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Nanocolloidal carriers of isotretinoin antimicrobial activity against propionibacterium acnes and dermatokinetic modeling 2013

Article
pubs.acs.org/molecularpharmaceutics

Nanocolloidal Carriers of Isotretinoin: Antimicrobial Activity against
Propionibacterium acnes and Dermatokinetic Modeling
Kaisar Raza,† Bhupinder Singh,†,‡ Saloni Singla,§ Sheetu Wadhwa,‡ Babita Garg,‡ Sanjay Chhibber,§
and Om Prakash Katare*,‡


UGC-Centre of Excellence in Applications of Nanomaterials, Nanoparticles and Nanocomposites, Panjab University, Chandigarh,
India 160014

Division of Pharmaceutics, University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh, India 160014
§
Department of Microbiology, Panjab University, Chandigarh, India 160014
ABSTRACT: Acne, a common skin disease in teenagers, is caused by
Propionibacterium acnes (P. acnes). Isotretinoin (ITR) is though reported
to have immense antiacne potential, yet there are hardly any reports
vouching its antimicrobial activity. The present study, therefore, was
undertaken to study the antimicrobial activity of ITR and evaluate the
effect of its encasement in nanocarriers on its minimum inhibitory

concentration (MIC). The nanocarriers were also evaluated for the skin
transport characteristics. MICs of pure drug and entrapped drug in
nanolipid carriers (ITR-NLCs) and in solid lipid nanoparticles (ITRSLNs) were determined by broth dilution method against clindamycin
phosphate as the reference antibiotic. It was observed that ITR
possessed marked antimicrobial activity against anaerobic pathogen, P.
acnes. Nanocarriers loaded with ITR, that is, SLNs and NLCs, enhanced
the antimicrobial activity even at lower concentrations vis-à-vis the drug alone and improved drug transport potential vis-à-vis the
commercial gel. The unique findings could be the result of effective adhesion of ITR-loaded nanocarriers to the bacterial
membranes and release of drug directly to the target. Besides establishing ITR as an antimicrobial agent against acne-causing
bacteria, the current work ratifies immense potential of nanocolloidal carriers like SLNs and NLCs to treat acne in a more
efficient manner.
KEYWORDS: minimum inhibitory concentration (MIC), solid lipid nanoparticles (SLNs), nanolipid carriers (NLCs),
Propionibacterium acnes, anaerobic pathogen, acne vulgaris, comedolytic

1. INTRODUCTION

Though acne is seldom associated with mortality, it
substantially affects the psychological and social status of the
affected population.6,7 Other concerns associated with acne are
the long tenure of treatment along with economic issues.8
Topical retinoids alone, or in combination with antibiotics, are
widely prescribed for the treatment of mild to moderate
acne.9,10 For severe acne, however, oral isotretinoin (ITR) is
frequently prescribed.11 Use of antibiotics alone for the
treatment of acne has yielded promising results.12−14 Nevertheless, issues of microbial resistance against antibiotics are of
great concern.15,16 It is also advised that clinicians should limit
the prescription of antibiotics, wherever and whenever
possible.15 Hence, there are adequate reasons to search for
alternative therapy or to improve the efficacy of the present
treatment approaches.

Acne vulgaris, commonly known as acne, affects around 85% of
teenagers, reaching its acme during adulthood.1,2 According to
WHO, acne is an inflammatory disease of the pilosebaceous
units in the skin of the face, neck, chest, and upper back.
Typically, it first appears during early puberty when androgenic
stimulation triggers excessive production of sebum and
abnormal follicular keratinization, colonization by a Grampositive bacterium (i.e., Propionibacterium acnes), and local
inflammation. P. acnes is one of the principal factors of acne
pathogenesis. P. acnes produces inflammation through the


production of extracellular products such as lipases, proteases,
hyaluronidases, and chemotactic factors.1 It is the predominant
microorganism of the infected pilosebaceous glands of acne
skin, as nearly as 107 viable organisms are reported to be
isolated from a single sebaceous unit.3,4 P. acnes typically grows
in the infrainfundibulum of the pilosebaceous units and digests
the oily substance produced by the sebaceous glands.3 Apart
from acne, P. acnes overgrowth is also reported to be associated
with diseases like toxic shock syndrome and endocarditis.2,5
© 2013 American Chemical Society

Received:
Revised:
Accepted:
Published:
1958

December 21, 2012
March 11, 2013
April 1, 2013
April 1, 2013
dx.doi.org/10.1021/mp300722f | Mol. Pharmaceutics 2013, 10, 1958−1963


Molecular Pharmaceutics

Article

dispersed in a portion of water along with Tween 80 (7.0 g)
and SMBS (0.5 g). Compritol (1.2 g) and IPM (0.2 g) were
melted at 70 °C. The lipid phase, aqueous phase, and drug
solution were mixed isothermally to obtain a clear microemulsion. The remaining portion of water (about 80 mL) was
cooled at 4 °C. The hot microemulsion formed was poured
into cold water previously maintained at 4 °C, and the
dispersion formed (100 mL) was stirred continuously for 20
min at 3000 rpm.23
2.3. Preparation of Bacteria. P. acnes (MTCC 1951)
(Microbial Type Culture Collection, IMTECH, Chandigarh,
India) was cultured on brain heart infusion media under
anaerobic conditions using Anaerogas Pack at 37 °C. Single
colonies were inoculated and incubated at 37 °C until reaching
around OD600 = 0.3 (logarithmic growth phase) under
anaerobic conditions. The bacteria were harvested by
centrifugation at 5000g for 10 min, washed with PBS, and
suspended in an appropriate amount of PBS.2
2.4. Antimicrobial Activity of ITR and Its Nanocarriers
against P. acnes. P. acnes was incubated in BHI broth with 1%
glucose for 48 h under anaerobic conditions and adjusted to
yield approximately 1 × 108 CFU/mL. 2-fold serial dilutions
were made in the broth over a range to give concentrations of
8−500 μg/mL of isotretinoin. In sterile 96-well microtiter
plates, 100 μL of the drug solution (in 40% dimethyl sulfoxide,
DMSO)/SLN-dispersion/NLC-dispersion/blank SLN-dispersion/blank NLC-dispersion/40% DMSO solution was diluted
with broth and added to wells containing 100 μL of bacterial
suspension in broth. To adjust the interference by nanoparticle
components, a parallel series of mixtures with uninoculated
broth was prepared. Triplicate samples were performed for each
test concentration. After incubation for 48 h at 37 °C under
anaerobic conditions, the quantum of microbial growth was
determined by absorbance at 600 nm using a microplate reader
(Biotek Instruments, Winooski, VT, USA). Aqueous solution of
clindamycin phosphate was also studied analogously to serve as
the control owing to its marked antimicrobial activity against P.
acnes.24 MIC was taken as the lowest concentration of a test
compound (i.e., ITR) which inhibited the growth of P. acnes.25
2.5. Drug Permeation Studies. Hairless Laca mice were
employed for the skin permeation studies on Franz diffusion
cells (M/s Permegear, Inc., PA, USA). After sacrificing the
animals, the hairs on the dorsal side of animals were removed.
The skin was harvested, freed of adhering fat layers, and
mounted on Franz diffusion cells having a cross-sectional area
of 3.142 cm2 and receptor volume of 30.0 mL. The diffusion
medium in the receptor compartment was composed of
Isotonic Palitzsch Buffer26 containing Tween 80 (2.7% w/w)
and ethanol (20.0% v/v). The assembly was maintained at 37 ±
1 °C with the help of thermo-regulated outer water jacket,
while the diffusion medium was stirred continuously using a
magnetic stirrer. Various formulations (ITR-SLN gel, ITR-NLC
gel, and commercial gel), each containing ITR equivalent to 0.6
mg, were applied onto the mice skin in the donor compartment.
Aliquots of 1 mL each were periodically withdrawn at
suitable time intervals from the sampling port and replaced with
an equal volume of fresh diffusion medium to maintain the
constant receptor volume. The samples were analyzed by a
validated reverse phase high-performance liquid chromatographic (RP-HPLC) method.27 ITR was quantified at 355 nm
using a liquid chromatograph LC-2010CHT (Schimadzu,
Japan), equipped with a UV detector and a software LC

Novel drug delivery systems are known to enhance the
overall performance of the drug at the target site vis-à-vis the
conventional dosage forms.17 Solid lipid nanoparticles (SLNs)
and nanolipid carriers (NLCs) are nanocolloidal systems
composed of biocompatible lipids.18,19 The biocompatible
lipid-based carriers can transport the drugs to the target site
and maintain drug concentrations higher than that with the
conventional formulations.2,20 These agents do not employ
solvents like dimethyl sulfoxide (DMSO), which are commonly
employed in other topical products as penetration enhancer(s)
or solubilizer(s). Such penetration enhancers are reported to
cause dermal irritation and at times, serious side effects.2
ITR is now-a-days also prescribed in the topical forms and is
believed to affect all of the causative mechanisms of acne,
including infestation by P. acnes.10 The hypothesis of ITR
antimicrobial activity against P. acnes has not yet been proved in
vitro or in vivo. Apart from this, it is a newly reported fact that
use of novel carrier systems can result in substantial
antimicrobial activity of the drug even at lower concentrations.2,21 Hence, the present study endeavors to establish the
antimicrobial activity of ITR against P. acnes and to study the
influence of encapsulation in nanocarriers SLNs and NLCs on
the skin transport characteristics, as well as on the minimum
inhibitory concentration (MIC). The developmental studies
have been published elsewhere along with antiacne activity,
biocompatibility, and biochemical evaluation.22,23

2. MATERIALS AND METHODS
2.1. Materials. Isotretinoin (ITR; M/s Ipca Laboratories
Ltd., Ratlam, India), phosphatidylcholine (PC; Phospholipon
90 G; M/s Phospholipid GmbH, Nattermannallee, Germany),
clindamycin phosphate (M/s Psycoremedies, Ludhiana, India),
and Compritol 888 ATO GF3123 (M/s Gattefosse, Hauptstrasse, Germany; M/s Colorcorn, Mumbai, India) were
provided ex-gratis by the respective organizations. Isopropyl
myristate (IPM; M/s LobaChemie, Mumbai, India), butylated
hydroxy toluene (BHT; M/s SD. Fine Chemicals Ltd.,
Mumbai, India), Anaerogas Pack 3.5 L (M/s Hi Media Lab.
Ltd., Mumbai, India), brain heart infusion media (BHI; M/s Hi
Media Lab. Ltd., Mumbai, India), and tocopherol acetate (M/s
E-Merck (India) Ltd., Mumbai, India were procured from the
corresponding sources. Microbial culture of P. acnes (MTCC
1951) was procured from Institute of Microbial Technology
(IMTECH), Chandigarh, India. All other chemicals employed
in various studies were of analytical grade and were used as
such. Ultrapure water (Milli-Q Integral system; M/s Merck
Millipore, Billerica, USA) was used throughout the study.
2.2. Preparation of ITR-Loaded Nanocarriers.
2.2.1. ITR-Loaded SLNs. The microemulsification method was
employed for the preparation of ITR-loaded SLNs.22 In brief,
ITR (50.0 mg) and the lipophilic antioxidant (BHT; 138.7 mg)
were dissolved in ethanol (6.0 g). The PC (208.5 mg) was
dispersed in a portion of water along with Tween 80 (6.5 g)
and sodium metabisulfite (SMBS; 0.5 g). Compritol (674.0
mg) was melted at 70 °C. The lipid phase, aqueous phase, and
drug solution were mixed at same temperature to give a clear
microemulsion. The hot microemulsion formed was poured
into the remaining portion of water (i.e., about 80% of the
total), previously cooled and maintained at 4 °C. The resulting
mixture (100 g) was stirred continuously at 3000 rpm for 20
min.23
2.2.2. ITR-Loaded NLCs. Drug (50.0 mg) and BHT (BHT;
138.7 mg) were dissolved in ethanol (5.0 g). PC (0.6 g) was
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Figure 1. TEM photomicrographs (a) ITR-loaded SLNs (2 000 000×); (b) ITR-loaded NLCs (1 000 000×).

solutions through a reverse phase C-18 column of length 150 ×
4.6 mm, 3 μ ODS1 Spherisorb (M/s Waters Corporation,
Milford, USA). The mobile phase consisted of glacial acetic
acid 1.96% v/v: methanol (27:73 v/v), delivered at a flow rate
of 1.0 mL/min. The permeation flux values were calculated for
all of the formulations by plotting the amount of drug released
per unit surface area vs time. The slope of the regressed line of
the straight portion of the graph was reported as the
permeation flux value.28
2.6. Skin Retention Studies. Skin mounted on the
diffusion cell was removed carefully after completion of
permeation studies. The formulation remaining adhered on
the skin was scrapped off carefully with the help of a spatula
and subsequently analyzed for drug content. The cleaned skin
tissue was washed thrice with ultrapure water and dried using
lint-free cotton swab. The skin was chopped into small pieces
and macerated in methanol (5 mL) for 24 h for complete drug
extraction to take place. After filtering the solution through a
membrane (0.45 μm), the filtrate was analyzed using the
validated RP-HPLC technique.
2.7. Dermatokinetic Modeling. The excised skins of
Wistar rats were used for the studies. The skin tissue was
prepared as discussed in section 2.5. Franz cell diffusion
assembly was used for the studies as discussed under
permeation studies with a dose of application equivalent to 1
mg of ITR. In this case, the whole skin was removed from the
Franz cell at the respective sampling times. The skin was
washed thrice to remove any adhering formulation and
subsequently soaked in hot water (60 °C) to detach the
epidermis from dermis.29,30 Both of the sections were chopped
into small pieces, separately, and macerated in methanol (5
mL) for 24 h for complete drug extraction to take place. After
filtering the solution through a membrane (0.45 μm), the
filtrate was analyzed using the validated RP-HPLC technique.
The obtained data were fitted into one-compartment open
model, as per the following equation (eq 1)
Cskin =

skin
K p·Cmax

(K p − Ke)

concentration achieved in skin, and Ke is the skin elimination
constant. Win-Nonlin Ver 5.0 software was employed to
compute various dermatokinetic parameters , namely, Kp, Cskin
max,
skin
(time
required
to
achieve
C
)
and
area
under
the
Ke, and Tskin
max
max
curve (AUC0−6h) using the Wagner−Nelson method. Data
analysis was accomplished using nonlinear function minimization employing Gauss-theorem algorithms built into the
software. Statistical validity of the results was discerned on
the basis of minimization of various model fitness parameters
like Akaike Information Criterion, Schwartz Criterion, sum of
squares due to residuals, and maximization of Pearsonian
correlation coefficient.
2.8. Estimation of Drug in Blood Portal. Six animals
(Healthy male Wistar rats, 9−10 weeks old, 280−320 g) each
were divided into three groups. Animals were caged and fed as
per the norms of Institutional Animal Ethics Committee. Group A
received marketed product while Group B and Group C
received ITR-SLN gel and ITR-NLC gel, respectively. Each
group received ITR equivalent to 0.6 g by topical application on
the shaven skin. After dose application, serial blood samples
(0.5 mL aliquots) were withdrawn from the tail vein at 0, 2, 4,
and 6 h postdosing. Plasma was harvested by centrifugation and
stored at −20 °C until analysis.31
2.9. Ethical Compliance. All animal protocols were duly
approved by Institutional Animal Ethics Committee, Panjab
University, Chandigarh, India (ref. letter no. CAH/09/70;
IAEC/170-175).
2.10. Statistical Analysis. Multiple comparisons were
made using one-way ANOVA followed by post hoc analysis
using Student’s t test. Statistical significance was considered at P
< 0.05.

3. RESULTS AND DISCUSSION
3.1. Formulation Details. Average particle size of both the
colloidal carriers (i.e., SLNs and NLCs) was found to be below
100 nm, i.e., 75.3 nm (SLNs) and 80.0 nm (NLCs),
respectively. Entrapment efficiency of these carriers was
found to be above 75%, i.e., 89.49% for SLNs and 78.60% for
NLCs. Both the carriers possessed significantly high magnitude
of zeta-potential, i.e., −22.4 mV for SLNs and −15.0 mV for
NLCs, the negative sign corroborating the anionic surface

(e−K pt − e−Ket )
(1)

where Cskin is the concentration of drug in skin at time t, Kp is
skin
the dermal permeation constant, Cmax
is the maximum
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components of the nanocarriers and better interaction of the
carriers with the skin components.17 The significant difference
(p < 0.001) in the drug transport characteristics of SLNs and
NLCs both may be assigned to the difference in the
composition and physicochemical properties of these lipidic
colloidal carriers.
3.4. Skin Retention Studies. Skin retention values (μg
cm−2 ± SD) from the studied formulations were observed as
follows:

charge on the colloidal carriers. ITR-loaded nanoconstructs
were observed to be spherical in nature, as confirmed by
transmission electron microscopic (TEM) photographs (Figure
1).
3.2. Antimicrobial Activity. The order of MIC values was
observed to be as follows:
ITR(125.0 μg/mL)
> ITR−SLNs
≈ ITR−NLCs(62.5 μg/mL)

ITR−NLCs(22.98 ± 1.98)
> ITR−SLNs(15.79 ± 1.32)

> clindamycin phosphate(31.75 μg/mL)

> commercial gel(9.78 ± 0.59)

It is clearly vivid from the results that ITR also possesses its
own antimicrobial activity with MIC value of 125.0 μg/mL visà-vis 31.75 μg/mL for the control antibiotic, i.e., clindamycin
phosphate. Interestingly, incorporation of ITR in the lipidbased nanocarriers resulted in marked 50% reduction of MIC
to 62.50 μg/mL for both ITR-SLNs and ITR-NLCs. This twofold enhancement in the antimicrobial activity of the nanoentrapped ITR can be ascribed to the better interaction of the
lipid-based nanocarriers with the bacterial cell wall, resulting in
increased contact time and sustained delivery of the drug to the
bacteria.2
3.3. Drug Permeation Studies. Figure 2 shows the drug
permeation profile of ITR from various studied systems.

Nanocolloidal carriers, namely, SLNs and NLCs, were able to
significantly (p < 0.001) enhance the skin deposition of ITR in
comparison to that of the commercial product. This can be
attributed to the fusion of the biocompatible drug loaded
carriers with the skin components and formation of micro drug
reservoirs.20 These micro drug-depots in the skin ensure the
sustained drug delivery to the target site in a controlled manner.
3.5. Dermatokinetic Modeling. Figures 3 and 4 show the
distribution of drug in epidermis and dermis of the Wistar rat

Figure 3. Graph showing the amount of drug present in the epidermis
of Wistar rats at various time points.

Figure 2. Drug permeation profile of ITR from various studied
formulations across Laca mice skin.

Percent drug permeation (±SD) after 24 h was found to
observe the following order:
ITR−SLN gel(84.91 ± 4.25)
> ITR−NLC gel(75.21 ± 3.76)
> commercial gel(43.88 ± 2.19)

The pattern of skin permeation flux values (μg cm−2 h−1 ± SD)
was found to observe the following:
ITR−SLN gel(19.75 ± 1.21)
> ITR−NLC gel(14.39 ± 1.37)
Figure 4. Graph showing the amount of drug present in the dermis of
Wistar rats at various time points.

> commercial gel(9.66 ± 0.89)

The extent of permeation, as reflected from the corresponding
permeation flux values, were found to be significantly (p <
0.001) greater in case of nanocarriers vis-à-vis the marketed
product. This can be attributed to the biocompatible

skin, respectively. Delivery of ITR in the skin layers by SLNs
and NLCs was found to be significantly greater (p < 0.05) than
that from the commercial gel. However, the drug delivery
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Molecular Pharmaceutics

Article

Table 1. Various Dermatokinetic Parameters (Mean ± SD) of ITR Topical Formulations in Epidermis and Dermis of Wistar
Rats (n = 6)
isotretinoin-SLNs

isotretinoin-NLCs

commercial gel

dermatokinetic parameters

epidermis

dermis

epidermis

dermis

epidermis

dermis

AUC0−6h (μg cm−2 h)
−2
Cskin
max (μg cm )
skin
Tmax (h)
Kp (h−1)
Ke (h−1)

176.99 ± 12.31
156.84 ± 7.23
0.35 ± 0.02
34.36 ± 2.97
1.03 ± 0.45

496.84 ± 31.09
109.62 ± 9.98
2.25 ± 0.02
0.89 ± 0.07
0.18 ± 0.01

192.89 ± 14.14
114.86 ± 9.65
0.55 ± 0.03
7.48 ± 0.79
0.98 ± 0.08

381.72 ± 20.12
88.97 ± 3.98
2.11 ± 0.02
0.67 ± 0.05
0.32 ± 0.02

142.31 ± 7.98
26.21 ± 0.04
0.75 ± 0.03
0.38 ± 0.02
0.33 ± 0.02

97.33 ± 7.81
25.93 ± 1.45
1.42 ± 0.08
1.46 ± 0.07
0.27 ± 0.01

edged. Generous help provided by Mr. Aman Singla in
dermatokinetic data treatment is highly appreciated.

potential in the skin layers by NLCs was observed to be
superior to that of SLNs. Table 1 gives the numeric values of
skin
AUC0−6h, Cskin
max, Tmax, skin penetration rate constant (Kp), and
skin elimination rate constant (Ke).
The nanocolloidal systems (SLNs and NLCs) significantly
enhanced the ITR delivery in the skin layers vis-à-vis the
commercial product (p < 0.001 each). AUC of ITR was also
found to be significantly greater (p < 0.001 each) in the dermis
than in the epidermis for both of the colloidal carriers. The
results, therefore, unequivocally vouch that the carriers
delivered ITR to the dermis layer quite efficiently ascertaining
adequate drug supply to the sebaceous glands, i.e., the afflicted
site of acne.
3.6. Estimation of Drug in Blood Portal. Plasma samples
of all of the studied groups were found to be devoid of retinoids
after single dose administration at the studied sampling
intervals. Therefore, the developed nanocarriers and the
marketed product possibly will not cause the side effects
related to the systemic distribution of ITR like psychological
disorders.31



4. CONCLUSIONS
The current studies successfully embarked upon the demonstration of the antimicrobial activity of ITR against the acne
causing pathogen, i.e., P. acnes. The findings indicate highly
encouraging results of the lipidic nanocarriers, i.e., SLNs and
NLCs, on the antimicrobial activity of ITR. These lipid-based
nanocarriers not only decreased the MIC of ITR but also
helped to retain the drug at the desired sites like various skin
layers. Therefore, lipid-based naocarriers hold immense
promise to enhance the efficacy and dermal delivery of
antimicrobials in a quite strategic manner. Significant findings
of the work can be rationally extrapolated to other similar drugs
and analogous lipidic carriers.



AUTHOR INFORMATION

Corresponding Author

*E-mail: drkatare@yahoo.com. Fax: 91-172-2541142.
Notes

The authors declare no competing financial interest.



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ACKNOWLEDGMENTS

Authors are thankful to M/s Ipca Laboratories, Ratlam, MP,
India for generously providing the gift samples of ITR where to
M/s Gattefosse, Germany along with M/s Colorcon, India and
M/s Phospholipid GmbH, Nattermannallee, Germany, for the
ex-gratis supply of Compritol 888 ATO GF3123 and
phospholipids, respectively. Financial grants obtained from
University Grants Commission (UGC), New Delhi, India and
M/s Ipca Laboratories, Mumbai, India are gratefully acknowl1962

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dx.doi.org/10.1021/mp300722f | Mol. Pharmaceutics 2013, 10, 1958−1963



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