European Journal of Pharmaceutical Sciences 48 (2013) 442452

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European Journal of Pharmaceutical Sciences

journal homepage: www.elsevier.com/locate/ejps

Preparation and characterization of lipid based nanosystems for topical delivery

of quercetin
Sonali Bose a,b, Bozena Michniak-Kohn a,
a
b

Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, 160 Frelinghuysen Road, Piscataway, NJ 08854, United States
Pharmaceutical and Analytical Development, Novartis Pharmaceuticals Corporation, One Health Plaza, East Hanover, NJ 07936, United States

a r t i c l e

i n f o

Article history:
Received 19 August 2012
Received in revised form 2 December 2012
Accepted 4 December 2012
Available online 13 December 2012
Keywords:
Solid lipid nanoparticles
Nanostructured lipid carriers
Sonication
Physical stability
Solid lipid
Oleic acid

a b s t r a c t
The main objective of this study was to evaluate the potential of lipid nanosystems for topical delivery of
the naturally occurring avonoid quercetin. These lipid based nanosystems were manufactured using a
solvent free probe ultrasonication process. Formulation factors such as the nature of the lipid (solid/combination of solid and liquid) in solid lipid nanoparticle (SLN) and nanostructured lipid carrier (NLC) systems and drug loading were evaluated to produce an optimum formulation with adequate physical
stability for up to 14 weeks at 28 C. The mean particle size of the optimized formulation was around
282 nm, with a zeta potential value of 36.57 2.67 mV, indicating the formation of a stable system.
Release studies showed a biphasic release prole, characterized by an initial burst release followed by
a more controlled release pattern from both SLN and NLC systems. The NLC system showed the highest
improvement in topical delivery of quercetin manifested by the amount of quercetin retained in full
thickness human skin, compared to a control formulation with similar composition and particle size in
the micrometer range. This study demonstrated the feasibility of nanostructured lipid carrier systems
for improved topical delivery of quercetin.
2012 Elsevier B.V. All rights reserved.

1. Introduction
Excessive exposure of the skin to environmental insults including sun and air pollution can lead to oxidative stress (OS) to the
skin. Under these conditions, reactive oxygen species (ROS) such
as singlet oxygen, hydroxyl radicals, superoxide radical-anions
and hydrogen peroxide can be generated by the photodynamic
reactions produced by endogenous cell photosensitizers. These
reactive oxygen species are capable of causing damage to cutaneous tissues (Pourzand and Tyrrell, 1999). They have also been reported to be involved in phototoxic reactions induced by the
interaction of UVA/visible light with drugs or chemical entities
(environmental or industrial) accumulated in the skin following
topical or systemic administration (Epstein, 1983). The use of topical antioxidants has been evaluated as a promising strategy for
prevention or alleviation of the biological effects of photo-oxidative stress produced in the skin by ROS.
Flavonoids are natural antioxidants derived from the plant
kingdom and are widely present in the human diet in the form of
numerous edible fruits and vegetables such as onions, apples, ber Corresponding author. Current address: Department of Pharmaceutics, 160
Frelinghuysen Road, Piscataway, NJ 08854, United States. Tel.: +1 732 445 3589;
fax: +1 732 445 5006.
E-mail address: michniak@biology.rutgers.edu (B. Michniak-Kohn).
0928-0987/$ - see front matter 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ejps.2012.12.005

ries and red grapes. The mechanism of the anti-oxidant action of

avonoids involve their reactivity with the ROS and their chelation
of transition metal ions responsible for oxygen activation via redox
reactions (Filipe et al., 2005). In a study investigating the structureactivity relationships of several avonoids (myricetin, quercetin, kaempferol, luteolin, apigenin and chrysin) and their antioxidant activity in a human dermal broblast model, correlations
were obtained between the anti-oxidative efcacy and the number
of OH-groups on the avonoid structure, with quercetin
(3,30 ,40 ,5,7-pentahydroxyavone) showing one of the highest activities. In the same study, quercetin was also observed to reduce the
expression of matrix metalloproteinase-1 (responsible for skin
wrinkling and loss of elasticity in both healthy and photoaged skin)
at both the mRNA and protein levels (Sim et al., 2007). This high
anti-radical/anti-oxidant activity shown by quercetin has been
attributed to the presence of three active functional groups in its
structure (Fig. 1): the ortho-dihydroxy (catechol) moiety in the B
ring, the C2C3 double bond in conjunction with a 4-oxo function
and the hydroxyl substitution at positions 3, 5 and 7 (Bors et al.,
1990; Saija et al., 1995).
The extremely low hydrophilicity of quercetin combined with
its extensive metabolism by the gut microorganisms result in minimal absorption from the gastrointestinal tract following its oral
administration, with no measurable plasma concentration reported in human volunteers from a 4 g oral dose (Gugler et al.,

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443

solvent based manufacturing method could be the failure to completely eliminate residual solvent (Grabnar and Kristl, 2011). The
objective of our study was to develop a solvent-free NLC formulation of quercetin using probe ultrasonication and evaluate its feasibility for topical delivery. The NLC system was fully
characterized for particle size, zeta potential, morphology and
crystallinity. Key properties such as physical/solid state stability
and in vitro drug release, that could affect formulation performance
in vivo, were compared between the two systems.

Fig. 1. Chemical structure of quercetin.

2. Materials and methods

2.1. Materials

1975). This limits the extension of the benecial effects of quercetin observed in in vitro studies to the in vivo or clinical level with an
oral delivery approach. Hence topical application of quercetin can
be a very attractive formulation approach, considering the fact that
topical application of quercetin has previously been shown to result in potent inhibition of UVB-induced oxidative skin damage
(Casagrande et al., 2006; Gonzalez et al., 2008).
Various formulation approaches including permeation enhancers (Olivella et al., 2007), ester based prodrugs (Montenegro
et al., 2007), microemulsion based approaches (Vicentini et al.,
2008; Kitagawa et al., 2009) and lecithinchitosan nanoparticles
(Tan et al., 2011) have been attempted to improve the penetration
of quercetin through the skin to facilitate topical/transdermal
delivery. Most of these studies have shown some degree of quercetin penetration into the skin, but no transdermal delivery has been
reported. However, since the efcacy of quercetin in delaying ultra-violet radiation mediated cell damage and eventual necrosis
mainly occurs in the epidermal layers of the skin, topical delivery
of quercetin without a sufcient degree of skin penetration should
be adequate to achieve the desired pharmacological action.
Lipid based nanosystems such as solid lipid nanoparticles
(SLNs) and nanostructured lipid carriers (NLCs) have been previously shown to improve the delivery of various actives such as glucocorticoids (Maia et al., 2000; Jensen et al., 2011), Vitamin A
(Jenning et al., 2000c) and betamethasone 17-valerate (Zhang
and Smith, 2011) to specic skin layers, with reported localization
in the upper layers of the skin. Such a formulation approach has
also been utilized by our group to develop solid lipid based nanosystems of quercetin and evaluate their feasibility for topical delivery using full thickness human skin (Bose et al., submitted for
publication). The optimized SLN formulation showed superior topical delivery of quercetin compared to the control formulation with
particle size in the micrometer range, with the differences being
statistically signicant (p < 0.05). However, an increase in the particle size was observed for samples placed on stability at 28 C
after 8 weeks. This increase in particle size could be attributed to
some degree of lipid transformation of the solid lipid (glyceryl
dibehenate) used in these nanoparticles over time, leading to the
formation of a highly ordered lipid structure resulting in drug
expulsion from the SLN system (Muller et al., 2002). The lipid
transformation was conrmed from the morphology of the nanoparticles visualized using TEM and also from X-ray diffraction
patterns.
Nanostructured lipid carriers (NLCs) are a second generation of
lipid based nanoparticles that are obtained by substituting a part of
the solid lipid used in the SLN formulation with a liquid lipid, in order to reduce the rigidity and ordered structure of the lipid matrix
and introduce imperfections in the matrix to minimize drug expulsion upon storage (Muller et al., 2002). Recently, the development
of NLCs of quercetin for topical delivery have been reported using a
solvent (chloroform/acetone) based emulsication technique
(Chen-yu et al., 2012). One of the major difculties of an organic

Highly pure (>99%) quercetin was obtained from Merck (Darmstadt, Germany). Compritol 888 (glyceryl dibehenate) was kindly
donated by Gattefoss (Paramus, NJ, USA). Tween 20 (polyoxyethylene derivative of sorbitan monolaurate), dioctyl sodium sulfosuccinate (DOSS) and oleic acid were purchased from SigmaAldrich
Corporation (St. Louis, MO, USA). HPLC or analytical grade of all
other solvents and reagents were used.
2.2. Preparation of nanosystems of quercetin
Nanostructured lipid carrier (NLC) systems of quercetin were
prepared using probe ultrasonication by melting 0.45 g of the solid
lipid (Compritol 888, glyceryl dibehenate) with 0.05 g of the liquid lipid (oleic acid) and either 0.025 g or 0.0125 g of quercetin
(corresponding to 0.05% or 0.025% drug loading in the system
respectively) at 85 C using a water bath. The heated mixture of
lipids and quercetin was then mixed with 20 mL of surfactant solution (composed of 2.5% Tween 20 and 0.1% DOSS) pre-heated to the
same temperature of 85 C. The mixture was then ultra-sonicated
at 85 C at a specic speed (power setting of four) for a pre-determined time interval (either 5 min or 30 min) using a Sonic Dismembrator Model 550 (Fisher Scientic, Pittsburgh, PA). The
primary product at the end of the sonication step was a nanoemulsion, since the processing temperature of 85 C was at least 10 C
higher than the melting point of the solid lipid and adequate to
maintain the system in the liquid state. At the end of the sonication
process, the nanoemulsion was dispersed into 30 mL of an ice-cold
surfactant solution maintained in an ice bath. The nal mixture
was then ultra-sonicated at a specic speed (power setting of 1)
for 10 min immersed in the ice-bath to promote the formation of
the solid lipid nanoparticles. The corresponding solid lipid nanoparticle (SLN) formulation was prepared in exactly the same way,
using 0.5 g of only the solid lipid (Compritol 888, glyceryl dibehenate) in the composition. All formulations were stored in the
refrigerator at 28 C till further analysis.
The non-homogenized control (for both SLN and NLC) formulation used in the skin penetration experiments were prepared by
mixing the lipid and quercetin solution heated to 85 C with the
surfactant solution heated to the same temperature. No ultrasonication step was performed on these non-homogenized control
formulations.
2.3. Freeze drying of nanoparticles
Samples of quercetin nanoparticles were freeze dried using a
Usifroid Freeze Dryer (Elancourt, France). A cooling rate of 1 C/
min was used to pre-cool the sample from room temperature to
50 C. The sample was then maintained at 50 C for 1 h, followed by primary and secondary drying steps as specied in
Table 1. Since the primary purpose of drying the nanoparticles
was to obtain a powder for further solid state characterization to

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S. Bose, B. Michniak-Kohn / European Journal of Pharmaceutical Sciences 48 (2013) 442452

Table 1
Parameters used for freeze-drying experiments.
Temperature range

Rate of cooling (C/min)

Hold time (min)

Primary drying cycle

50 C to 45 C
45 C to 35 C
35 C to 30 C
30 C to 25 C
25 C to 20 C

1
1
1
1
1

5
480
480
360
360

Secondary drying cycle

20 C to 10 C
10 C to 0 C
0 C to 10 C
10 C to 25 C
25 C to 30 C

1
1
1
1
1

600
600
480
720
960

evaluate crystallinity, no matrix formers were added to the solution prior to freeze drying.
2.4. Characterization of nanoparticles
2.4.1. Particle size measurement
The particle size of NLC systems was measured using photon
correlation spectroscopy (PCS) on a Delsa Nano C particle size
analyzer (Beckman Coulter, Brea, CA, USA) at 25 C and at a light
scattering angle of 90. The sample (undiluted) was poured into a
disposable plastic cuvette, the cuvette manually shaken for about
10 s and then placed inside the sample holder of the instrument.
Once the intensity of the sample was within the range recommended by the instrument, analysis was performed to obtain the
particle size and the polydispersity index (PI). All measurements
were performed in triplicate. All reported particle size data refer
to intensity weighted distributions.
2.4.2. Zeta potential measurement
The surface charge on the nanoparticles was quantied by measuring the zeta potential using a Delsa Nano C (Beckman Coulter,
Brea, CA, USA). An appropriately diluted nanoparticle solution was
used for the measurement, under an applied electric eld of 16 V/
cm. Dilutions were performed with distilled water adjusted to a
conductivity of 50 lS/cm by addition of 0.9% (m/v) sodium chloride. All reported values are the mean of three separate
measurements.

2.4.5. Modulated differential scanning calorimetry (MDSC)

Modulated differential scanning calorimetry (MDSC) was performed using a differential scanning calorimeter Q1000DSC (TA
Instruments, New Castle, DE, USA). Instrument calibration was carried out using Indium supplied by TA instruments. The sample
(10 mg) was placed in an aluminum DSC pan, covered with a
lid containing pinholes. An empty pan with pinholes in the lid
was used as the reference. The weights of the reference and sample
pans were accurately recorded. The sample cell was equilibrated at
0 C for 8 min, then heated to 320 C under an atmosphere of nitrogen using modulation conditions of 0.5 C for every 60 s with an
underlying heating rate of 2 C/min.
2.4.6. Release study
The release of quercetin from NLC systems was determined by a
dialysis based method using Slide-A-Lyzer MINI Dialysis devices,
2 mL volume, 10 K MWCO (Thermo Scientic, Rockford, IL, USA). A
mixture of doubly distilled water and absolute alcohol in the ratio
of 65:35% v/v (Li et al., 2009) was used as the release medium. The
receptor medium had adequate solubility of quercetin (0.358 mg/
mL) to ensure sufcient sink conditions throughout the study. 2 mL
of the quercetin formulation and 44.5 mL of the release medium
were added to the donor and receptor compartments of the dialysis device respectively (n = 3). The experiment was performed at
37 C and at a stirring speed of 100 RPM. At each sampling time
point (2, 4, 6, 8, 24 and 30 h), 1 mL of receptor medium was withdrawn and replaced with fresh medium. The withdrawn samples
were analyzed using the HPLC based method described in
Section 2.4.7.
2.4.7. Detection of quercetin by HPLC
Quercetin concentrations were determined using an Agilent
HPLC 1100 system (Agilent Technologies Inc., Santa Clara, CA,
USA) consisting of a standard quarternary pump, diode array
detector, an autosampler and vacuum degasser (Model G1311A)
run by Chemstation software version B.03.01. A mixture of 80%
methanol and 20% water (pH adjusted to 3.72 with glacial acetic
acid) was used as the mobile phase. Chromatographic separation
was achieved using a Phalanx C18, 250 mm  4.6 mm, 5 lm column (Higgins Analytical, Mountain View, CA, USA) and an isocratic
method with the following parameters: Injection volume of 20 lL,
ow rate of 1.0 mL/min, column temperature of 30 C, detection
wavelength of 370 nm and a run time of 8 min. External standards
in the mobile phase were prepared at concentrations of 0.1, 0.5, 1,
5, 10, 25, 50 and 100 lg/mL and used to determine the linearity
(R2 = 0.999) and the limit of detection (0.5 lg/mL).

2.4.3. Morphology
Transmission electron microscopy (TEM) was used to conrm
the morphology of the nanoparticles using a FEI Tecnai G2 BioTwin
transmission electron microscope tted with a SIS Morada digital
camera system (Fei Corporation, Hillsboro, OR, USA). Formvar
coated copper grids (200 mesh) were oated on top of 510 lL of
liquid sample for approximately 30 min, then rinsed with ltered
HPLC grade water and allowed to dry at room temperature. Images
were captured at magnications ranging from 4800 to 150,000.

2.4.8. Stability study

The physical stability of selected NLC systems was monitored
for up to 8 weeks at 28 C. Physical stability testing of the most
promising variant was continued till 14 weeks at 28 C, at which
time the particle size and zeta potential was measured. All reported particle size and zeta potential data are the mean of three
separate measurements.

2.4.4. X-ray powder diffraction (XRPD)

XRPD analysis was performed using a Bruker D8 Advance (Bruker-AXS, Karlsruhe, Germany) controlled by Diffrac plus XRD commander software. Samples (analyzed within 2 weeks after
manufacturing) were prepared by spreading freeze dried powder
samples on PMMA specimen holder rings from Bruker. All samples
were scanned from 2 to 40 2h using the following parameters:
scanning rate of 2/min with 0.02 step size and 0.6 s/step at
40 KV and 40 mA, divergence and anti-scattering slits set to 1
and a stage rotation speed of 30 rpm. EVA Part 11 version
14.0.0.0 software was used for data analysis.

2.4.9. In vitro permeation study using human skin

In vitro permeation studies (n = 56 for each formulation) were
carried out using full thickness human skin obtained from New
York Presbyterian Hospital (New York, NY, USA). Vertical Franz diffusion cells (PermeGear, Inc., Hellertown, PA, USA) with a diffusion
area of 0.64 cm2 and a receptor compartment volume of 5.1 mL
were used. The skin was thawed for 30 min, hydrated by immersing in PBS solution for 60 min at 37 C, cut into appropriate sized
sections and mounted on the Franz diffusion cell, with the stratum
corneum facing the donor compartment (where the formulation
was applied) and the dermis facing the receptor compartment.

S. Bose, B. Michniak-Kohn / European Journal of Pharmaceutical Sciences 48 (2013) 442452

PBS solution (pH 7.4) containing 1% Tween 20 maintained at a temperature of 37 0.1 C and a stirring speed of 600 RPM was used as
the receptor medium. 0.5 mL of formulation (NLC or control) was
added to the donor compartment and occluded with Paralm to
prevent evaporation. For the preliminary experiments, 300 lL of
sample was withdrawn at pre-determined time intervals (2, 4, 6,
8 and 24 h) from the receptor compartment and replaced by an
equal volume of fresh receptor media maintained at 37 C. The
samples were stored in the refrigerator prior to HPLC analysis.
At the end of the experiment (24 h), a glass transfer pipette was
used to remove the formulation from the donor compartment. The
surface of the skin was thoroughly washed with distilled water to
remove any excess formulation (Tan et al., 2011) and allowed to
dry at ambient temperature. The area of the skin in contact with
the formulation (corresponding to the diffusion area of the Franz
cell) was then punched out, weighed accurately and cut into ne
pieces. 1 mL of methanol was added to the skin pieces and homogenized using a Polytron PT 1035 homogenizer (Kinematica, Inc.,
Bohemia, NY, USA). The homogenized residue was sonicated for
60 min at 37 C using a VWR Ultrasonic B5500A-DTH sonicator
(VWR International, LLC, Radnor, PA, USA), followed by centrifugation at 4000 RPM for 30 min using an AllegraTM 6R centrifuge
(Beckman Coulter, Brea, CA, USA). The supernatant obtained after
centrifugation was collected and analyzed using the HPLC method
described in Section 2.4.7, with an increased injection volume of
50 lL.

2.4.10. Statistical analysis

All particle size, zeta potential and in vitro release rate measurements were performed in triplicate. Means and standard deviations were calculated using Microsoft Excel 2010. Mean values
were compared using the Students t-test using Statgraphics Plus
5.1 (Statpoint Technologies Inc., Warrenton, VA), with differences
being considered as signicant at a level of p < 0.05.

3. Results and discussion

3.1. Rationale for selection of liquid lipid in NLC system
Systematic screening of formulation and process variables was
carried out to develop a lipid based nanosystem comprising of 5%
quercetin (based on the total amount of lipid, corresponding to
0.05% drug loading in the overall system), 2.5% Tween 20 and
0.1% dioctyl sodium sulfosuccinate (DOSS) in a lipid matrix composed of Compritol 888 (glyceryl dibehenate) (Bose et al., submitted for publication). This system was used as the starting point for
the evaluation of NLC systems of quercetin which will be discussed
in this section. 10% of the solid lipid (Compritol 888) was substituted with a liquid lipid (oleic acid) in the NLC systems. Oleic acid
is a well-known skin permeation enhancer and has been reported
to enhance the skin delivery of a number of compounds such as
voriconazole (Song et al., 2012), estradiol (El Maghraby et al.,
2004), 5-uorouracil (Yamane et al., 1995) and tranilast (Murakami et al., 1998). Its permeation enhancing effect is attributed to
the reduction of the phase transition temperature of the lipids
present in the skin, thereby increasing the uidity of these structures (Golden et al., 1987; Francoeur et al., 1990). It has also been
suggested that oleic acid might exist as a separate phase (pool)
within the lipids of the stratum corneum (Ongpipattanakul et al.,
1991). The selection of oleic acid as the liquid lipid was based on
literature references citing its extensive use in NLC systems (Tiwari
and Pathak, 2011; Pardeike et al., 2011; Kuo and Chung, 2011) with
specic application to topical delivery (Silva et al., 2009; Lombardi
Borgia et al., 2005).

445

3.2. Effect of process parameters on physical stability of quercetin NLC

systems
The duration of the rst sonication step in the manufacturing
process is considered to be a critical processing parameter since
it involves the input of energy into the system, resulting in more
efcient breakage of coarse emulsion droplets into the nanometer
range and subsequent particle size reduction. To conrm the applicability of the same for the production of NLC systems, two different sonication times (5 min and 30 min) at a power setting of four
were evaluated for the rst sonication step at 85 C. The particle
size data for these two batches at the initial time point and after
1 week at 28 C are shown in Fig. 2.
The polydispersity index (PI) values obtained for these batches
were as follows: 0.285 and 0.250 for the initial and 1 week sample
for the NLC batch manufactured using a sonication time of 30 min
and 0.122 and 0.293 for the initial and 1 week sample for the NLC
batch manufactured using a sonication time of 5 min. For the batch
sonicated for 30 min, the difference in the particle size (both D50
and D90) between the initial sample and the sample kept for
1 week at 28 C was not considered to be statistically signicant
(p > 0.05). However statistically signicant differences (p < 0.05)
in both D50 and D90 values between the initial sample and the
1 week sample were observed for the batch sonicated for 5 min.
These results conrmed the criticality of using a longer sonication
time of 30 min for the rst sonication step (homogenization step)
in order to generate a physically stable nanosystem with a narrow
particle size distribution. The improved short term physical stability of the NLC batch manufactured using a sonication time of
30 min can be explained by the higher input of energy into the system during the longer sonication time resulting in more efcient
breakage of coarse emulsion droplets into the nanometer range
and subsequent particle size reduction. This observation is consistent with other reports in the literature, where higher sonication
times were found to be more efcient in generating nanoparticles
using a probe sonication method (Siekmann and Westesen, 1994;
Das et al., 2011). The zeta potential values for the initial samples
of
batches
sonicated
for
30 min
and
5 min
were
35.64 1.13 mV and 34.52 2.50 mV respectively, which were
not statistically signicant (p > 0.05). Based on the short term
physical stability data, a sonication time of 30 min for the rst sonication/homogenization step was selected for the manufacturing of
future quercetin lipid based nanosystems using the probe ultrasonication process.
3.3. Effect of oleic acid on physical stability of quercetin lipid based
nanosystems
In order to evaluate the effect of oleic acid on the physical stability of quercetin lipid based nanosystems, the particle size data of
batches with and without oleic acid (NLC and SLN systems respectively) was compared. The data is shown in Fig. 3. No statistically
signicant differences (p > 0.05) were observed between the SLN
and NLC formulations with 0.05% quercetin loading for D50 and
D90 values at the initial time point and after 8 weeks at 28 C.
The zeta potential values for the SLN and NLC batches were
35.83 2.11 mV and 35.64 1.13 mV respectively, which were
not found to be statistically signicant (p > 0.05). However, an increase in particle size for both the SLN and NLC formulations was
observed between the initial and 8 week samples. This is similar
to observations reported by Mitri et al. (2011), where increase in
particle size for both SLN and NLC formulations was observed for
up to 7 days after production, beyond which no signicant change
in particle size was observed from day 7 till day 30. The lack of any
observed differences between the particle size and zeta potential
values of SLN and NLC batches may be due to the low amount of

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S. Bose, B. Michniak-Kohn / European Journal of Pharmaceutical Sciences 48 (2013) 442452

Fig. 2. Effect of sonication time on physical stability of quercetin NLC systems (n = 3 measurements); PI refers to the polydispersity index.

Fig. 3. Effect of oleic acid on physical stability of quercetin lipid based nanosystems (n = 3 measurements); PI refers to the polydispersity index.

oleic acid (10%) used in the formulation. This is consistent with reports in the literature where no signicant difference in initial particle size (volume average diameter) was observed between NileRed loaded SLN and NLC formulations containing 515% of oleic
acid. Signicant reduction in particle size was only observed when
the amount of oleic acid in the formulation was increased to 30% or
higher, which was attributed to the reduced viscosity of the NLC
system at higher concentrations of oleic acid which facilitated
the reduction of surface tension to generate smaller particles (Hu
et al., 2005). Teeranachaideekul et al. (2007) reported increase in
oil content to have no effect on the initial particle size of Q10loaded NLC systems. Other researchers (Ying et al., 2008; You
et al., 2007) have also reported no signicant effect of oleic acid
on NLC particle size, at low oil concentrations (up to 10% w/w).

In our study, higher concentrations (greater than 10%) of the liquid lipid (oleic acid) were not investigated since an increase in the
concentration of the liquid lipid has been associated with a reduction in the occlusion factor (Teeranachaideekul et al., 2008), which
is one of the major aspects of topical application of lipid based
nanosystems. Also, higher concentrations of oleic acid in NLC systems were not evaluated since the acidic nature of fatty acids have
been associated with dermal side effects including extraction of
the stratum corneum lipids and damage to viable epidermal cells
(Sintov et al., 1999; Touitou et al., 2002). However, the use of oleic
acid in concentrations of up to 10% in topical formulations have
been reported to have no irritating effects on the skin of animal
species such as guinea pigs (Yu et al., 2010) or rabbits (Moreira
et al., 2010).

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447

Fig. 4. Effect of drug loading on the physical stability of quercetin NLC systems (n = 3 measurements).

3.4. Effect of drug loading on physical stability of quercetin NLC

systems
Most NLC systems described in the literature mention a relatively low drug loading, with loading values of 0.025% reported
for triamcinolone acetonide (Araujo et al., 2010), 0.03% reported
for urbiprofen (Luo et al., 2011) and 0.04% reported for ketoprofen
(Cirri et al., 2012). Although the batch with 5% quercetin (based on
the total amount of lipid, corresponding to 0.05% drug loading in
the NLC system) loading demonstrated good short term physical
stability with no statistically signicant differences in particle size
between the initial and 1 week samples, a lower drug loading of
2.5% quercetin (based on the total amount of lipid, corresponding
to 0.025% drug loading in the NLC system) was evaluated to determine if further improvement in physical stability of the nanosystem could be achieved by reducing the drug loading. All other
components of the formulations were kept constant, so that any
difference in particle size could be attributed solely to the drug
loading. The effect of drug loading (0.025% and 0.05%) on the physical stability of quercetin NLC systems is shown in Fig. 4.
No statistically signicant differences (p > 0.05) in D50 and D90
values were observed for initial samples of NLC batches with the
two different drug loading. However, for samples kept at 28 C
for 8 weeks, the differences in the D50 and D90 values for batches
with 0.025% and 0.05% drug loading were statistically signicant
(p < 0.05). The increase in particle size for the batch with 0.05%
drug loading can probably be attributed to insufcient quantity
of stabilizer in the formulation. When the drug loading is reduced
to 0.025%, the quantity of stabilizer incorporated in the formulation becomes more adequate to impart long term stabilization as
evidenced from the particle size data. The zeta potential values
for the batches with 0.025% and 0.05% drug loading were
36.57 2.67 mV and 35.64 1.13 mV respectively, which were
not statistically signicant (p > 0.05). The particle size data showed
better long term physical stability (up to 8 weeks at 28 C) for the
NLC batch with the lower (0.025%) quercetin loading, which was
selected for further evaluation. In order to ensure that the high
temperature (85 C) used during the manufacturing process did
not compromise the chemical stability of quercetin, the NLC batch

with 0.025% drug loading was also analyzed for quercetin content.
98% of quercetin could be recovered from the formulation. This
indicated that the manufacturing temperature did not induce any
degradation of quercetin and conrmed the validity of the production process.

3.5. Long term physical stability of selected NLC formulation

The NLC formulation with 0.025% quercetin loading was placed
on stability at 28 C. At pre-determined time points, the particle
size of the NLC system was measured to assess the physical stability of the system. At the end of the stability study (after 14 weeks),
the zeta potential of the batch was measured and compared to the
initial sample. The mean particle size and zeta potential results are
shown in Table 2. Very slight increase in the mean particle size
(<15 nm) was observed for the sample kept on stability for
14 weeks at 28 C compared to the initial particle size. Also, no
statistically signicant changes were observed in the zeta potential
value between the initial and 14 weeks sample. The zeta potential
value was around 36 mV that indicated good physical stability of
the system. Physical instabilities of lipid nanoparticles such as
agglomeration, aggregation and gelation are normally associated
with a reduction in the absolute zeta potential value. In general,
higher zeta potential values (either positive or negative) tend to
stabilize particle suspensions by inducing repulsion between similarly charged particles and thereby reduce their potential to
agglomerate (Muller et al., 2002; Gonzalez-Mira et al., 2011). Since
no signicant changes in the absolute zeta potential values were
observed between initial and stability samples kept for more than
3 months, good physical stability of the NLC system beyond the
stability study period of 14 weeks can be predicted. Lipid nanoparticle systems with up to 3 years of physical stability have previously been reported (Freitas and Mller, 1998).
The overall particle size distribution (D50 and D90) of the selected NLC formulation placed on stability is shown in Fig. 5. Differences in D50 and D90 were not found to be statistically
signicant (p > 0.05) between initial and 14 week or between the
8 week and 14 week samples.

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S. Bose, B. Michniak-Kohn / European Journal of Pharmaceutical Sciences 48 (2013) 442452

Table 2
Mean particle size and zeta potential of selected NLC formulation upon stability.

Time point

Particle size, mean SD (n = 3)

PIa

Zeta potential (mV), mean SD (n = 3)

Initial
8 weeks, 28 C
14 weeks, 28 C

281.9 2.9
309.8 3.5
294.6 6.7

0.306
0.310
0.315

36.57 2.67
Not measured
36.88 1.61

PI refers to polydispersity index.

Fig. 5. Long term physical stability of selected NLC formulation (n = 3 measurements).

3.6. Morphology of selected NLC formulation

NLC morphology was observed using transmission electron
microscopy (TEM). The TEM image of quercetin NLC with 0.025%
drug loading showed spherical particles in the nanometer range.
It is worthwhile to mention here that TEM images project threedimensional particles in a two-dimensional manner. It is quite possible that the NLC particles are not truly spherical in nature but

rather platelet like particles adhering with their at side on the

surface resulting in an overall round appearance when viewed
from the top in the TEM image (Jores et al., 2004; Esposito et al.,
2008). The mean particle size obtained from the TEM measurements was around 240 nm which was slightly lower than the mean
particle size obtained from the PCS measurements (282 nm). This
difference can be explained by the difference in the sample preparation step between the two techniques.

Fig. 6. X-ray diffraction patterns: (A): Compritol 888 bulk material, (B): freeze dried NLC batch, 0.025% drug loading, (C) freeze dried NLC batch, 0.05% drug loading, and (D):
freeze dried SLN batch, 0.05% drug loading.

S. Bose, B. Michniak-Kohn / European Journal of Pharmaceutical Sciences 48 (2013) 442452

449

3.7. Solid state characterization of quercetin NLC systems: X-ray

diffraction (XRD) data
The X-ray diffraction patterns are shown in Fig. 6. The diffractogram labelled A shows the diffraction pattern of neat Compritol
888, with peaks characteristic of the orthorhombic b0 form of triglycerides (Chapman, 1962). In the freeze dried NLC formulations
with 0.025% and 0.05% drug loading (labelled as B and C respectively), an additional peak is observed at 2h values between 18
and 20. This peak has been associated with the bi polymorph of
the lipid (Jenning et al., 2000a). The appearance of this peak between 2h values of 18 and 20 indicates the onset of a partial polymorphic transformation of glyceryl behenate from the metastable
b0 to the more stable bi form, as had been previously observed with
the freeze dried SLN formulation (diffractogram labelled D).
Aggregation of glyceryl behenate based nanoparticles are usually accompanied by a transformation from the b0 (metastable)
polymorph to the more stable bi polymorph (Jenning et al.,
2000a). However, for dispersions with 10% glyceryl behenate content (comparable to concentrations used for our experiments), reports of nanoparticle aggregation have been associated with a
signicant increase in the D90 (from 0.77 0.01 lm to
23.34 0.19 lm) accompanied by gelling of all samples within a
time frame of 5 days (Freitas and Mller, 1999). Upon reduction
of the lipid concentration from 10% to 2%, the authors reported improved stability of the dispersion with only a slight change
(0.1 lm) in the D90 values. An increase in particle size of around
0.1 lm (based on the D90 values) was observed for our experiments
(Fig. 5) which is similar to the observations of Freitas et al. for dispersions with 2% lipid content. Hence, our solid lipid nanosystems
can be concluded to be relatively stable systems, even at higher lipid concentrations (10%) compared to what has been previously
reported in the literature. Additionally, polymorphic transformation of the lipid was found to be complete only for samples which
had undergone complete conversion to a solid gel (Freitas and Mller, 1999). Based on the fact that the solid lipid nanosystems developed in our studies still retained their liquid state with no visible
gelling observed in the system, it is believed that only a partial
polymorphic transformation of the solid lipid (glyceryl behenate)
occurred in these samples during the time frame studied.
3.8. Solid state characterization of quercetin NLC systems: Differential
scanning calorimetry (DSC) data
Fig. 7 shows the modulated DSC scans of the quercetin SLN and
NLC formulations, with focus on the melting endotherm of Compritol 888. Bulk Compritol 888 melts between 63 C and 77 C,
with a melting point at 72.2 C. A slight depression in the melting
point of Compritol 888 to around 68 C was observed for all the
lipid based nanoparticle systems which is similar to values previously reported in the literature (Hamdani et al., 2003; Freitas and
Mller, 1999). This depression in melting point can be attributed
to the particle size in the nanometer range, the high specic surface area of the particles, the presence of surfactants in the formulation and the lipid polymorphism occurring from crystallization
(Jenning et al., 2000b). In all the SLN and NLC formulations, the
melting event of quercetin (starting around 315 C) could not be
detected (data not shown). This could be due to the presence of
quercetin in a dissolved state in the lipid matrix. During the production procedure of both SLN and NLCs, quercetin was dissolved
in the molten lipid matrix. Cooling the dispersion in an ice bath
led to the formation of SLN or NLCs depending on the composition.
The absence of any melting event of quercetin in the SLN and NLC
formulations could be due to the existence of quercetin as a supercooled melt, in the amorphous state or in a molecularly dispersed
state within the lipid matrix. Even if a small fraction of quercetin is

Fig. 7. Modulated DSC scans; (A): Freeze dried SLN, 0.05% drug loading, (B): freeze
dried NLC, 0.05% drug loading, and (C): freeze dried NLC, 0.025% drug loading.

present in the nanosystem as undissolved material/in the crystalline state, the relatively low drug loading of quercetin in the nanosystems would render it improbable to detect the melting event of
any such fraction using a DSC based technique.
3.9. Release of quercetin from lipid nanosystems
The release of quercetin from the SLN and NLC formulations
compared to a control formulation of quercetin in propylene glycol
solution are shown in Fig. 8. The formulations with 0.05% quercetin
loading were used to obtain a direct comparison of the release of
quercetin from formulations with and without oleic acid. Quercetin from the NLC formulation (with oleic acid) was released at a
faster rate compared to the SLN formulation (without oleic acid).
This is consistent with literature references comparing the release
rates from SLN and NLC formulations (Hu et al., 2005; Souto et al.,
2004). The release prole from both SLN and NLC formulations
were biphasic, with an initial burst release (55% from the NLC formulation compared to 45% from the SLN formulation after 2 h)
followed by controlled release for up to 30 h. Equilibrium was attained within 24 h, which was conrmed by the plateau observed
in the release prole between 24 and 30 h. The incorporation of the
liquid lipid (oleic acid) in the NLC formulation reduced the viscosity of the lipid matrix, leading to faster diffusion and release of the
drug from the NLC system compared to the SLN system that was
composed solely of solid lipids.
Statistical analysis showed signicant differences in the rate of
release of quercetin from the control formulation compared to both
the SLN and NLC formulations (p < 0.05). Also, statistically signicant differences in quercetin release rates were observed between
the SLN and NLC formulations at the earlier time points up to 6 h
(p < 0.05). Beyond 6 h, the differences in release rates between the
SLN and NLC systems were not statistically signicant (p > 0.05).
3.10. Topical delivery of quercetin from lipid nanosystems
In most in vitro permeation studies, the feasibility of topical
delivery of quercetin from the optimized formulation has been

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S. Bose, B. Michniak-Kohn / European Journal of Pharmaceutical Sciences 48 (2013) 442452

Fig. 8. Release of quercetin from lipid based nanosystems (n = 3).

evaluated using skin from animal species such as pig (Olivella

et al., 2007; Vicentini et al., 2008), guinea-pig (Kitagawa et al.,
2009) and mouse (Tan et al., 2011; Chen-yu et al., 2012). In one
study, human skin was used to evaluate the in vitro permeability
of quercetin from ester prodrugs; however only epidermal membranes (obtained by physical separation of the epidermis and dermis) were used for the experiments (Montenegro et al., 2007).
Since in many instances the results from skin permeation experiments conducted with animal skin cannot be extrapolated to human skin, full thickness dermatomed human skin was used in
our experiments to evaluate the feasibility of topical delivery of
quercetin from the developed lipid based nanosystems.
In vitro skin permeation studies were carried out with the SLN
and NLC formulations of quercetin, the main difference between
the formulations being the presence of oleic acid in the NLC formulation. The corresponding non-homogenized formulations (with
and without oleic acid, containing the same amount of lipid as in

the SLN and NLC formulations), with particle size in the micrometer
range, were used as the control formulations. Sampling from the
receptor media was carried out at 2, 4, 6, 8 and 24 h. However, no
quercetin could be detected in the receptor media, indicating the
lack of any transdermal delivery. This observation is consistent with
reports from other studies with quercetin (Vicentini et al., 2008;
Kitagawa et al., 2009), including one study from a chitosanlecithin
based nanoparticle formulation (Tan et al., 2011). The NLC formulation (with oleic acid) showed the highest amount of quercetin retained in the skin at the end of the 24 h experimental period
(Fig. 9). The difference in the amount of quercetin retained in the
skin was not found to be statistically signicant (p > 0.05) between
the NLC and the SLN formulation. However, tighter standard deviation values were observed from the NLC formulation which could
possibly be attributed to the improved physical stability of this formulation compared to the SLN formulation. Statistically signicant
differences (p < 0.05) were observed between the lipid based

Fig. 9. Topical delivery of quercetin from lipid based nanosystems (n = 45).

S. Bose, B. Michniak-Kohn / European Journal of Pharmaceutical Sciences 48 (2013) 442452

nanosystems with particle size in the nanometer range and their

corresponding control formulations with particle size in the
micrometer range (with and without oleic acid).
The superior topical delivery of quercetin observed from both
the lipid based nanosystems compared to the control formulation
(particles in the micrometer range) can be possibly explained by
the increased surface area/contact surface of the active compound
with the skin corneocytes, higher occlusive effect and increased
hydration of the stratum corneum that has been associated in general with lipid nanoparticles (Muller et al., 2007). The absence of
any transdermal delivery of quercetin from the lipid nanoparticles
is also consistent with literature reports of lipid nanoparticles not
considered to penetrate the horny layer (Schafer-Korting et al.,
2007). Also, in this study, the presence of oleic acid in the NLC formulation was not observed to have any signicant effect on the
skin accumulation of quercetin. Although oleic acid has been reported to increase the absorption of many active compounds such
as salicylic acid (28-fold increase) and 5-uorouracil (50-fold increase) (Goodman and Barry, 1989), there are also some literature
references where it has been shown to have no enhancement effect
on the absorption of actives such as urbiprofen (Fang et al., 2003)
and hexyl nicotinate (Tanojo et al., 1999) which is consistent with
our observation.
4. Conclusions
In this study, a part of the solid lipid from the SLN formulation
was substituted with a liquid lipid (oleic acid) to produce NLC systems of quercetin using the probe ultrasonication method. Long
term stability study carried out with the NLC system with 0.025%
drug loading showed excellent physical stability of the system
based on particle size and zeta potential measurement values.
TEM measurements showed spherical particles in the nanometer
range for the quercetin NLC system. The onset of a partial transformation of glyceryl dibehenate from the metastable b0 to the more
stable bi form was conrmed via XRD and DSC experiments. In vitro
release studies showed a biphasic release prole, characterized by
an initial burst release followed by a more controlled release pattern from both SLN and NLC systems. The reduction in the viscosity
of the NLC system due to the incorporation of the liquid lipid resulted in faster diffusion and release of quercetin from the NLC system. Superior topical delivery of quercetin was observed from both
the lipid based nanosystems (SLN and NLC) compared to the control formulation (particles in the micrometer range), although the
differences in the efcacy of topical delivery between the SLN
and NLC systems were not found to be statistically signicant.
However, the results of the in vitro skin permeation experiments
showed a tighter standard deviation for the NLC formulation which
could be related to the improved physical stability of this formulation compared to the SLN formulation. Hence, the NLC formulation
is considered to be the most promising for further development as
a topical delivery system of quercetin. Future studies will focus on
the feasibility of high pressure homogenization, a widely used
manufacturing process for lipid nanosystems, to manufacture
these quercetin NLC systems.
Acknowledgements
The authors would like to acknowledge Yuechao Du and Reena
Nadpara for providing assistance during batch manufacturing and
Dr. Paul Takishtov for providing lab access to the instrumentation.
The authors would also like to thank Danielle Lazarra for help with
the skin permeation experiments, Karen Killary for the TEM images
and Marilyn Alvine for the XRD measurements.

451

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