNEDDS formulations. These
possessing the high solubilities and emulsification for further phase diagrams play important role in studying phase
study. behaviour of the formed nanoemulsions [16, 28]. A simple
ternary phase diagram comprises oil, water, and Smix, where
Table 2. Solubility of PLB in various oils, surfactants and each corner in the phase diagram represents 100% of that
co-surfactants. particular component. Based on the data obtained from the
solubility study and preliminary screening of surfactants,
Vehicle Solubility Vehicle Solubility Capmul® MCM was used as the oil phase, Tween® 20 and
Tween® 80 as surfactants and PG and PEG 400 as co-
(mg/ml) (mg/ml)
surfactant for constructing different phase diagrams. The
Oleic acid 112.00±1.23 Cremophor® 24.89±1.74
pseudo ternary phase diagrams of oil (Capmul® MCM),
RH 40, surfactant (Tween® 20, Tween® 80) and co-surfactant (PG,
Arachis Oil 109.44±2.24 Tween® 20 34.10±1.50 PEG 400) were constructed with different surfactant/co-
surfactant ratio of 1:1, 2:1 and 3:1. Fig. 1 shows the
Sesame oil 87.46±2.35 Tween® 80 29.12±1.96
constructed phase diagrams, where the translucent and low
Cinnamon oil 73.86±1.57 Span® 80 2.30±0.64 viscosity nanoemulsion area is presented as shaded area in the
Labrafac® PG 55.51±2.22 PEG 400 8.33±1.46 phase diagrams. It was clearly shown that the use of PEG 400
Olive oil 34.45±0.816 PEG 200 4.72±2.25 as co-surfactant at Smix ratio (1:1) with Tween® 20 could not
form clear isotropic nanoemulsion region while at Smix ratio
Castor oil 21.47±2.52 Propylene 15.79±1.41
(2:1) and (3:1) could form nanoemulsion but with not more
glycol than 20% oil. Employing Tween® 80 could not form any
Capmul® 128.10±2.64 Glycerol 1.27±0.56 nanoemulsion region with PEG 400 as co-surfactant. The use
MCM of PG as co-surfactant gave clear isotropic regions employing
Data expressed as mean ± SD (n = 3). not more than 30% of oil for all constructed phase diagrams
except for Tween® 20/PG (3:1) system which could emulsify
6
up to 40% of oil. Tween® 20 showed larger self emulsification not undergo precipitation following phase separation with
region compared to Tween® 80, due its higher HLB and hence infinite dilution in the GI fluids. It is observed more
more hydrophilic nature. Higher HLB value is required for prominently with drugs having poor aqueous solubility or
forming a good o/w emulsion [20] as higher hydrophilicity nanoemulsion, which undergoes phase transition [16]. The
property favors faster emulsification of the oil–surfactant SNEDDS should disperse completely and quickly when
mixture in contact with water [22]. Self-nanoemulsification is subjected to dilution under mild agitation of GIT due to
spontaneous and the resulting dispersion is thermodynamically peristaltic activity [16, 27].
stable [24]. Free energy of nanoemulsion formation depends
on the extent to which the surfactant lowers the surface Figure 1. Pseudo-ternary phase diagrams of Capmul®
tension of the oil–water interface and the change in dispersion MCM, Tween® 20, Tween® 80, PEG 400 and PG at Smix
entropy [38], and the present results demonstrated that ratios 1:1, 2:1 and 3:1 indicating the clear o/w
increasing surfactant proportion (Smix 1:1, 2:1 and 3:1) led to nanoemulsion region as shaded regions.
a more favorable formation of nanoemulsion. The surfactant
forms a layer around oil globule in such a way that polar head
lies toward aqueous and non-polar tail pull out oil and thereby
reduces surface tension between oil phase and aqueous phase
[40]. Further, increasing the concentration of surfactant
increased the spontaneity of the self emulsification process
[22]. The increase in co-surfactant decreases the region of
emulsion formation, as co-surfactants have very little effect on
reducing the interfacial tension directly rather they help the
surfactants to reduce the interfacial tension [20].
Thermodynamic stability studies

The main difference between emulsions and nano emulsions is
kinetic stability, reflecting the thermodynamic stability of the
two systems. SNEDDS undergoes in situ solubilisation to
form nano emulsion system, and it should have stability such
that it does not undergo precipitation, creaming or cracking.
However, in many cases, prolonged storage might cause the
drug to precipitate from the nanoemulsion; seed crystals start
to appear and might grow to large crystalline materials that
will precipitate out at the bottom of the vessel [16]. Therefore,
to check the stability, formulation was exposed to It has been reported that self-emulsification mechanism
centrifugation study, heating and cooling cycle and freeze involves the erosion of a fine cloud of small droplets from the
thawing cycle in order to eliminate the metastable ones. monolayer around emulsion droplets, rather than progressive
Twenty two formulations passed the test (Table 4) i.e. there reduction in droplet size [16]. The ease of emulsification was
was no sign of phase separation, turbidity or drug precipitation suggested to be related to the ease of water penetration into the
observed. Formulation 5, 10 and 15, containing Capmul colloidal or gel phases formed on the surface of the droplet
MCM in a concentration more than 25% with Tween® 20 and [42]. Accordingly, dispersibility study in double distilled
and formulation 22, 25 and 28 containing concentration more water was conducted. In the present study, double distilled
than 15% with Tween® 80 were unstable and showed phase water was used as a diluent for self-nano emulsification test
separation as well as turbidity, which may be due to the because it is well known that there is no significant difference
coagulation of the internal phase which led to phase separation in the SNEDDS prepared using pharmaceutically acceptable
[41]. The stable formulations were further tested for surfactants, dispersed in either water or GI fluids [38]. All
dispersibility. SNEDDS that passed this test either in grade A or B were
selected for further investigation, because grade A or B
Dispersibility study formulations will remain as SNEDDS when dispersed in GI
As SNEDDSs are released in the lumen of the gastrointestinal fluids [46]. It was clearly revealed that only SNEDDSs
tract, it disperses to form a fine nanoemulsion with the aid of prepared using Tween® 20 as the surfactant i.e. Formulation
GI fluid. Thus, it is important that formed nanoemulsion does 1, 2, 3, 6, 7, 8, 11, 12, 13, 18 and 19 could pass the test (Table
7
4). This might be explained by the higher HLB value (16.7) emulsification. The reason that can be put forward is the
possessed by Tween® 20 and consequently higher increase in interfacial tension between larger volume of oil
hydrophilicity compared to Tween® 80 (15.0). Higher HLB and aqueous phase with a net decrease in surfactant system,
value is required for forming a good o/w emulsion [20] since which makes the emulsification time longer [44]. Pouton
higher hydrophilicity property favors faster emulsification of found that the formulations containing more hydrophilic
the oil–surfactant mixture in contact with water [22], in components was superior in self-nanoemulsifying ability and
addition to reduced free energy of the system [43]. It was also provided smaller droplets than those containing more
observed that the increase in oil concentration in formulation lipophilic components [5].
4, 5, 9, 10, 14 and 15, and decrease in Tween® 20
concentration in formulation 16 and 17 required more time for
Table 4. Evaluation of thermodynamic stability and dispersion study, globule size, self-emulsification time and %
transmittance of PLB loaded SNEDDS formulations.
Formulation Centrifugation Heating Freeze Dispersion Globule Self %

code and thaw size emulsification transmittance
cooling (nm) time (sec) (%)
Grade Inference
1 Passed Passed Passed A Passed 52.16 ± 1.10 20.66 ± 2.51 99.28 ± 0.5
3 Passed Passed Passed A Passed 200.26±1.85 40.33 ± 1.52 87.08 ± 1.21
4 Passed Passed Passed E Fail - - -
5 Fail Fail Fail - - - - -
9 Passed Passed Passed D Fail - - -
14 Passed Passed Passed C Fail - - -
19 Passed Passed Passed B Passed 315.56±2.45 115.34 ± 1.65 78.23 ± 2.04
20 Passed Passed Passed C Fail - - -
22 Fail Fail Fail E Fail - - -
Data expressed as mean ± SD (n = 3).
8
Robustness to dilution nanoemulsification. The transmittance values (Table 4) ranges
Uniform emulsion formation from SNEDDS is very important from 78.23 ± 2.04 % to 99.28 ± 0.50%. High transmittance
at different dilutions because drugs may precipitate at higher value showed formulation, which contain PG than PEG 400.
dilution in vivo which affects the drug absorption significantly
[27, 37]. SNEDDS formulations were exposed to different Self emulsification time
folds of dilution in different media to mimic the in vivo Self emulsification time of PLB loaded SNEDDS found to be
conditions where the formulation would encounter gradual in ranges 17.00 ± 1.00 sec to 115.34 ± 1.65 sec (Table 4). It is
dilution. Hence, each formulation was subjected to 50, 100 observed that SNEDDS containing PG emulsify within 1 min
and 1000 times dilution in double distilled water, 0.1 N HCl, while for SNEDDS containing PEG 400 more than 1 min it
and phosphate buffer pH 7.4. Even after 24 h, all formulations, indicates spontaneous nanoemulsion formation on contact
which passed dipersion study, showed no signs of with physiological fluids observed in SNEDDS containing
precipitation, cloudiness or separation, which ensured the PG.
stability of the reconstituted emulsion. This reveals that the
SNEDDS were robust to dilutions by fluids of different pH. CONCLUSION
These findings will ensure the prospect of uniform drug
From the ternary phase diagrams, thermodynamic stability
release profile in vivo.
study and characterization it was observed that Capmul®
MCM (10% to 20% concentration) Tween® 20 and PG were
Characterization of formulations
found to be the best suitable oil, surfactant and co-surfactant
Globule size
respectively. The study helped in arriving at a suitable
Droplet size is one of the most important characteristics of
working range for concentration of surfactant and co-
nanoemulsion for stability evaluation and a critical step in the
surfactant. Further optimization can be done using a suitable
pathway of enhancing drug bioavailability [27]. Smaller
DOE.
droplet size leads to larger interfacial surface area for drug
absorption and hence may lead to more rapid absorption and
improved bioavailability [16]. Hence, the droplet size of the ACKNOWLEDGEMENT
nanoemulsion may govern the effective drug release [22]. The The authors acknowledge the help of funding by Babasaheb
droplet sizes of formulations are given in Table 4. The average Ambedkar Research Training Institute (BARTI) Pune in the
droplet size of the formulations at 100 times dilution ranged form of JRF and IMCD (Mumbai, India) for providing
from 50.4 ± 1.01 to 315.56 ± 2.45 nm, which indicated that generous gift sample of Capmul® MCM.
emulsion droplets are in nanometric range. The formulation,
which contain PG as co-surfactant showed small globule size
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Indo Global Journal of Pharmaceutical Sciences, 2020; 10(1): 1-11

INDO GLOBAL JOURNAL OF

Effect of Surfactants and Co-surfactants on Phase Behaviour and

Tween® 20 Tween®80 PG PEG 400

Thermodynamic stability studies

Formulation Centrifugation Heating Freeze Dispersion Globule Self %

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