Journal Pre-proof

Development of Microemulsion Based Topical Ivermectin Formulations:

Pre-Formulation and Formulation Studies

Surajit Das (Conceptualization) (Methodology) (Validation) (Formal

analysis) (Investigation) (Resources) (Data curation) (Writing -
original draft) (Writing - review and editing) (Visualization)
(Supervision) (Project administration) (Funding acquisition), Lee Sie
Huey (Methodology) (Validation) (Formal analysis) (Project
administration), Vernissa Dilys Chia Chye Ting (Methodology)
(Validation), Pui Shan Chow (Conceptualization) (Funding
acquisition) (Writing - review and editing), Calum Macbeath
(Funding acquisition) (Writing - review and editing), Yuanjie Liu
(Funding acquisition) (Validation) (Writing - review and editing),
George Shlieout (Conceptualization) (Funding acquisition) (Writing -
review and editing)

PII: S0927-7765(20)30053-9
DOI: https://doi.org/10.1016/j.colsurfb.2020.110823
Reference: COLSUB 110823

To appear in: Colloids and Surfaces B: Biointerfaces

Received Date: 21 October 2019

Revised Date: 9 January 2020
Accepted Date: 21 January 2020

Please cite this article as: Das S, Huey LS, Chye Ting VDC, Chow PS, Macbeath C, Liu Y,
Shlieout G, Development of Microemulsion Based Topical Ivermectin Formulations:
Pre-Formulation and Formulation Studies, Colloids and Surfaces B: Biointerfaces (2020),
doi: https://doi.org/10.1016/j.colsurfb.2020.110823
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© 2020 Published by Elsevier.

Development of Microemulsion Based Topical Ivermectin Formulations: Pre-

Formulation and Formulation Studies

Surajit Das a†, Lee Sie Huey a, Vernissa Dilys Chia Chye Ting a, Pui Shan Chow a, Calum

Macbeatha, Yuanjie Liub,George Shlieoutc

a Institute of Chemical and Engineering Sciences, A*STAR (Agency for Science, Technology

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and Research), 1 Pesek Road, Jurong Island, Singapore 627833, Singapore
bAbbott Laboratories (S) Pte Ltd, 1 Pesek Road, Jurong Island, Singapore 627833, Singapore

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cAbbott Laboratories GmbH, Freundallee 9A, 30173 Hannover, Germany

Corresponding author

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Institute of Chemical and Engineering Sciences, A*STAR (Agency for Science, Technology and

Research), 1 Pesek Road, Jurong Island, Singapore 627833

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† Tel: (65) 6796 3719, Fax: (65) 6316 6183, E-mail: surajit_das@ices.a-star.edu.sg;

surajitdas1982@yahoo.com
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Short statistical summary of the article

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Number of words: 5,522

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Number of tables: 1

Number of figures: 5

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 Graphical abstract

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Highlights
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 Tea tree oil and ethyl butanoate were suitable oils for ivermectin microemulsions

 Type and % of co-surfactants showed huge influence on microemulsion area (1φ)

 Ivermectin-loaded stable microemulsion formulations were successfully formulated

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 Ivermectin permeation of was faster from microemulsion gels than Soolantra cream
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Abstract

The aim of this work was to develop microemulsions and microemulsion gels which can

be used as vehicles for the topical delivery of ivermectin. Tea tree oil and ethyl butanoate were

found to be suitable for ivermectin-loaded microemulsion formulations due to the higher

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solubility of ivermectin in these two oils than other tested oils. The pseudo-ternary phase

diagrams were constructed based on these selected oils and combination of different

surfactant/co-surfactant at different ratios. Ivermectin-loaded stable microemulsions and

microemulsion gels were successfully formulated based on the selected compositions from the

phase diagrams. Ivermectin-loaded microemulsions showed spherical nano-droplets dispersed

in the continuous phase (via cryogenic field emission scanning electron microscope image) and

the particle size was less than 100 nm (via dynamic light scattering measurement). Ethyl

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butanoate based microemulsion appeared to be the best microemulsion formulation considering

the stability and permeation profiles while tea tree oil based microemulsion showed the best

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stability profile. Overall, microemulsion gel formulations exhibited better stability profiles than

their microemulsion counterparts. All microemulsion gel formulations demonstrated significantly

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faster in vitro membrane permeation (release) rate of ivermectin than Soolantra cream
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(reference marketed product by Galderma, USA).The developed microemulsion and

microemulsion gel formulations appear to be promising vehicles for topical delivery of

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ivermectin.
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Keywords: microemulsion; phase diagram; ivermectin; stability; permeation.

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1. Introduction

Several attempts have been made to incorporate poorly water soluble (or hydrophobic)

drugs in the topical liquid/semi-solid formulations. Most common approach is to make

emulsion based formulations (e.g., cream) where the poorly water soluble drug is

dissolved in the oil phase before making the emulsion [1, 2]. Other relatively less

common approaches are to encapsulated hydrophobic drugs in polymeric nanoparticles

[3] or lipid based nanoparticles [4-6] or nanoemulsion [7] or liposomes [3] prior to their

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addition in the liquid/semi-solid formulations. Microemulsion is one of the potential

technologies to formulate poorly water soluble drugs in liquid/semi-solid formulations [8-

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11]. Microemulsion can improve the solubility of hydrophobic drugs [12, 13]. Unlike

emulsion, microemulsion is thermodynamically stable which enables longer shelf life

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than emulsion [14]. It is also macroscopically homogeneous and optically transparent
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[15]. Furthermore, microemulsion is easy to prepare and scale-up as high energy input is

not required for production, hence cost-effective [8, 16, 17]. It is composed of oil phase,
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aqueous phase and surfactant phase (mixture of surfactant and co-surfactant) [10, 18,

19]. Generally, there are three most common microemulsion microstructures: i) oil-in-
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water or O/W (where oil nano-droplets are dispersed in continuous aqueous phase), ii)

water-in-oil or W/O (where aqueous nano-droplets are dispersed in continuous oil phase)

and iii) bi-continuous (where aqueous and oil phases are inter-dispersed) [19].
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Surfactant and co-surfactant form interfacial layer between oil and water phases, which
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helps to stabilize the microemulsion system [11]. Microemulsions can solubilise the

poorly water soluble drugs and improve the permeation of the drug molecules through

skin, hence improve the bioavailability [9, 10, 16, 19]. Microemulsion can also improve

stability of the drugs by encapsulating the drug molecules in the nano-droplets [12, 13,

20]. However, often gelling agent is required to add in the microemulsion to increase

viscosity as microemulsion is difficult to apply on the skin due to its high fluidity [17].

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Ivermectin is a mixture of 22, 23-dihydroavermectin B1a and 22, 23-dihydroavermectin

B1b. It is a poorly soluble compound that belongs to BCS class II drug [21]. It is a broad-

spectrum anthelmintic drug, which is used to treat different types of parasite infestations,

such as scabies [22], rosacea [15], head lice [23], trichuriasis [24], river blindness

(onchocerciasis) [25], lymphatic filariasis [26], andstrongyloidiasis [18]. Oral ivermectin

exhibits side effects especially at high doses [22]. Therefore topical treatment can be

beneficial to avoid the systemic side effects. Furthermore, parasites for scabies,

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onchocerciasis also reside in the deep skin layer (epidermis and dermis) and

subcutaneous tissues [27, 28]. Topical microemulsion based formulation with high skin

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penetration is expected to be more effective than emulsion based cream formulations in

these cases. It may also reduce the required dose of ivermectin than the marketed

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ivermectin topical formulations (emulsion based). One previous study demonstrated
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better characteristics of microemulsion than micelle and ternary solvent system as

transdermal carrier of ivermectin, which reduced the barrier function of the stratum
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corneum and increased concentration gradient toward the skin [29].

The aim of this study was to develop microemulsions and microemulsion gels which can
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be used as vehicles for the topical delivery of ivermectin. First, suitable oils were

screened, which have good ivermectin solubilisation property. Then, pseudo-ternary

phase diagrams were constructed using different surfactant and co-surfactant

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compositions to determine the microemulsion regions. After that, ivermectin-loaded

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microemulsions and microemulsion gels were formulated based on the microemulsion

regions in the phase diagrams. Ivermectin-loaded microemulsions were investigated

under cryogenic scanning electron microscope. The stability of the formulations were

also evaluated at different storage conditions for three months. Finally the in vitro

membrane permeation (or release) test was conducted on the ivermectin-loaded

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microemulsion gel formulations and compared with Soolantra 1% ivermectin cream

(marketed product by Galderma, USA).

2. Materials and Methods

2.1. Materials

Ivermectin was purchased from Zhejiang Hishun Pharmaceutical Co. Ltd (China). Ethyl-n-

butanoate (ethyl butanoate), tea tree oil, Tween® 80, diethylene glycol monoethyl ether (DGME),

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oleic acid, isopropyl palmitate (IPP), triacetin, ethyl-n-octanoate, ethyl-n-oleate, eucalyptus oil,

paraffin oil, rosemary oil, safflower oil and soyabean oil were bought from Sigma (USA).

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Methanol and acetonitrile were obtained from Duksan Pure Chemicals (South Korea). Absolute

ethanol, propylene glycol (PG) and isopropyl myristate (IPM) was purchased from VWR

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Chemicals (USA). SepiplusTM 400 was a gift sample from Seppic (France). LabrafacTM Lipophile
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WL 1349 (medium chain triglycerides) and LabrafacTM PG (Propylene glycol

dicaprolate/dicaprate) were provided by Gattefossé (France). Ultra-pure water from Millipore-Q®

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Gradient A10TM system (Millipore; France) was used in this study.

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2.2. HPLC assay of ivermectin

HPLC analysis of ivermectin was conducted as per USP method with slight modification. Agilent

HPLC (1100 series, Agilent Technologies; USA) and ZORBAX Eclipse XDB-C18 column (4.6
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mm x 15 cm, 5 µm, Agilent Technologies; USA) were used. Mixture of acetonitrile (53.0%),
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methanol (27.5%) and de-ionized (DI) water (19.5%) was used as mobile phase. Flow rate and

injection volume were set at 2 mL/min and 20 μL, respectively. Column temperature and

detection wavelength were set at 25 °C and 245 nm, respectively. Standard solutions of

ivermectin were prepared by dissolving ivermectin powder in methanol.

2.3. Screening of oils for microemulsions

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The solubility of ivermectin in different oils was determined by dissolving excess amount of

ivermectin to 3 mL of each oil in 5 mL stopper vials. Each sample was initially shaken using a

vortex mixer (Vortexer, Heathrow Scientific; USA) and then stirred at room temperature for 72 h

with a magnetic stirrer at 300 rpm. The sample was then filtered through a 0.45 µm PTFE

syringe filter (Agilent Technologies; USA). The concentration of ivermectin in the filtrate was

determined by HPLC at 245 nm after appropriate dilution with methanol. The oils with good

solubilising capacity for ivermectin were selected for the development of ivermectin-loaded

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microemulsions.

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2.4. Identification of microemulsion region

Pseudo-ternary phase diagrams were constructed using water titration method to identify the

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microemulsion regions of the tested systems [30]. Briefly, surfactant phase was prepared by
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mixing accurately weighed surfactant (Tween® 80) and co-surfactant (ethanol, propylene glycol,

DGME). Oil was accurately weighed in the glass vial (oil phase). Surfactant phase was added in
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the oil phase in the glass vials at surfactant phase to oil phase weight ratios of 1:9, 2:8, 3:7, 4:6,

5:5, 6:4, 7:3, 8:2, and 9:1. The mixtures were homogeneously mixed by a vortex mixer
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(Vortexer, Heathrow Scientific; USA). These surfactant-oil mixtures were continuously titrated by

adding 10 µL water using micropipette and mixed well by a vortex mixer. The titration was

continued until the transparent and homogeneous dispersion turned turbid (end point), signalling
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the transition from microemulsion to coarse emulsion. The amount of water required to turn the
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mixture slightly turbid was recorded. The % of each component (oil phase, surfactant phase and

water) was calculated and plotted in a ternary plot to generate the phase diagram.

2.5. Microemulsion and microemulsion-gel formulations of Ivermectin

As ivermectin is insoluble in water but soluble in selected oils, a few microemulsion

compositions were selected from the phase diagrams where microemulsion structures are most

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likely O/W type (significantly lower portion of oil phase than water phase). In case of W/O type

microemulsion, ivermectin remains in continuous oil phase which is not nano-sized. The aim

was to encapsulate ivermectin in the oil nano-droplets of these O/W type microemulsion

systems. Nano size droplets are expected to improve ivermectin penetration into skin.

Ivermectin-loaded microemulsions were prepared as follows (Table 1). First, oil phase was

prepared by dissolving ivermectin in the oil under magnetic stirring (IKA; Germany). Surfactant

phase was prepared separately by mixing surfactant and co-surfactant using a magnetic stirrer.

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Surfactant phase was then homogeneously mixed with the oil phase to produce a transparent

mixture. DI water was then added and mixed well using a magnetic stirrer to produce

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microemulsion which is a transparent homogeneous mixture.

In the preparation of microemulsion gel formulation, 2% of the DI water in the microemulsion

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was replaced by an equivalent amount of thickener (Table 1). SepiplusTM 400 (Polyacrylate-13
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and Polyisobutene and Polysorbate 20) was used as thickener due to its good electrolyte

resistance power [31]. The total % of DI water and thickener in the microemulsion gel is the
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same as the % of DI water in the corresponding microemulsion. The thickener was slowly added

to the microemulsion while stirring at 100 rpm for 15 min with an overhead stirrer (IKA;
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Germany).
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Table 1: Compositions of ivermectin-loaded microemulsions and microemulsion gels.

Amount (% w/w)

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Ingredients
IME-3 IME-4 IME-5 IME-6 IME-9 IME-10 IMEG-3 IMEG-4 IMEG-5 IMEG-6 IMEG-9 IMEG-10
Oil Phase Ethyl 5.00 - - 5.00 - 4.00 5.00 - - 5.00 - 4.00

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butanoate
Tea tree oil - 8.00 5.00 - 4.00 - - 8.00 5.00 - 4.00 -
Ivermectin 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00

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Surfactant Tween® 80 16.67 16.00 11.67 20.00 15.33 12.00 16.67 16.00 11.67 20.00 15.33 12.00
Phase Ethanol 8.33 - - - - - 8.33 - - - - -
DGME
Propylene
glycol
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-
16.00
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23.33
10.00
-
-
30.67
24.00
- -
16.00
- 23.33
10.00
- 30.67
24.00
-
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Water DI water 69.00 59.00 59.00 64.00 49.00 59.00 67.00 57.00 57.00 62.00 47.00 57.00
Phase
Thickener SepiplusTM - - - - - - 2.00 2.00 2.00 2.00 2.00 2.00
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400
Total 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00
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IME = Ivermectin microemulsion

IMEG = Ivermectin microemulsion gel

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2.6. Particle size and polydispersity index

Particle size (z-average diameter) and polydispersity index (PdI) of the microemulsion

formulations were measured by Malvern Zetasizer Nano ZS (Malvern Instruments, UK) at 25 °C

using dynamic light scattering (DLS) technique [32]. Size and PdI of the microemulsions were

measured without diluting the samples as dilution with water will disrupt the microemulsion

structure. Briefly, microemulsion was poured in a glass cuvette. The cuvette was then inserted

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into the cuvette holder of Zetasizer Nano ZS and analysed by DTS v 6.12 software (Malvern

Instruments, UK).

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2.7. Cryogenic Scanning Electron Microscopy

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Cryogenic Scanning Electron Microscope is a great tool to investigate the morphology of

aqueous based liquid/semi-solid formulations at close to their original state [33]. In this study,
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cryogenic field emission scanning electron microscope (cryo-FESEM) was used to investigate
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the morphology of the microemulsion formulation. Briefly, few drops of the microemulsion were

deposited onto a copper rivet and frozen in liquid nitrogen at less than -196 °C. This frozen

sample was stored in liquid nitrogen and quickly transferred into the cryo preparation
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chamber(Alto CT2500, Gatan, UK) under vacuum. The frozen sample was then etched with a

knife at -95 °C on the cryo stage and sputter coated with platinum for 180 s. The coated sample
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was then inserted into the specimen stage of the cryo-FESEM (JEOL JSM-6700F, Japan) at -

140 °C and analysed at an excitation voltage of 5.0 kV.

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2.8. Stability

The ivermectin contents in the fresh formulations (immediately after preparation) were analyzed

by HPLC. Ivermectin-loaded microemulsion and microemulsion gel formulations were stored at

5 °C, 25 °C, 40 °C/75% RH (relative humidity) chambers. Physical stability of the microemulsion

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formulations were evaluated via appearance check (phase separation, color change).

Furthermore, any changes in particle size and PdI of the microemulsions were also evaluated

during stability test period. Chemical stabilities of ivermectin in the formulations were analyzed

by HPLC after one week, one month and three months of storage at the above mentioned

stability conditions. HPLC samples were prepared by dissolving the formulations in methanol

[4]. Briefly, accurately weighed formulations were first added in the volumetric flasks and then

methanol was added. The flasks were sonicated for 30 min for complete extraction of ivermectin

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from the formulations and finally filtered through 0.45 µ PTFE syringe filters (Fisher Scientific;

USA) into the HPLC vials. Product colors of the stability samples were also visually compared

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with the fresh samples.

2.9. Ivermectin permeation through membrane

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Franz diffusion cells (Teledyne Hanson Research, 6-cell manual diffusion test system, standard

7 ml vertical diffusion cell; USA) and synthetic polysulfone membranes (Tuffryn® PALL Life
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Science, HT-450, P/N 66221, 0.45 μm pore size, 25 mm diameter; USA) were used to measure

the in vitro membrane permeation of ivermectin from the microemulsion gel formulations.
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Double walled Franz diffusion cells were placed on a multistage magnetic stirrer and connected

with a water circulator (Julabo; Germany). Hydro-alcoholic solution (70 % (v/v) phosphate buffer

saline (PBS; pH 7.4) and 30 % (v/v) methanol) was used as receptor fluid to maintain sink
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condition. Receptor fluid was placed in the receptor chamber. Temperature of the water
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circulator was set at 33 °C so that the temperature of receptor fluid was maintained at 32 ±

0.5 °C. The membrane was placed in between donor chamber and receptor chamber. The

donor chamber was filled with ivermectin-loaded microemulsion gel or blank microemulsion gel

or reference standard (Soolantra® 1% ivermectin cream; Galderma, USA). Speed of magnetic

stirrer was set at 300 rpm. Samples from the receptor fluid were collected through sampling port

at 0.5, 1, 2, 3, 4, 5 and 6 h for HPLC analysis of permeated ivermectin. Topical gel type

11
products do not stay on the skin for more than few hours (less than 6h). Therefore permeation

study of the gel formulations were conducted till 6h. During sample collections, magnetic stirrer

was switched off, 2 mL fresh receptor fluid was injected from the bottom port, first 1 mL sample

was discarded and next 1 mL sample was collected in the HPLC vial from the top sampling port

and finally magnetic stirrer was again switched on. The integrity of the membranes was

confirmed under microscope after completion of permeation study.

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3. Results and Discussion

3.1. HPLC assay

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HPLC assay method for ivermectin was developed and validated. Retention time of ivermectin

was ~18.2 min. Limit of detection (LOD) and limit of quantification (LOQ) of ivermectin were 1

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µg/mL and 2.5 µg/mL, respectively. The calibration curve was linear (r2 = 0.9995) within 2.5 –
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500 µg/mL ivermectin concentration.
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3.2. Solubility of Ivermectin

The solubility data indicate that ivermectin has the highest solubility in tea tree oil followed by in
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ethylbutanoate among the oils tested. The solubility profile of ivermectin in different oils can be

found in Supplementary Figure 1. Therefore, these two oils were selected to prepare

microemulsions and construct pseudo-ternary phase diagrams.

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3.3. Phase diagram

Figure 1A represents the pseudo-ternary phase diagram where ethyl butanoate, Tween® 80 and

ethanol were used as oil phase, surfactant and co-surfactant, respectively. Three compositions

of surfactant phase at surfactant to co-surfactant ratios of 1:2, 1:1 and 2:1 were investigated to

identify their impact on microemulsion region (1φ). The data showed differences in

microemulsion regions (especially towards oil rich region) at different surfactant/co-surfactant

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ratios. Surfactant phase containing Tween® 80 and ethanol at 2:1 ratio was selected for

ivermectin-loaded microemulsion as this ratio showed slightly larger microemulsion area at

water rich region compared to the other two surfactant/co-surfactant ratios.

Figure 1B represents the pseudo-ternary phase diagram where ethyl butanoate, Tween® 80 and

DGME were used as oil phase, surfactant and co-surfactant, respectively. Three compositions

of surfactant phase at surfactant to co-surfactant ratios of 1:2, 1:1 and 2:1 were investigated to

identify their impact on microemulsion region (1φ). The data showed no significant difference in

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microemulsion regions between surfactant to co-surfactant ratios of 1:2 and 1:1. However,

surfactant to co-surfactant ratios of 2:1 showed higher microemulsion region towards water rich

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region and lower microemulsion region towards oil rich region than other 2 surfactant phase

compositions. That is why surfactant phase containing Tween® 80 and DGME at 2:1 ratio was

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selected for ivermectin loading. Tween® 80 to DGME ratio of 1:2 was also selected in one
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formulation to reduce Tween® 80 level in the formulation.
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A.

0 Tween 80: EtOH (1:2)

100 Tween 80: EtOH (1:1)
Tween 80: EtOH (2:1)
20
80
ME (1f)

Tw
40

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r

ee
te

60
Wa

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0/E
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60

H
40

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80
20
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100
0
0 20 40 60 80 100
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Ethyl Butanoate
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B.

0 Tween 80: DGME (1:2)

100 Tween 80: DGME (1:1)
Tween 80: DGME (2:1)
20
80
ME (1f)

Tw
40

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te

ee
60
Wa

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0/D

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60

GM
40

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80
20
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100
0
0 20 40 60 80 100
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Ethyl Butanoate

Figure 1: The pseudo-ternary phase diagram of A. ethyl butanoate / (Tween® 80 + ethanol) /

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water system at room temperature with different weight ratio of Tween® 80 and ethanol (EtOH).

B. ethyl butanoate / (Tween® 80 + DGME) / water system at room temperature with different
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weight ratio of Tween® 80 and DGME. ME (1φ) represents single-phase microemulsion region.
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Figure 2A represents the pseudo-ternary phase diagram where tea tree oil (TTO), Tween® 80

and DGME were used as oil phase, surfactant and co-surfactant, respectively. Three

compositions of surfactant phase at surfactant to co-surfactant ratios of 1:2, 1:1 and 2:1 were

investigated to identify their impact on microemulsion region (1φ). The data showed wider

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microemulsion regions towards water rich region for Tween® 80 to DGME ratio of 1:1 than 1:2

and 2:1. Therefore, Tween® 80 to DGME ratio of 1:1 was selected for ivermectin loading.

Figure 2B represents the pseudo-ternary phase diagram where TTO, Tween® 80 and propylene

glycol (PG) were used as oil phase, surfactant and co-surfactant, respectively. Three

compositions of surfactant phase at surfactant to co-surfactant ratios of 1:2, 1:1 and 2:1 were

investigated to identify their impact on microemulsion region (1φ). The data for different

surfactant to co-surfactant ratios showed no significant difference towards water rich region.

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Tween® 80 to PG ratio of 1:2 was selected for ivermectin loading as PG is more compatible with

skin than Tween® 80.

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A.

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0 Tween 80/DGME (1:2)
100 Tween 80/DGME (1:1)
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Tween 80/DGME (2:1)

20
80
ME (1f)
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Tw

40
ter

ee

60
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Wa

0/D
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60
GM

40
E
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80
20

100
0
0 20 40 60 80 100
TTO

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B.

0 Tween 80: PG (1:2)

100 Tween 80: PG (1:1)
Tween 80: PG (2:1)

20
80
ME (1f)
40

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Tw
r

60
te

ee
Wa

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0/P

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60

G
40

-p
80
20
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100
0
0 20 40 60 80 100
lP

TTO

Figure 2: The pseudo-ternary phase diagram of A. tea tree oil (TTO) / (Tween® 80 + DGME) /
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water system at room temperature with different weight ratio of Tween® 80 and DGME. B. tea

tree oil (TTO) / (Tween® 80 + propylene glycol) / water system at room temperature with
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different weight ratio of Tween® 80 and propylene glycol (PG). ME (1φ) represents single-phase

microemulsion region.
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3.4. Particle size and polydispersity

Particle size of all ivermectin loaded microemulsions were within 18 – 54 nm (Figure 3A). IME-3

and IME-6 showed the lowest particle size while IME-9 showed largest particle size.

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Polydispersity index (PdI) of all ivermectin loaded microemulsions were within 0.3 – 0.5 range

(Figure 3B). IME-5 and IME-6 showed the narrowest and widest size distribution, respectively.

A.

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B.
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Figure 3: Particle size (A) and PdI (B) of the ivermectin-loaded microemulsions during stability

study. 3M @ 5°C, 3M @ 25°C and 3M @ 40°C in the graphs indicate the data after three

months storage at 5 °C,25 °C,40 °C/75% RH, respectively. Data represent mean ± SD (n = 3).

3.5. Cryo-FESEM

Cryo-FESEM is a useful tool to distinguish between bi-continuous or droplet-like microstructure

of the microemulsions. IME-3 was chosen for cryo-FESEM study as this microemulsion appears

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to be the best formulation considering stability and release profiles (to be discussed later).

Droplet-like microstructures can be observed in the cryo-FESEM image. Supplementary Figure

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2 demonstrates the microstructure of the microemulsion formulation. IME-3 was considered as

O/W type microemulsion due to the presence of significantly higher proportion of water than oil

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in its composition (Table 1). The droplet size in Supplementary Figure 2 appears to be slightly
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larger than the value obtained from the DLS measurement (Figure 3A). This may be due to the

expansion of the aqueous phase during freezing process of Cryo-FESEM, which can change
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the dimension of droplets [34].

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3.6. Stability

Stability of ivermectin-loaded microemulsions were monitored at 5 °C (to check stability at low

temperature), 25 °C (to check stability at room temperature) and 40 °C/75% RH (to check
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stability at accelerated condition) for three months. All microemulsions were physically stable at
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all storage conditions (Figure 3 and Supplementary Figure 3) except IME-5 which showed

phase separation at 40 °C/75% RH after just a week (Supplementary Figure 3D) and at 25 °C

within three months (Supplementary Figure 3F). This indicates that the amount surfactant/co-

surfactant was not enough in IME-5 to maintain the microemulsion structure at high

temperature. Slight decrease in sizes of IME-3, IME-9 and IME-10 were observed at 40 °C/75%

RH after 3 months. However, the changes in these sizes did not impact microemulsion

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structures. At 40 °C/75% RH, IME-6 and IME-10 showed slight color change after one month

(Supplementary Figure 3D) and the color became darker after three months, especially for IME-

6 (Supplementary Figure 3G). Other microemulsion samples did not exhibit significant

discoloration after one month in all storage conditions (including 40 °C/75% RH) and exhibited

minor color change after three months at 40 °C/75% RH only. Generally, there is a higher

chance of precipitation of dissolved drug when stored at 5 °C than at 25 °C and 40 °C as the

solubility of the drug decreases with decreasing temperature. However, no precipitation of

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ivermectin crystal was observed in all microemulsions even after three months at any storage

conditions (including 5 °C), which indicates that all microemulsions were able to keep ivermectin

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in dissolved state at wide temperature range (5 – 40 °C). In comparison to initial particle size

and polydispersity index (PdI), there were no huge deviation of particle size and PdI of all

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ivermectin-loaded microemulsions (except IME-5 where phase separation occurred) after three
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months storage at all stability conditions (Figure 3).

IME-4 showed the best chemical stability of ivermectin (>98% after three months) followed by
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IME-3 and IME-9 (>93% after one month and >85% after three months) under all storage

conditions (Figure 4A-C). Although IME-6 showed good ivermectin stability (>90%) at 5 °C and
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25 °C for three months, it showed moderate stability (~86%) after one month and poor stability

(~59%) after three months at 40 °C/75% RH. IME-10 showed good ivermectin stability (>90%)

after one month and moderate stability (>80%) after three months at 5 °C and 25 °C. However,
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it showed poor ivermectin stability (~68% after one month and ~48% after three months) at
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40 °C/75% RH. IME-5 showed >98% ivermectin stability at 5 °C and 25 °C after one month and

at 5 °C after three months. As mentioned earlier, phase separation of IME-5 was observed after

one week at 40 °C/75% RH and after three months at 25 °C. Therefore, ivermectin stability

assay was not conducted on this sample. Overall, poor ivermectin stability in the

microemulsions (IME-6 and IME-10) was observed at 40 °C/75% RH when ethyl butanoate and

DGME were used as oil and co-surfactant, respectively.

20
Stability of ivermectin-loaded microemulsion gels were also monitored at 5 °C, 25 °C and

40 °C/75% RH for three months. All microemulsion gels were physically stable (no phase

separation) at all storage conditions (Supplementary Figure 4). There was no visible

discoloration in any of the samples after one month at all storage conditions and after three

months at 5 °C, 25 °C. However, IMEG-6 and IMEG-9 turned to light yellow (IMEG-6 > IMEG-9)

after three months at 40 °C/75% RH. All other microemulsion gels showed negligible color

change after three months at 40 °C/75% RH. Precipitation of ivermectin crystal was not

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observed even after three months at any storage conditions including low temperature (5 °C).

IMEG-3, IMEG-4 and IMEG-5 showed very good chemical stability of ivermectin (>96%) at all

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storage conditions even after three months (Figure 4D-F). IMEG-10 showed moderate stability

(>85%) at all storage conditions for three months. IMEG-6 and IMEG-9 showed relatively lower

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stability after three months, especially at 40 °C/75% RH (78% – 80%). Overall, chemical stability
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of ivermectin in the microemulsion systems increased after addition of thickener (microemulsion

gels).
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A.
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 B.

D.
C.
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 F.
E.
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Figure 4: Stability of ivermectin-loaded microemulsions at different storage conditions: A. 5 °C,

B. 25 °C (excluded three months data point of IME-5 as phase separation was observed at

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three months), C. 40 °C/75% RH (excluded IME-5 as phase separation was observed at one1
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week). Stability of ivermectin-loaded microemulsion gels at different storage conditions: D. 5 °C,

E. 25 °C, F. 40 °C/75% RH. Data represent mean ± SD (n = 3).

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3.7. Ivermectin permeation through membrane

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Permeation of ivermectin from microemulsion gel formulations (contain 1% ivermectin) through

polysulfone membranes (0.45 µm pore size) was evaluated by Franz diffusion cells. Soolantra

(1% ivermectin cream for topical use) was used as the reference standard. In case of Soolantra,
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ivermectin in the receptor fluid was below detection limit in the first three hours and only
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detectable at very low levels thereafter (Figure 5). Ivermectin permeation was significantly better

from all microemulsion gel formulations. Among the microemulsion gel formulations, IMEG-3

showed the highest permeation rate followed by IMEG-10 and IMEG-6. IMEG-4 and IMEG-5

showed the lowest permeation among the tested microemulsion gels. Ivermectin permeation

from IMEG-9 was equivalent to IMEG-6 in the first hour but subsequently became equivalent to

IMEG-4 and IMEG-5. The result indicates that microemulsion systems containing ethyl

24
butanoate exhibited higher ivermectin permeation than the microemulsion systems containing

tea tree oil (although composition of surfactant phase was different which was decided from the

phase diagrams). Among the ethyl butanoate based microemulsion gels, Tween® 80 and

ethanol (IMEG-3) combination showed faster permeation than Tween® 80 and DGME

combinations (IMEG-6 and IMEG-10). Higher permeation from IMEG-9 in the first hour than

other TTO based system was probably due to higher amount of surfactant and co-surfactant

combination.

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Figure 5: Permeation of ivermectin from microemulsion gel formulations through synthetic

membrane. Data represent mean ± SD (n = 3).

4. Conclusion

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Selection of oils, surfactants, co-surfactants and construction of phase diagram(s) are

prerequisites for the development of microemulsion based formulations of any drug. As

ivermectin (drug) has poor aqueous solubility, the oils were selected based on their

solubilisation capability so that small amount of oil used in the microemulsion

formulations can dissolve 1% ivermectin. Ethyl butanoate and tea tree oil were found to

be suitable for this purpose. Phase diagrams were successfully constructed on these oils

using different surfactant/co-surfactant compositions and microemulsion regions were

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identified. Stable ivermectin-loaded microemulsion and microemulsion gel formulations

were prepared based on the selected compositions from the phase diagrams. The

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microemulsion gel formulations showed significantly higher membrane permeation of

ivermectin (i.e., higher drug release from the microemulsion-gels) than marketed product

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(Soolantra 1% ivermectin cream). The outcome of this study can be utilized for the
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following three purposes. i) Significantly lower level of ivermectin content in the

microemulsion gel formulations may provide equivalent or better efficacy than Soolantra
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due to higher membrane permeation. This will reduce cost and may reduce side effects

of the product. In fact, clinical study of 1%, 0.3% and 0.1% ivermectin creams were
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performed for the registration of Soolantra [35]. 1% ivermectin cream was selected

because 0.3% and 0.1% ivermectin creams did not show the desired efficacy. ii) Some

parasites (e.g., parasites for scabies, onchocerciasis) also reside inside the skin
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(epidermis and dermis) and subcutaneous tissues. Penetration of drug through stratum
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corneum can help to act on those parasites. Therefore, the microemulsion based

formulations should be more effective (due to higher membrane permeation of

ivermectin) than Soolantra for topical treatment of parasites. iii) High membrane

permeation (or drug release) of these microemulsion based formulations can be utilized

to develop topical/transdermal delivery systems of other BCS class II drugs for systemic

actions.

26
Notes

The authors declare no competing financial interest.

Author Contributions

All authors have contributed in this work. The final version of the manuscript has been

approved by all authors.

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Credit Author Statement

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Surajit Das: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Resources, Data

Curation, Writing - Original Draft, Writing - Review & Editing, Visualization, Supervision, Project
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administration, Funding acquisition.
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Lee Sie Huey: Methodology, Validation, Formal analysis, Project administration.

Vernissa Dilys Chia Chye Ting: Methodology, Validation.

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Pui Shan Chow: Conceptualization, Funding acquisition, Writing - Review & Editing.
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Calum Macbeath: Funding acquisition, Writing - Review & Editing.

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Yuanjie Liu: Funding acquisition, Validation, Writing - Review & Editing.

George Shlieout: Conceptualization, Funding acquisition, Writing - Review & Editing.

27
Declaration of interests

The authors declare that they have no known competing financialinterestsor personal relationships that
could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by a joint collaborative grant between A*STAR (Agency

for Science, Technology and Research), Singapore and Abbott Laboratories. We would

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like to thank Mr. Ng Jun Wei and MS. Inez Kwek for their technical assistance.

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