Novel isotretinoin microemulsion-based gel for targeted topical therapy of acne

15 Pages·2015·1.38 MB·English

by  Mrunali R. Patel

ApplNanosci(2016)6:539–553
DOI10.1007/s13204-015-0457-z
ORIGINAL ARTICLE
Novel isotretinoin microemulsion-based gel for targeted topical
therapy of acne: formulation consideration, skin retention
and skin irritation studies
Mrunali R. Patel1 • Rashmin B. Patel2 • Jolly R. Parikh3 • Bharat G. Patel4
Received:2April2015/Accepted:2May2015/Publishedonline:16May2015
(cid:2)TheAuthor(s)2015.ThisarticleispublishedwithopenaccessatSpringerlink.com
Abstract Isotretinoin was formulated in novel mi- isotretinoin, indicating its potential in improving topical
croemulsion-based gel formulation with the aim of im- delivery of isotretinoin. The developed microemulsion-
provingitssolubility,skintolerability,therapeuticefficacy, based gel may be a potential drug delivery vehicle for
skin-targeting efficiency and patient compliance. Mi- targeted topical delivery of isotretinoin in the treatment of
croemulsion was formulated by the spontaneous mi- acne.
croemulsification method using 8 % isopropyl myristate,
24 % Labrasol, 8 % plurol oleique and 60 % water as an Keywords Acne (cid:2) Isotretinoin (cid:2) Microemulsion (cid:2) Skin
external phase. All plain and isotretinoin-loaded mi- irritation study (cid:2) Skin retention study (cid:2) Topical drug
croemulsions were clear and showed physicochemical pa- delivery
rameters for the desired topical delivery and stability. The
permeation profiles of isotretinoin through rat skin from
selected microemulsion formulation followed zero-order Introduction
kinetics. Microemulsion-based gel was prepared by incor-
porating Carbopol(cid:3)971 in optimized microemulsion for- Acne, a cutaneous disorder of multifactorial origin, is a
mulation having suitable skin permeation rate and skin disease whose cause and severity depend on the relation-
uptake. Microemulsion-based gel showed desired physi- ship between hormones, keratinization, sebum and bacte-
cochemical parameters and demonstrated advantage over ria. The objectives of acne therapy usually include
marketed formulation in improving the skin tolerability of controlling acne lesions, preventing scarring and
minimizing morbidity (Russell 2000; Zaenglein 2008).
Isotretinoin(ITTN),aderivativeofretinoicacid(13-cis-
& MrunaliR.Patel retinoic acid), is the most effective compound with po-
[email protected] tential to suppress acne over the long term (Zaenglein
2008; Patel et al. 2011a). It appears to derive its effec-
1 DepartmentofPharmaceuticsandPharmaceutical
tiveness from increased production of the antimicrobial
Technology,IndukakaIpcowalaCollegeofPharmacy,
NewVallabhVidyanagar388121,Gujarat,India protein neutrophil gelatinase-associated lipocalin in the
2 DepartmentofPharmaceuticalChemistryandQuality skin reducing sebum levels and in turn reducing levels of
Assurance,A.R.CollegeofPharmacyandG.H.Patel Propionibacterium acnes (P. acnes). An ongoing trial in
InstituteofPharmacy,VallabhVidynagar388120,Gujarat, patients with antibiotic-resistant P. acnes indicates that
India
ITTN is highly effective; treatment of P. acnes may well
3 DepartmentofPharmaceuticsandPharmaceutical become a new indication for this drug (Zaenglein 2008).
Technology,A.R.CollegeofPharmacyandG.H.Patel
So, ITTN, reducing the growth of P.acnes in a secondary
InstituteofPharmacy,VallabhVidynagar388120,Gujarat,
manner, was selected for further study. However despite
India
these interesting features, extremely low solubility limits
4 DepartmentofPharmacology,RamanbhaiPatelCollegeof
ITTN incorporation into an acceptable vehicle and its tol-
Pharmacy,CharotarUniversityofScienceandTechnology,
Changa388421,Gujarat,India erability results in either discontinuation of treatment or
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540 ApplNanosci(2016)6:539–553
poor compliance in patients (Zaenglein 2008; Patel et al. Methocel KM premium hydroxypropyl methylcellulose
2011a). Therefore, it is prudent to improve the topical (Colorcon Asia Pvt. Ltd., Goa, India) were procured on
delivery and reduce adverse effects of ITTN using an ap- gratis. Isopropyl myristate was purchased from National
propriate carrier having the desired skin-targeting proper- Chemicals (Vadodara, India). Ethanol was purchased from
tiesthatmayreducetheadversereactionssubstantiallyand Baroda Chemical Ind. Ltd. (Dabhoi, India). Double dis-
improve users’ compliance to the novel regimen. tilled water was used throughout the study. All other che-
The conventional anti-acne formulations could be less micals and solvents were of analytical reagent grade and
efficacious due to insignificant skin penetration properties. were used as received without further purification.
Incomparison,thenoveldrugdeliverysystemsofrecently
available anti-acne formulations have potential to reduce Methods
the adverse effects without reducing the efficacy. Mi-
croemulsion(ME) offers apowerfulway fordrugdelivery Solubility determination
ascolloidaldrugcarrier,duetoitsversatilityandfavorable
advantages(BachhavandPatravale2009;Djordjevic2004; The solubility of ITTN was determined in various oils,
Patel et al. 2009, 2011b, 2013a, b, c). The application of emulsifiers, and co-emulsifiers. In this experiment, excess
ME systems to skin distributes the anti-acne amountofITTN wasadded in1 mLofeach excipient and
agent(s) steadily and reduce irritation. These systems have mixed thoroughly using vortexer at ambient temperature
also been shown to promote follicular targeting and thus (Patel et al. 2009). The mixed samples were kept for 72 h
create high local concentrations ofthe active compound in to allow equilibration. Then the samples were centrifuged
the pilosebaceous unit and efficient penetration in hair at 50009g for 30 min to separate the undissolved drug.
follicles (Zaenglein 2008; Patel et al. 2011a). Thus, MEs Supernatants were diluted and analyzed by the HPTLC
could play a pivotal role in improving topical delivery of method(Pateletal.2011a)toestimatetheconcentrationof
anti-acne agents by enhancing their dermal localization soluble ITTN. Excipients in which ITTN showed good or
with concomitant reduction in their adverse effects. ME- excellent solubility were chosen for preparing MEs.
basedgels(MBG)areknowntoprovidefasterdrugrelease
than conventional formulations (Bachhav and Patravale Construction of phase diagrams
2009). A ME-based gel was thus developed and evaluated
using a suitable polymer that is capable of modifying the Thepseudo-ternaryphasediagramswereconstructedusing
rheological behavior. watertitrationmethodatambienttemperature.Threephase
TofindoutinnovativewaysforadministeringITTNand diagramswereconstructedwiththe1:1,2:1and3:1weight
assuaging their disadvantages, the present study investi- ratios of emulsifier/co-emulsifier mixture (Emix) of
gated the development of ME-based gel containing ITTN. Labrasol (LAB) and plurol oleique (PO), respectively. For
ItwashypothesizedthattheoilycorepresentaroundITTN each phase diagram at a specific emulsifier/co-emulsifier
may result in the reduction of irritation when presented in mixingratio(Km),theratioofisopropylmyristate(IPM)to
theformofME.Thecombinationofeffects,viz.equaland/ the Emixwasvaried as1:9, 2:8, 3:7, 4:6,5:5, 6:4, 7:3,8:2
or enhanced effectiveness, reduction in adverse reactions and 9:1. Spontaneous formation of ME was evaluated by
and regimen adherence may provide a better platform of the addition of a known amount of water at once to a
novel drug delivery with improved therapeutic efficacy. known amount of oil, emulsifier and co-emulsifier with
Thus, the present study focuses on novel formulation controlled stirring. The ease offormationof clear ME was
consideration, characterization and evaluation of skin-tar- taken as the criteria of spontaneity of microemulsification
geting efficiency of ITTN when loaded with MBG. (Patel et al. 2009, 2011b, 2013a, b, c).
Preparation of microemulsions
Materials and methods
Six potential ME formulations, which were different from
Materials each other by water volume fraction, were selected and
formulated at Km 3:1 by the spontaneous microemulsifi-
ITTN was procured as gratis samples from Astron Re- cation method. A calculated amount of ITTN (0.05 % w/
search Ltd. (Ahmedabad, India). Labrasol, plurol oleique w) was added to the oily phase of ME and magnetically
(Gattefosse, Saint-Priest, France), Carbopol 980 NF, Car- stirred until dissolved, followed by addition of Emix in a
bopol 971P NF, Carbopol 974P NF, (Lubrizol Advanced fixedproportiontoproduceaclearmixture.Then,adefined
Materials India Pvt. Ltd., Mumbai, India), Methocel E50 proportionofwater wasadded andstirredtoproduceclear
LVpremiumhydroxypropylmethylcelluloseEPgradeand ME containing ITTN (Patel et al. 2013c).
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ApplNanosci(2016)6:539–553 541
Preparation of microemulsion-based gel In vitro skin permeation study
Carbopol(cid:3) 971P NF was selected as the gel matrix to The in vitro skin permeation study was carried out under
prepareME-basedgel(MBG).Itwasentirelyswollenupon the guidelines proposed by the Committee for the Purpose
slowmixinginthewateranditspHwasadjustedbyadding of Control and Supervision of Experiments on Animals
50 % w/w triethanolamine (TEA). MBG was obtained by (CPCSEA) and all the study protocols were approved by
mixing the swollen gel in water with the oily phase the Local Institutional Animal Ethics Committee (IAEC).
(Bachhav and Patravale 2009; Chen et al. 2007). The abdominal skins obtained from male Wistar rats
weighing 230 ± 20 g (age, 6–8 weeks) were used for
Characterization of formulations in vitro permeation experiments of the prepared formula-
tions. After hair was shaved carefully with an electric
Theaveragedropletsizeandpolydispersityindex(PDI)of clipper,theskinwasexcisedfromtheabdominalregionof
ME were measured by photon correlation spectroscopy each killed rat and the subcutaneous fat and other extra-
(PCS) with in-built Zetasizer (Nano ZS, Malvern Instru- neous tissues were removed without damaging the epi-
ments, UK) at 633 nm. Helium–neon gas laser having in- dermal surface. The excised rat skins were rinsed with
tensity of 4mW was the light source. The droplet size was physiological saline and the thickness of each skin tissue
calculated using Stokes–Einstein relationship by Zetasizer was kept almost similar (Patel et al. 2009, 2011b).
Software. Electrophoretic mobility (lm/s) was measured The excised skins were then placed in a refrigerator at
using small volume disposable zeta cell and converted to 4 (cid:4)C and then used for permeation experiments. The skin
zeta potential by in-built software using Helmholtz–S- membranes were first hydrated for 30 min in buffer solu-
moluchowski equation. All determinations were made in tion (pH 7.4) at room temperature to remove extraneous
triplicate.TEMwasusedtocharacterizethemicrostructure debrisandleachableenzymes.Thepermeationexperiments
ofITTN-loadedME.TheTEMimageswereobtainedusing were performed using Franz diffusion cells fitted with ex-
a Tecnai G2 20 TEM (Philips, Holland). ITTN content in cised rat skins, such that the dermal side of the skin was
the formulation was assayed using an HPTLC (Patel et al. exposed to the receptor fluid and the stratum corneum re-
2011a). The percent transmittance of the formulation was mained in contact with the donor compartment. The ef-
measuredusingacolorimeter(DigitalColorimeter,D-801, fective permeation area was 3.14 cm2 (20 mm-diameter
Photocon, India) at 570–590 nm. The isotropic nature of orifice), and the receptor compartment was filled with
the ME formulations was verified using cross-polarized 12 mL of physiological saline:ethanol (7:3 v/v) for ensur-
light microscopy (Polarizing Microscope RPL-55 Series, ing pseudo-sink conditions by increasing the solubility of
Radical Instruments, India). The pH value of the formu- ITTN in the receiving phase. The temperature of the re-
lationwas determinedusingadigital pHmeter (HI98107, ceiver chamber containing 12 mL of receptor media was
Hanna Instruments, India) which was standardized using controlled at 32 ± 1 (cid:4)C using circulating equibath (Model
pH4and7buffersbeforeuse.Theelectricconductivityof 8506, Medica Instrument Mfg. CO, Mumbai, India). The
ME was measured using a conductivity meter (Equip- diffusion medium was continuously stirred with a Teflon-
Tronics, EQ—664, Mumbai, India) equipped with an in- coatedmagneticbarataconstantrate,inawaythattherat
built magnetic stirrer. The viscosity of ME was measured skin surface just flushed the diffusion fluid. The formula-
using a Brookfield Viscometer LVDV-IIIU (Brookfield tion (1 g) was gently placed in a donor chamber and then
Engineering LABS, Stoughton, USA) with spindle SC 18 the diffusion cells were covered with an aluminum foil to
at100 rpmusinganintervalof30 s.Arheologicalstudyof prevent light exposure. At 1, 2, 4, 6, 8, 10 and 12 h, ali-
theMBGwas performed usingaviscometerwithHelipath quots of 1 mL sample were withdrawn from the receptor
stand (Patel et al. 2009, 2011b, 2013a, b, c). compartmentandreplacedimmediatelywithanequivalent
volume of receptor fluid. The concentration of ITTN in
Determinationofspreadabilityofmicroemulsion-basedgel receptor fluid was analyzed using the HPTLC method
(Patel et al. 2011a). Each experiment was performed in
The spreadability of the gel was determined by placing triplicate. Cumulative corrections were made to obtain the
0.5 ggelwithinapre-markedcircleof1 cmdiameterona total amount of ITTN permeated at each time interval.
glass plate over which a second glass plate was placed. A Thepermeationratewasdeterminedusingtheequation,
weight of 500 g was allowed to rest on the upper glass J = 1/A(dQ/dt) = C K , where J is the permeation
ss ss 0 p ss
plate for 5 min. The increase in the diameter due to rate at steady state (lg/cm2/h), A the effective diffusion
spreadingofthegelswasnotedandthemeandiameterwas area (cm2), (dQ/dt)ss the amount of drug that permeated
taken by repeating the experiment three times (Bachhav throughtheskinperunittimeatsteadystate(lg/h),C the
0
and Patravale 2009). applied drug concentration in the donor compartment (lg/
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542 ApplNanosci(2016)6:539–553
mL) and K the permeability coefficient (cm/h). The av- Histopathological investigation
p
erage cumulative amount of drug that permeated per unit
surface area of the skin was plotted versus time. The per- The rat abdominal skin was mounted on Franz diffusion
meation rate of ITTN at steady state (J , lg/cm2/h) cell.TheoptimizedME,MBG(1 g)andmarketedgelwere
ss
throughratskinwascalculatedfromtheslopeofthelinear applied similar to method for permeation study and the
portion of the cumulative amount that permeated through effects were compared against control. A piece of fresh
the rat skins per unit area versus time plot. To obtain the excised untreated skin sample was used as control. The
permeability coefficient K (cm/h), the following equation skin was fixed in 10 % neutral formalin for 24 h and then
p
was used: K = J /C (Patel et al. 2009, 2011b). cut vertically against the surface at the central region
p ss 0
(4 mm width). Each section was dehydrated using graded
Stability studies solutions of ethanol and then embedded in paraffin wax.
Tissues were divided into small pieces and stained with
The stability of ME formulations was studied through hematoxylin and eosin. The sections were observed under
various parameters such as clarity, phase separation ob- 1009 magnifications and photographed (Patel et al. 2009,
servation, droplet size determinations, viscosity measure- 2011b, 2013a, b, c).
ments, conductivity measurements, pH and zeta potential
determinations for 6 months at room temperature. To Skin irritation testing (Draize patch test)
estimate metastable systems, the selected ME vehicles
were centrifuged (Remi Lab, Mumbai) at 50009g for The irritation potential of the ME-based ITTN gel in
30 min. Besides the physicochemical properties, the che- comparison to marketed ITTN gel was evaluated by car-
mical stability of the investigated drug in the vehicle ryingouttheDraizepatchtestonrabbits.Animalcareand
plays a major role. Therefore, the drug content was ana- handling throughout the experimental procedure were
lyzed at defined time intervals during the observation performed in accordance with the CPCSEA guidelines.
period of 6 months. During the observation period, the White New Zealand rabbits weighing 2.5–3 kg were ac-
formulations were stored at room temperature in tubes to climatized before the beginning of the study.
simulate patient usage conditions. During stability studies, Animals were divided into four groups (n = 3) as
the samples were taken at fixed time intervals (Patel et al. follows:
2011b). Group 1: no application (control).
Group 2: marketed formulation (Sotret(cid:3) gel containing
Infrared study 0.05 % w/w ITTN, Ranbaxy).
Group 3: MBG without ITTN (placebo gel).
The infrared (IR) spectra of ITTN, unloaded ME, opti- Group 4: MBG containing ITTN (0.05 %, w/w).
mized ITTN-ME and ITTN-MBG were taken using an IR Thebackoftherabbitswereclippedfreeofhair24 hprior
spectrophotometer (Spectrum GX FT-IR, Perkin Elmer, to the application of the formulations. 0.5 g formulations
Norwalk, CT). The unloaded ME, ITTN-ME and ITTN- were applied on the hair-free skin of rabbits by uniform
MBG were spread as a thin layer on potassium bromide spreadingwithintheareaof4 cm2.Theskinwasobserved
cell and then scanned between 4000 and 400 cm-1. The foranyvisiblechangesuchaserythema(redness)at24,48
resulting IR spectra of ITTN and plain ME were then and72 haftertheapplicationofvariousformulations.The
compared with ITTN-ME and ITTN-MBG to detect any meanerythemascoreswererecorded(rangingfrom0to4)
possible interaction between the drug and different com- depending on the degree of erythema as follows: no ery-
ponents used (Patel et al. 2013c). thema = 0, slight erythema (barely perceptible-light
pink) = 1,moderateerythema(darkpink) = 2,moderateto
Skin retention study severe erythema (light red) = 3 and severe erythema (ex-
tremeredness) = 4(Draizeetal.1944;Shahetal.2007).
Skin retention study was performed to analyze the content
of the drug in the skin. At the end of the in vitro skin Statistical analysis
permeation study, the skin samples were washed with
waterandmethanolonbothsidesandcarefullydried.Then All the skin permeation studies were carried out in tripli-
a defined amount of methanol was added to each piece of cateandthedatawereexpressedasthemeanvalue ± SD.
skin. The samples were vortexed for 10 min to extract its Statistical data were analyzed by one-way analysis of
drug content and stirred overnight. The samples were variance (ANOVA). A multiple comparison test was used
analyzed by HPTLC (Patel et al. 2011a) after centrifuga- to compare different formulations and P\0.05 was con-
tion (Bachhav and Patravale 2009). sidered to be significant.
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ApplNanosci(2016)6:539–553 543
Fig.1 Thepseudoternary
phasediagramsoftheisopropyl
myristate,Labrasol/plurol
oleique(Emix),wateratthe1:1
(X),2:1(Y)and3:1(Z)weight
ratiosofEmix(Km)atambient
temperature.Theone-phase
regionisdesignatedby1Uand
themultiphaseregionbyMU.
Thedilutionline(a,b)atoil/
Emixof0.25representsthe
investigatedsystems’
compositions(I toI )
1 6
Results and discussion vehicles for topical application (Alvarez-Figueroa and
Blanco-M´endez 2001;Djekicand Primorac 2008). Also, it
Development and evaluation of microemulsion has been reported that LAB-based systems incorporating
fatty acid esters in an oil phase have a large ME region in
The solubility of ITTN in various oils, emulsifiers and co- theirphasediagrams.TofacilitatetheformulationofLAB-
emulsifiers was analyzed toscreen for componentsof ME. based MEs, PO has been used as co-emulsifier (Fanun
The solubility of ITTN was found to be highest in IPM 2008).Ithasalsobeenreportedthatsolubilizationofwater
(42.92 ± 3.97 mg/gm), a low molecular volume fatty acid is easier in systems containing LAB as emulsifier and PO
ester and one of the most often used oils along with long as a co-emulsifier. The highest solubility of ITTN was
chaintriglyceridesandmediumchaintriglycerides forME found in PO (10.04 ± 3.54 mg/gm) among the co-emul-
(Subramanian et al. 2005). It has been reported that the sifiersstudied.Fromtheseresults,IPM,LABandPOwere
large solubilizing capacity of ME leads to larger concen- subsequently used as the oil phase, emulsifier and co-
tration gradienttowardtheskin,providingsuperiordermal emulsifier for the formulation of MEs containing ITTN in
flux,forwhichhighsolubilityofITTNinoilyphasecanbe this study.
an added advantage to increase it in ME. Also, literature The pseudoternary phase diagrams of the system of
reveals that IPM acts as a chemical enhancer by disorder- IPM, LAB, PO and water were constructed to find out the
ing the prearranged lipids of the horny layer. To study the existence range of MEs. The phase diagrams are repre-
influence of oil on ME characteristics and penetration-en- sented in Fig. 1. The phase boundary was determined by
hancingability,IPMwaschosenforthepreparationofME- observing the changes of the sample appearance from
containing ITTN (Patel et al. 2009, 2011b, 2013a, b, c; turbidtotransparentorfromtransparenttoturbidbasedon
Subramanian et al. 2005). visualidentification.Theisotropicandlow-viscosityregion
The solubility of ITTN was determined in different is presented in the phase diagram as the one-phase ME
emulsifiers to find a suitable emulsifier for the preparation region (1U) and the remainder of the phase diagram rep-
of ME. The highest solubility of ITTN was found in LAB resents the turbid region, designated as multiphase (MU)
(86.81 ± 4.82 mg/gm) which is a non-ionic emulsifier conventional emulsions. From a microstructural point of
usedasapharmaceuticalexcipientforthesolubilizationof view, MEs may sometimes be envisaged as emulsifier ag-
hydrophobic drugs. There have been several studies in- gregratesofwaterdispersed(solubilized)inoil(w/o)oroil
volving low-irritant LAB-based MEs as drug delivery dispersed in water (o/w). When the ME region extends
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544 ApplNanosci(2016)6:539–553
Table1 Compositionoftheselectedplainmicroemulsionformulationsandtheirphysicochemicalparameters
MEs MEcompositions Particle Polydispersity Zetapotentiala Percentage pHa Conductivitya Viscositya
(%w/w) sizea(nm) Indexa (mV) transmittancea (lS/cm) (cp)
(%T)
IPM LAB PO Water
I 18 54 18 10 85.9±3.7 0.261±0.021 -27.86±2.32 98.04±0.75 6.30±0.01 2.4±0.1 120±0.05
1
I 16 48 16 20 82.7±1.8 0.159±0.058 -31.55±1.62 98.26±0.66 6.49±0.05 6.7±0.2 112±0.06
2
I 14 42 14 30 70.1±1.7 0.321±0.026 -33.67±2.10 98.05±0.65 6.50±0.04 13.8±0.6 92±0.03
3
I 12 36 12 40 50.1±1.2 0.278±0.014 -32.18±1.89 98.47±0.62 6.62±0.03 29.5±0.3 75±0.08
4
I 10 30 10 50 34.9±1.2 0.198±0.025 -38.76±2.41 98.97±0.56 6.28±0.02 50.8±0.5 80±0.02
5
I 08 24 08 60 22.4±0.2 0.127±0.015 -37.98±2.05 99.17±0.67 6.67±0.06 68.6±0.4 38±0.07
6
a Mean±SEM,n=3
fromanoil-richregion(i.e.,w/oME)toawater-richregion the range of 22.4–85.9 nm, while drug-loaded vehicles
(i.e., o/w ME), the transition between these two regions have a globule size in the range of 16.9–72.3 nm. Two
passes through a bicontinuous isotropic region (Fanun explanations, (1) undissolved drug acting as a emulsifier
2008).TheareaofMEisotropicregionchangedslightlyin and (2) reduction in emulsifier mobility, are offered in the
size with Km in the order of: 3:1[2:1[1:1. The phase literature for the decrease in droplet size with the addition
study revealed that the maximum solubilization of water of the drug due to deposition of drug particles at the in-
was achieved in ME systems when Km was 3:1 and so it terface of ME (Biruss and Valenta 2008; Djekic and Pri-
was fixed at 3:1 for further studies. morac2008;Fanun2008;Kantarcietal.2007).Ithasbeen
Themaximumpercentageofwaterinthephasediagram reported that the lowest conductivity plots extend to the
withIPMasanoilphase(Fig. 1z)was86.94 % w/wforthe largest droplet radius. The size of the droplet of unloaded
ratioofoiltoEmixof0.11withthecontentofEmixatKm ME with low conductivity values was greater than that of
= 3:1of9.89 % w/wand76 % w/wforratioofoiltoEmix the ITTN-loaded formulation with higher conductivity
of 0.25 with content of Emix at Km = 3:1 of 20 % w/w. values (Patel et al. 2009, 2011b, 2013a, b, c). All the for-
The obtained results suggested that the maximum solubi- mulations had droplets in the nano range, which is very
lizationofwater(Wmax)wasincreasedwithdecreaseinthe well evident from the low PDI values for both unloaded
amphiphilecontentintheratioofoiltoEmixintheorderof and ITTN-loaded vehicles as described in Tables 1 and 2.
0.11[0.25[0.42[0.6[1[1.5[2.3[9.TheW AlthoughthePDIvaluesofallformulationswereverylow,
max
was achieved at a ratio of oil to Emix less than 0.25. IPM indicating uniformity of droplet size within each formula-
wassolubilizedcompletelywithLABandtheW ofIPM/ tion, it was least for I (0.122 ± 0.012). An emulsifier can
max 6
LABmixturewasfoundtobehigher.Itwasconsideredthat stabilize the emulsion, not only just by forming a me-
IPM, due to its preferable chemical structure, probably chanical barrier, but also by producing an electrical (elec-
penetrated the interfacial layer and interacted with LAB, trostatic) barrier or surface charge. The electrical surface
increasing its efficiency in solubilizing water. Hence, the charge of the droplets is produced by the ionization of
obtained results indicated that LAB showed a good effi- interfacial film-forming components. The surface potential
ciency and water-solubilizing capacity in the presence of and the resulting zeta potential of emulsion droplets will
IPM. These observations were quite consistent with previ- depend onthe extent ofionizationofemulsifiers.ForMEs
ouslyreportedresults(DjekicandPrimorac2008). withnon-ionicemulsifier,thezeta potentialcan beusedto
Further six potential ME vehicles, I –I , for IPM as analyze the charge of the system (Biruss and Valenta
1 6
an oil phase (Table 1) were selected at a ratio of oil to 2008). The zeta potential of plain and ITTN-loaded ME
Emix of 0.25 along the dilution line A–B in Fig. 1z. The formulationsare presentedinTables 1and2,respectively.
entire drug-loaded ME formulations containing ITTN The zeta potential values of all the formulations are less
were prepared in the same ratios and the proportion of than -30 mv. It can be seen that the ME system is
ITTN was kept constant at 0.05 % w/w for all the negatively charged and on addition of ITTN, the zeta po-
batches. tentialvaluesintheMEdidnotchangesignificantly(Patel
The average particle size of the plain MEs and drug- et al. 2013a, b, c). The % T values of plain and ITTN-
loaded ME were determined. The results of particle size loaded ME formulations were between 98 and 100 %
analysisofMEformulationcontainingIPMasanoilphase (Tables 1 and 2). Hence, all the plain and medicated ME
are shown in Table 1 for plain MEs and Table 2 for drug- formulations were found to be optically clear. The TEM
loaded MEs. The unloaded vehicles have a globule size in imaging of ITTN-loaded ME formulation is shown in
123
ApplNanosci(2016)6:539–553 545
9 2 8 1 2 5 Fig. 2.TheTEMimagesrevealedthattheparticlesizewas
b 0 0 0 0 0 0
osity ±0. ±0. ±0. ±0. ±0. ±0. in the nanometric range and that particle had nearly
Visc(cp) 125 116 96 77 82 40 scpohrdearinccael mwoitrhphpoalrotgicylewsiitzheouatnaaglygsriesg.aFtiiognurweh3ichshioswins athce-
particle size distribution by intensity of optimized ITTN-
b
vity 0.1 0.8 0.6 0.5 0.3 0.5 loaded ME formulation. The ITTN content of ME formu-
uctim) ± ± ± ± ± ± lationswasfoundtobeintherangeof99.12–99.98 % w/w
dc 9 9 5 8 5 7
Conl(S/ 2. 13. 31. 67. 128. 205. osafmtphleestwheeorreeteicxaalmivnaeldueby(0o.0c5ul%ar win/swp)ect(iToanblien2a).crTohses
polarizer for sample homogeneity and birefringence. All
8 7 3 1 4 2
0 0 0 0 0 0
0. 0. 0. 0. 0. 0. MEformulationsappearedcompletelydarkwhenobserved
± ± ± ± ± ±
under cross polarizer which confirmed its optically
bpH 6.38 6.55 6.65 6.70 6.41 6.89 isotropic nature (Draize etal. 1944;Shahet al. 2007).The
rate of diffusion of a drug is dependent on the pH of the
T) formulation. It is one of the factors influencing skin
%
( penetration(Pateletal.2013a,b,c).ThepHvaluesforME
bce 63 35 51 58 41 52 formulations were found in the range of 6.28–6.67
ntagemittan ±0. ±0. ±0. ±0. ±0. ±0. (Table 1).TheincorporatedITTNdidnotaffecttheoptical
Percetrans 98.00 98.17 98.67 98.35 98.83 99.14 tpeHxtuvraeluoefstohfethMeEMfEorfmourmlautiloantisonasndsigdnidifincoatntilnyfl(uTeanbcleet2h)e.
Electricalconductivityisastructure-sensitivepropertyand
bnt 28 20 67 45 85 87 therearesomestudiesreportedonthesystemsofnon-ionic
arameters Drugconte(%w/w) 99.12±2. 99.28±2. 99.78±1. 98.56±2. 99.46±1. 99.98±1. etsimmviautlyllsiafimmeeorasus.nuAtreolmtfhaeoqnuutgehwouerserepeolerdctoetnrdoeliynwteilti(htNeorauaCttul)riner,ceostprhpoeonrscaiobtinlnedgufoca-r
p charge transport due to its effect on phase behavior and
physicochemical bZetapotential(mV) -24.56±2.35 -29.69±1.75 -34.42±2.04 -31.15±2.33 -39.42±2.31 -36.99±2.03 n sTaowtmfhraueitptecshrtiuinnpvrvohaoenillls-ueptimisorgoneeapixtceefhrrdnitabiaceiMttstuieorEodenf.eMf(FloUeirEcgwmtusr,,ruoe%rlcaeo4tsniawuodln/dtuwisicns)tgpicvlcoiaehnnyatbspanehigtnhhaeiaesnveogiinnosflernt,puohaienenrn-aciestoeilpoeniocntiec-f.
ulationsandtheir Polydispersitybindex 0.241±0.033 0.138±0.056 0.243±0.027 0.267±0.016 0.186±0.021 0.122±0.012 w/wofisotretinoi twcvroaiicnltuhadelutshccioteninvdidinituccyacrettieinavcsriereteysiatnr(sirecwd,tealcdteoSwrn/ctcaimontenu)rtooemufnsotMl,byti.EhliTetfyhov,erawmllouhuweillsaetcoihoofinngedshl.uecvcAttairllvoiucinetagysl
m
for be 5%
croemulsion Particlesiz(nm) 72.3±0.4 68.5±0.3 62.2±0.4 44.5±0.7 28.3±0.5 16.9±0.3 contains0.0
g-loadedmi aw) Water 10 20 30 40 50 60 additionally
thedru (%w/ PO 18 16 14 12 10 08 ulation
Table2Compositionof MEMEcompositions IPMLAB 1854I1 I16482 I14423 I12364 I10305 I08246 aEachdrug-loadedformbnMean±SEM,=3 Fig.2 TheTEMimageofisotretinoin-loadedmicroemulsion
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546 ApplNanosci(2016)6:539–553
Fig.3 Theparticlesize
distributionbyintensityof
optimizedisotretinoin-loaded
microemulsion
Fig.4 aElectricalconductivity
(r)andbviscosity(g)asa
functionofwaterphasevolume
fraction(Uw)inthesystemwith
oil/Emix0.25andKm3:1for
microemulsionformulations
suggest that the system undergoes a structural inversion relatively moderate rate up to a maximum Uw = 60 %
from w/o to o/w MEs. These changes have been attributed w/w. This shows that the conductivity of the system was
to the occurrence of a percolation transition. In the per- notsignificantlyaffectedbyfurtheradditionofwater.After
colation threshold model, the conductivity remains low up reaching the maximum value, the conductivity of the sys-
to a certain critical volume fraction of water (Uc) as the temwasslightlydecreasedbyfurtheradditionofthewater
w/o droplets surrounded by continuous oil phase are iso- phase. This suggested the further dilution of o/w ME with
lated from each other, thus contributing very little to the added water which decreased the concentration of the
electricalconductance. When thevolume fraction ofwater dispersedoildroplets.Thus,ther–Uwcurveillustratesthe
reaches the percolation threshold (Uc = Up), clusters of occurrence of the three structural regions: w/o
some conductive droplets which begin to contact each (Uw\10 % w/w), non-spherical w/o-bicontinuous-non-
other are formed. Beyond Up the conductivity increases spherical o/w (10 % w/w\Uw\50 % w/w) and o/w
sharply and rapidly as the number of clusters increases. (Uw[50 % w/w). The conductivity results obtained for
Thishasbeenattributedtotheformationofwatercylinders plain and ITTN-loaded ME formulations (Tables 1 and 2)
or channels in an oil phase due to the sticky collisions showed thatloadingITTN and the addition ofwater phase
through transient merging of spherical microdroplets of into the formulations had no negative effects on system
water phase in the w/o ME (Fanun 2008). As shown in stability. When an unstable emulsion system and phase
Fig. 4a, the electrical conductivity of the oily mixtures separation occurs, the conductivity values are greatly re-
containingIPMwasalmostzeroaslongasUwwassmaller duced (Kantarci et al. 2007). However, the conductivity
thanthecriticalvolumefraction(Uc)10 % w/w.However values for unloaded vehicles were about two to three fac-
whenthewatercontentwasraisedaboveUc = 10 % w/w, tors lower than those of ITTN-loaded formulations. The
the value of conductivity increases fast up to variation of viscosity as a function of water content along
Uw = 50 % w/w. This phenomenon is known as percola- the dilution line (A–B) for the system IPM/LAB/PO/water
tion and the critical Uw at which it occurs is known as (Fig. 1)ispresentedinFig. 4b.Allthetestedsampleswere
percolation threshold (Up = 10 % w/w). At liquids of low viscosity. Three regions were observed in
Uw[50 % w/w, it increases continuously, but with this curve. In the first region, where the water volume
123
ApplNanosci(2016)6:539–553 547
contentisbelow10 % w/w,theviscosityincreases.Thena increaseofwaterphasevolumefractionintheselectedoil/
sharpdecreaseisobservedatwatervolumefractionsabove emulsifier/co-emulsifier mixture.
10 % w/w. For water contents between 10 and 40 % w/w, In vitro skin permeation studies were conducted to
theviscosityoftheMEsystems decreasesslowly.Aslight measure the rate and extent of compound transport across
increase in viscosity is also observed with increase in the the skin. Figure 5(A) shows the release profiles of ITTN
water volume fraction from 40 to 55 % w/w. A sudden from the ME formulations. The cumulative amount of
decrease in the viscosity is observed with increase in the ITTN that had permeated through rat skin (lg/cm2) was
water volume fraction above 55 % w/w. The viscosity is plotted as a function of time (hours). It is possible to cal-
sensitive to interactions and it is rather an interesting culate the steady-state flux (J ) obtained from the slope of
ss
technique to study interactions in systems of known the linear portion (2–8 h) of the graph (Bachhav and Pa-
structure. The change in viscosity with the increase in travale 2009; Djordjevic 2004; Patel et al. 2009, 2011b;
water volume fractions should be correlated to structural Shahet al.2007).Thepermeationparametersofthetested
effectsduetointerfacialpacking(BirussandValenta2008; ME formulations are presented in Table 3.
Fanun2008;Kantarcietal.2007).Forwatercontentbelow AsteadyincreaseofITTNinthereceptorchamberwith
10 % w/w, the increase in viscosity indicates increase of time was observed. The permeation profiles of MEs fol-
volume fraction of dispersed phaseand reflects a transfor- lowed zero-order kinetics. The depletion of ITTN in the
mationofsystemmicrostructureduetogrowthofspherical external phase because of the permeation into the skin can
droplets into larger non-spherical droplets by an attractive be supplemented by the release of the ITTN from the in-
interaction as a function of Uw. The position of the max- ternal phase. Thus, the zero-order release kinetics and
imumontheg-Uwcurveisatwaterphaseofabout10 % sustained and controlled delivery of ITTN were obtained
w/w The reduction in the viscosity with the increase in (BachhavandPatravale2009;Djordjevic2004;Pateletal.
.
water content above 10 % w/w to about 60 % w/w seems 2009, 2011b, 2013a).
to be attributed to the fact that the emulsifiers move from When all the formulations containing IPM as an oil
the bulk to the interface to cover the water and oil in a phasewereevaluatedaccordingtothereleaserateofITTN,
bicontinuous structure. The small increase in the dynamic I showedthehighestfluxvalue(0.0380 ± 0.0057lg/cm2/
6
viscosity for water contents above 40 % w/w suggests a h) and I showed the lowest flux value (0.0318 ± 0.0021
1
structural transformation from a bicontinuous structure to lg/cm2/h). However, there was no significant difference
oil in water structure. This phenomenon can also be ex-
plained in terms of a decrease in the hydrophobic interac- Table3 The permeation parameters of isotretinoin-loaded mi-
tion of the emulsifier tails. High water contents cause a croemulsion formulation containing isopropyl myristate as an oil
reduction in the inter-droplet interactions and reduction in phase
the viscosity. The relatively low viscosity values indicate Formulation J (lg/cm2/h) K (cm/h)
ss p
that the MEs formulated are composed of individual
I 0.0318±0.0021 6.36910-5±0.10910-6
spherical droplets or bicontinuous structures and no ani-
I 0.0320±0.0013 6.40910-5±0.08910-6
sometric aggregates are present (Patel et al. 2011b). The 2
I 0.0340±0.0034 6.80910-5±0.12910-6
results of viscosity measurements for plain and ITTN- 3
I 0.0355±0.0045 7.10910-5±0.16910-6
loaded ME formulations (Tables 1 and 2) indicate that the 4
I 0.0368±0.0029 7.36910-5±0.14910-6
viscosityvaluesoftheITTN-loadedMEswerehigherthan 5
I 0.0380±0.0057 7.60910-5±0.18910-6
the unloaded vehicles. The conductivity and viscosity data 6
confirmed the continuous structural transitions during the Mean±SEM,n=3
Fig.5 Invitropermeation
profilesofisotretinointhrough
ratskinfromamicroemulsion
formulations,bmarketedgel
andmicroemulsion-basedgel
(ITTN-MBG)(mean±SEM,
n=3)
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548 ApplNanosci(2016)6:539–553
between flux valuesof all the formulations. From Table 3, Development and evaluation of microemulsion-
it can be concluded that the permeation rate of ITTN in- based gel
creases with decrease in the concentration of oil and Emix
and increase in the concentration of water (Bachhav and The formulation I (containing 8 % w/w IPM, 32 % w/w
6
Patravale 2009; Djordjevic 2004; Patel et al. 2009, 2011b, Emix, 60 % w/w water) having appropriate physicochem-
2013a). ical parameters and higher permeation parameters was
Due to low viscosity of MEs, the mobility of drugs in considered as the optimized formulation. The MBG of the
MEsismorefacileandthisisimportantforthepermeation optimized formulation was developed using a suitable
into skin. So the water content of ME formulations was polymer capable of modifying the rheological behaviour.
varied between 10 and 60 %, as there exists a really tight Various gelling agents, namely hydroxypropyl methylcel-
relationship between the hydration effect on the stratum lulose(MethocelK4 M)andnon-benzenegradeCarbopol(cid:3)
corneumanddermalpermeation(BirussandValenta2008; polymers (Carbopol(cid:3) 971P NF, Carbopol(cid:3) 974P NF and
Fanun 2008; Kantarci et al. 2007). Carbopol(cid:3)980PNF)wereevaluatedfortheirabilitytogel
It was concluded that the content of Emix in ME af- optimized ITTN-ME. Also, different concentrations of the
fected the skin permeation rate of ITTN significantly. As above-mentioned gelling agents were tried. The suitable
the content of Emix was decreased from 72 to 32 % w/w, gelling agent was selected on the basis of compatibility
the skin permeation rate of ITTN was also increased. This with ME structure, feel and ease of spreadability. When
may be due to increased thermodynamic activity of the hydroxypropyl methylcellulose (Methocel K4 M) and
drug in the ME at the lower content of Emix, as ITTN is Carbopol(cid:3) 974P NF were added to ME to prepare MBG,
soluble in Emix. To evaluate the effect of oil phase on the only an ivory-white gel was obtained indicating that the
permeation rates, different MEs containing IPM concen- structurewasdisturbed.Soitwasconcludedthattheywere
tration from 8–18 % w/w were compared. The ME I not a suitable gel matrix for this ME system. The incor-
6
(containing 8 % w/w oil phase) showed the highest per- poration of Carbopol(cid:3) 980P NF and Carbopol(cid:3) 971P NF
meation rates. The increase in oil phase in the vehicles intoME couldincreasethe viscosity andalso maintain the
resultedindecreaseofpermeationrate.Itisconsideredthat ME structure. Further, since MBG containing Carbopol(cid:3)
ahighcontentofEmixintheMEs(I ,I ,I ,I ,andI )may 971P NF had the most appropriate fluidity and spread-
1 2 3 4 5
make the effect of oil on the skin permeation less pro- ability for topical administration, it was selected as an
nounced. Also, the high solubility of drug in the oil phase optimum gel matrix for optimized ME (Bachhav and Pa-
could lead to an unfavorable partition from the oil phase travale 2009).
(Patel et al. 2009, 2011b, 2013a, b, c). Thus, it was con- Briefly, the MBG of I was prepared as shown in
6
cluded that the high permeation of MEs may be attributed Table 4. The influence of the method of addition of Car-
to the combined effects of both the lipophilic and hy- bopol(cid:3) 971P NF on the formation of MBG was investi-
drophilic domains of MEs. Large microparticles ([10–20 gated. Carbopol(cid:3) 971P NF was directly added into ME or
lm) do not penetrate the skin and remain on the stratum the aqueous phase of ME, respectively (Bachhav and Pa-
corneum, but particles of\3 lm are randomly distributed travale 2009).
in the stratum corneum and penetrate the skin (Shah et al. As per the above observations, it can be concluded that
2007) When the particle size is very small, the number of thegel networkincreasedtheviscosityofthe system,with
vesicles that can interact on a fixed area of stratum cor- noinfluenceonthespontaneousdispersionofoilyphasein
neummaybeincreased,therebyincreasingitsefficiencyin aqueous phase or the spontaneous formation of ME. The
thepercutaneousuptakeofdrugs.Thelowerpermeationof orderofadditionofCarbopol(cid:3)971PNFhadnosignificant
ITTN from ME containing larger size particles as com- influenceontheformationofMBG,butmightinfluencethe
paredtoMEwithsmallerparticlesmaybeexplainedbythe homogenized swelling of Carbopol(cid:3) 971P NF. In a
above-mentioned facts (Fanun 2008).
MEshowedgoodshelfstabilityandnophaseseparation, Table4 Composition of microemulsion-based gel containing
cloudiness or any flocculation or precipitation for isotretinoin
6 months. These samples also showed no sign of phase
Components Content
separation under stress when subjected to centrifugation at
Isotretinoin(%w/w) 0.05
50009g for 30 min. Thus centrifuge tests showed that all
Carbopol(cid:3)971PNF(%w/w) 2
MEs had good physical stability. All physicochemical pa-
Isopropylmyristate(%w/w) 8
rameters were determined at weekly intervals for the first
Labrasol(%w/w) 24
month and monthly interval for the subsequent months.
Plurololeique(%w/w) 8
However, no significant change in the physicochemical
Water(g) Quantitysufficienttoproduce100gofgel
parameters was observed (data not shown).
123