AAPS PharmSciTech ( # 2018)

DOI: 10.1208/s12249-018-0955-x

Research Article

Formulation and Evaluation of Organogels Containing Hyaluronan

Microparticles for Topical Delivery of Caffeine

Erol Eli Simsolo,1 İpek Eroğlu,2 Sakine Tuncay Tanrıverdi,1 and Özgen Özer1,3

Received 6 November 2017; accepted 6 January 2018

Abstract. Cellulite is a dermal disorder including the extracellular matrix, the lymphatic
and microcirculatory systems and the adipose tissue. Caffeine is used as the active moiety
depending its preventive effect on localization of fat in the cellular structure. Hyaluronic acid
(hyaluronan-HA) is a natural constituent of skin that generates formation and poliferation of
new cells having a remarkable moisturizing ability. The aim of this study is to formulate HA
microparticles loaded with caffeine via spray-drying method. Resulting microparticle
formulations (33.97 ± 0.3 μm, span < 2, 88.56 ± 0.42% encapsulation efficiency) were
distributed in lecithin organogels to maintain the proper viscosity for topical application.
Following the characterization and cell culture studies, in vitro drug release and ex vivo
permeation studies were performed. The accumulated amount of caffeine was twice higher
than the aqueous solution for the microparticle-loaded organogels at 24 h (8262,673 μg/
cm2versus 4676,691 μg/cm2). It was related to the sustained behaviour of caffeine release
from the microparticles. As a result, lecithin organogel containing HA-encapsulated
microparticles could be considered as suitable candidate formulations for efficient topical
drug delivery system of caffeine. In addition to that, synergistic effect of this combination
appears as a promising approach for long-acting treatment of cellulite.
KEY WORDS: caffeine; hyaluronan microparticles; organogel; cellulite; skin permeation.

INTRODUCTION the skin, which also presents potent antioxidant properties.

The basic terminology that underlies cellulite involves
multiple parameters such as the systems of microcirculation
and lymphatics, extracellular matrix and existence of high
amounts of fat that over accumulates into the dermis
structure of the skin. Dermal damage, which mainly appears
on the thighs and buttocks, occurs by virtue of the hypertro-
phy of the subcutaneous fat cells that bulges into the dermis
and decrease in elasticity of the skin (1). Cellulite has been
treated with different techniques such as laser heat treating,
high energy radial shockwaves, radiofrequency energy, dry
brushing and mesotherapy. Usage of effective topical creams
contains coffeine, xanthines, hydroxycitrate, epigallocatechin
gallate, linoleic acid, retinol etc. ameliorate the dermal
damage by stimulating fibroblast (and keratinocyte) activity
and decreasing adipocyte activity at the cellular level (2,3).
Caffeine is widely used in the treatment of cellulite as
topical formulations for inhibition of the formation of fats in
1
Faculty of Pharmacy, Department of Pharmaceutical Technology,
Ege University, 35100, Izmir, Turkey.
2 provides sustained release of the active pharmaceutical ingredi-
Faculty of Pharmacy, Department of Basic Pharmaceutical Sciences,
Hacettepe University, 06100, Ankara, Turkey.
3
To whom correspondence should be addressed. (e–mail:
ozgen.ozer@ege.edu.tr)
Caffeine improves the microcirculatory blood flow, which
accelerates the drainage of the lymph system from fatty tissue
and activates lipolysis in contrast with inhibiting lipogenesis
(1,4). The site of action for caffeine is in the adipocytes
located in the hypodermis and caffeine plays an active role in
adipocyte lipolysis involving effects on phosphodiesterase
(PDE) inhibition, cyclic adenosine monophosphate (cAMP)
levels, lymphatic drainage and lipase activity. The inhibition
of PDE activity increases the cAMP concentration in cells,
which stimulates the degradation of fats during lipolysis and
also elevates blood pressure (5). Caffeine is not metabolized
during the transport through the skin, and its low logP value
(− 0.07) allows generating high fluxes through the skin. Many
dosage forms (emulsion, gel, micro/nanoparticle, liposome
etc.) have been formulated in order to reach caffeine to the
active site at the effective concentration. In recent years,
sustained release approaches have attracted attention for
overcoming problems associated with the skin barrier prop-
erties and for improving the drug delivery (6,7).
Microparticles have been studied over the past years for
dermal drug delivery due to the polymeric structures that
ents (APIs) and to behave as drug reservoir in the skin (8).
Although there exists mant methods for the preparation of
microparticles, spray-drying method is generally preferred due to

1530-9932/18/0000-0001/0 # 2018 American Association of Pharmaceutical Scientists


having narrow particle size distribution, satisfactory level of
encapsulation efficiency, high product yiled and better stability of
the microparticles (9). Hyaluronan (HA), which is an endoge-
nous and naturally occurring non-sulphated glycosaminoglycan,
is also one of the glycosaminoglycan components of skin’s extra
cellular matrix. Its degradation stimulates new cell formation and
proliferates skin hydration. During wound healing process, HA is
produced in large amounts; therefore, it has many tissue
engineering and viscosupplementation applications (10–12). HA
microparticles have been used recently to improve the physical
properties of powders for inhaled or ocular therapies. High
molecular weight HA was previously proven to prevent
bronchoconstriction induced in asthmatics because of its high
molecular weight results in anti-inflammatory effects in mammals
(13–16). Spray-dried HA microparticles have also been evaluated
as possible materials for bone supplementation (17–19). On the
other hand, its usage on topical applications to modulate the drug
release properties has not been extensively studied. In one of the
studies, HA microparticles were investigated for the maintenance
of HA for a longer time in dermis and particle texturing feel. The
authors have suspended HA microparticles in hydrogels and
resulted that these systems may be suitable candidates for dermal
biphasic fillers (20). In another study by Babo et al., HA
microparticles have been prepared by spraying the solution of
HA into a dehydration/crosslinking bath containing isopropyl
alcohol. The resulting microparticles were well characterised with
respect to the formulation parameters. It was concluded that
these systems might be classifies as suitable candidates for
regenerative medicine applications depending on providing
biochemical cues for endogenous cell migration and proliferation
as well as maintaining a controlled release at the site of action
(21). Therefore, it is a clear fact that a lacking area is existing in
the field of HA microparticle administration for dermal delivery
of chemical agents.
Particulate formulations have been formulated in a
matrix, such as creams, gels, etc. to facilitate drug delivery
across the epidermis after topical application, to enhance
drug absorption and to obtain the suitable viscosity for topical
application (22). Organogel may be classified as a member of
semi-solid formulations, having viscoelastic properties. The
amphiphilic nature of organogels is the major factor enhanc-
ing dissolution characteristics of of various drug classes.
Apolar liquid structures of organolgels exist in three-
dimensional structure. Lecithin or phosphatidylcholine is a
cell component isolated from soya beans or eggs and purified
to show excellent gelation in non-polar solvents when
combined with water. Lecithin results in isotropic reverse
micelle solutions upon mixing with the organic solvents, and
after adding small quantities of polar solvents, these
cyclindrical micelles constantly get larger they form a gel
structure network (23). Lecithin organogels (LO) can form a
heat-stable, visco-elastic, optically transparent and non-
birefringent micellar system. The moisture-insensitive nature
of the gels makes them resistant to microbial contamination.
It can dissolve both hydrophilic and lipophilic drugs and
hence acts as an effective vehicle to deliver wide variety of
drugs across the skin. It serves as an organic medium to
enhance dermal permeation of poorly permeable drugs by
effectively partitioning into the skin (24–26).
Simsolo et al.
The purpose of this study is to formulate and optimize
microparticles in organogel system for the synergistic effect,
which is intended for a long-term treatment of cellulite. For
this purpose, caffeine-loaded biodegradable microparticle
formulations were prepared by spray-drying method using
HA, which is a natural constituent of skin that generates
formation and proliferation of new cells having a remarkable
moisterizing ability. The physicochemical properties of the
microparticle formulations were characterized and
cytotoxicity/proliferation characteristics were investigated.
Prepared microparticle formulations were distributed in
lecithin organogels to facilitate drug delivery across the
epidermis after topical application and also to give the proper
viscosity for topical application. Characterization (pH, viscos-
ity, rheological, mechanical, stability), in vitro release and
ex vivo permeation studies were conducted to evaluate
organogels potential for the topical treatment of cellulite
with caffeine.
MATERIALS AND METHODS
Materials
HA sodium salt 95% (MW: 1000 kDa) was obtained
from Acros Organics (Belgium). Caffeine was obtained from
Boehringer Ingelheim (Germany). Soybean lecithin (Lipoid
S100) was obtained from Lipoid AG (Germany) as a gift. All
reagents, which are used in the experimental parts, were
purchased from Sigma-Aldrich (France). The other chemicals
were from Merck (Germany), and they were used without
further purification.
Preparation of Microparticles
Microparticle formulations were prepared by using
BUCHI B-290 mini spray dryer (BUCHI Labortechnik AG,
Flawil, Switzerland), in which the solution was sprayed
through a 0.7-mm diameter two-fluid nozzle (10,14,27). The
spray-drying parameters were set as 600 L/h air flow rate; 50–
55 mL/min feed-flow rate; 180°C inlet temperature 100%
aspiration power; and these parameters were kept constant
throughout the experiments. HA, the polymeric material for
microparticles, was firstly dissolved in water (0.5% w/v).
Afterwards, the solution was introduced in the pump of the
spray dryer under continuous stirring over a magnetic stirrer.
For the loaded formulations, after complete dissolution of
HA, caffeine was added at the ratio of 0.1% (w/v). The
resulting microparticles were collected from the vessel and
stored in amber vials at room temperature in a vacuum
desiccator. The preparation efficiency was calculated by the
ratio of resulting microparticles to the ratio of solid materials
used for preparation.
CHARACTERIZATION OF MICROPARTICLES
Particle Size Measurement and Morphology
Particle size distribution of the microparticle formula-
tions was measured by Mastersizer 2000 (Malvern Instru-
ments, France), in which the basic principle depends on light
diffraction. Each sample was measured six times. Morphology
Organogels Containing Hyaluronan Microparticles Loaded with Caffeine
Fig. 1. SEM images of caffeine-loaded microparticle (× 1.60 K,
3.00 kV, 3.2 mm)
of the microparticles was investigated by scanning electron
microscopy (SEM) equipped with a LEO1530 microscope
(LEO Electron Microscopy Inc., Thornwood, USA).
Drug Loading and Encapsulation Efficiency
For this purpose, firstly 10 mg of microparticles was
accurately weighed and added into falcon tubes containing
25 mL of phosphate buffer saline (PBS) solution. Afterwards,
falcon tubes were vortexed for a duration time of 3 min and
finally centrifuged for 15 min at 5000 rpm. The supernatant
was withdrawn, and for quantification of caffeine, this
solution was injected to ultra-performance liquid
Fig. 2. DSC thermograms of a caffeine; b HA; c physical mixture of caffeine and unloaded microparticles; d unloaded microparticles; e caffeine-
loaded microparticles
chromatography (UPLC) system. The UPLC system
consisted of a C18 column (ACE 5-C18 100 mm × 2.1 mm)
with 10-μL sample loop. Acetonitrile/phosphoric acid (v/v;
25:75) was used as the mobile phase with a flow rate of
300 μL/min. UV detector was set at 272 nm wavelength. The
partial validation of the analytical method has been done
through the parameters of linearity, precision, accuracy,
specificity, selectivity and stability.
Thermal Analysis
Thermal characteristics of pure caffeine, HA,
unloaded microparticles, caffeine-loaded microparticles,
physical mixture of caffeine and unloaded microparticles
were carried out using a differential scanning calorimetry
(DSC) (Perkin Elmer DSC-8000). Measurements were
performed under nitrogen flow, having a flow rate of
20 mL/min and evaluated within the temperature range of
20–350°C over three replicates.
Cytotoxicity Assay
Indirect cytotoxicity method has been used for the
investigation of any possible effect of the microparticles
on cell viability. Human dermal fibroblast cells were
seeded onto 96-well plates with a density of 5 × 103 cells/
cm2. These cells were incubated at 37°C in a humid
atmosphere containing 5% CO2 for 24 h for the adhesion
of the cells. After 24 h, the cells were incubated for 24 h
with 5.55, 16.66, 50 and 150 μM of the formulations or
caffeine solution calculated by their caffeine
concentration. Vehicle only, negative (medium only) and
positive (15 mM of TritonX-100) controls were also
included in every experiment. After removing the

Fig. 3. Cytotoxicity evaluation from the MTT assay: concentrations
are the final concentration of API in the incubation media. Bars
represent Bmean ± SD^ from four separate studies (n = 4). The
numbers present on top of the bars are % cell viability compared to
the control values. *p < 0.05, ** p < 0.005 compared to the control
values
medium, cells were washed with PBS. Medium of 100 μL
and 20 μL of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyltetrazolium bromide) solution (5 mg/mL in PBS)
were added to each as well. After incubation for another
4 h, the medium was removed, and 100 μL of dimethyl
sulfoxide (DMSO) was added to each well in order to
dissolve the formazan crystals. The absorbance of the
purple solution was measured at 490 nm in microplate
reader. The cell viability results are expressed in terms of
percentage with respect to the untreated cells existing in
the pure medium. Acceptance criteria were set as 90% of
greater for the absence of cytotoxic effects.
Preparation of Organogel Formulations
Organogels were prepared by dissolving lecithin (25.6 g)
in isopropylmyristate (52.8 mL) at 40°C with constant stirring
at 200 rpm to obtain a homogeneous mixture. Distilled water
of 1.6 mL and glycerol mixture (1:1) were added to this
mixture, through a micropipette, and stirring was continued
Fig. 4. Macroscopic appearance of organogel formulations (1,
organogel; 2, caffeine-loaded organogel; 3, caffeine microparticle-
loaded organogel)
Simsolo et al.
for 15 min. Thereafter, the stirring was stopped and the
samples were allowed to cool and set aside to obtain clear,
homogenous organogels at room temperature (24). After
preparation of blank organogel (O–B), caffeine-loaded
organogel (O–C) and microparticle loaded organogels (O–
MC) were also prepared by the addition of 0.1% caffeine or
the same amount caffeine containing microparticle,
respectively.
CHARACTERIZATION OF ORGANOGEL
FORMULATIONS
Measurement of the pH Value
The pH value of the organogel formulations was done by
using a digital pH meter by inserting the probe into the
organogel formulations (Beckman Coulter, USA).
Viscosity and Rheological Properties
Brookfield viscometer (Model DV-III) was used to
measure the viscosity of the organogel formulations at 25 ±
1°C. The spindle number was 29 and it was rotated at the rate
of 25 rpm. For the quantification of rheological properties,
HAAKE rheometer (Thermo Fisher Scientific, Waltham,
MA, USA) was used. Following the determination of linear
viscoeleastic region, oscillatory analysis of the formulations
has been investigated, which was also followed by frequency
sweep analysis within the range of 0.1–10.0 Hz via application
of a constant stress. The measurement was expressed over the
results of three replicate analyses, and then, the storage
modulus (G′) and loss modulus (G′) were determined using
HAAKE RheoWin software (Thermo Fisher Scientific,
USA).
Mechanical Properties
For the determination of mechanical properties, we
have investigated hardness, adhesiveness cohesiveness,
elasticity and compressibility of the formulations with
texture analyser. (TA.XTplus; Stable Micro Systems, UK).
The analyser was equipped with 5 kg of load cell. The
Fig. 5. The modulus plotted versus frequency of microparticle-loaded
organogel
Organogels Containing Hyaluronan Microparticles Loaded with Caffeine

Table I. Mechanical properties of organogel formulations

Formula Hardness Adhesiveness Compressibility Cohesiveness ± Elasticity ±

(N) ± SD (N.mm) ± SD (N.mm) ± SD SD SD

O–B 0.011 ± 0.002 0.026 ± 0.002 0.190 ± 0.006 0.761 ± 0.013 0.908 ± 0.049
O–C 0.025 ± 0.001 0.036 ± 0.001 0.186 ± 0.002 0.836 ± 0.009 0.836 ± 0.025
O–MC 0.018 ± 0.002 0.042 ± 0.002 0.175 ± 0.004 0.811 ± 0.013 0.884 ± 0.049

formulations are filled in a beaker and the probe was
immersed into the beakers for a 15-mm deepness with a rate
of 2 mm/s. There was a lag time of 15 s between two
consecutive immersions. A minimum number of five repli-
cate was done for the analyses with a fresh sample at room
temperature. Texture Exponent software (version 3.0.5.0)
was used for the data collection and necessary calculations,
which is included in the device configuration by the
manufacturer. The mechanical parameters for each formu-
lation were defined by the force versus time plotting.
Stability of Formulations
Stability of microparticle and organogel formulations
were evaluated according to the International Conference
on Harmonisation (ICH) Q1A stability conditions at 25 ±
2°C and 60 ± 5% relative humidity and 40 ± 2°C and 75 ±
5% relative humidity (TK 252 Test Cabinet; Nüve) in
tightly closed glass vials for 6 months (28). Microparticles
were evaluated periodically in terms of particle size
distribution and encapsulation efficiency parameters; on
the other hand, organogels were evaluated for visual
inspection (phase separation and transparency), pH and
viscosity during 6 months.
In Vitro Drug Release from Formulations
Caffeine, caffeine-loaded microparticles (MC),
caffeine-loaded organogel (O–C) and microparticle-loaded
organogel (O–MC) formulations were used for in vitro
drug release studies. Caffeine release was determined by
the dialysis bag method (cutoff 12 kDa Spectra/Por®,
Canada). A total of 200 mL pH 7.4 phosphate-buffered
saline (PBS) was used as medium. Caffeine in 1 mL PBS
or MC (that amount corresponds to 10 mg caffeine
encapsulated in microparticles) in 1 mL PBS or caffeine
in 1 mL organogel or MC (that amount corresponds to
10 mg caffeine encapsulated in microparticles) in 1 mL
organogel were placed in the dialysis bag. Afterwards, they
were placed in a water bath at 37 ± 0.5°C over constant
magnetic stirring (200 rpm) and tightly covered for
preventing evaporation during release studies (n = 6).
Certain amount of samples was withdrawn and immedi-
ately replaced with fresh medium in order to maintain sink
conditions. Fore mentioned analytical method has been
used for the quantification of caffeine amount in the
samples.
The release profiles were evaluated kinetically by zero-
order, first-order, Higuchi (square root of time), Hixson-
Crowell (cube-root) and Weibull (RRSBW) models. The
determination coefficients (r 2) and the residuals were
analysed by GraphPad software. The best model was chosen
to analyse the release profiles of caffeine from microparticle-
loaded organogel (O–MC) formulation.
Experiments for Determination of Skin Permeation
The ex vivo permeation experiments of caffeine solution,
caffeine-loaded organogel (O–C) and microparticle loaded
organogel (O–MC) were performed using Franz-type diffu-
sion cells across rat abdominal skin. Wistar Albino rats used
for the study were approved by the Local Animal Ethical
Committee. Receptor compartment (5 mL) was phosphate
buffer saline (PBS, pH:7.4) and the solution was maintained
to keep its temperature at 37 C ± 0.5°C during the experi-
ments. At predetermined time points, samples of 300 μL were
collected up to to 24 h. Infinite dose regimen was applied in
all experiments. The amount of caffeine in the permeation
medium was determined by the validated UPLC method.

Table II. Stability evaluations of microparticles

Storage Time Particle Span Encapsulation

conditions (month) size (μm) efficiency (%)

25°C 1 35.97 ± 0.8 1.975 ± 0.07 90.56 ± 0.12

60% R.H. 3 37.42 ± 1.1 1.908 ± 0.05 86.38 ± 0.44
6 37.28 ± 0.9 1.978 ± 0.08 88.42 ± 0.56
40°C 1 34.12 ± 0.9 1.828 ± 0.09 87.45 ± 0.28
75% R.H. 3 36.22 ± 1.2 1.972 ± 0.04 85.92 ± 0.32
6 37.47 ± 0.5 1.872 ± 0.04 85.56 ± 0.45
 Simsolo et al.

Table III. Stability evaluations of organogel formulations (6 months)

Storage conditions Formulation code Macroscopic aspect pH Viscosity

(cP)

25°C O–B Transparent, yellowish, homogeneous 5.75 4790 ± 18

60% R.H. O–C Non-transparent, yellowish, homogeneous 5.86 4825 ± 14
O–MC Particulate, yellowish, homogeneous 6.05 4862 ± 16
40°C O–B Transparent, yellowish, homogeneous 5.75 4569 ± 14
75% R.H. O–C Non-transparent, yellowish, homogeneous 5.86 4512 ± 10
O–MC Particulate, yellowish, homogeneous 6.05 4659 ± 20

Statistical Analysis
The statistical comparison of the results was investigated
by ANOVA for the existence of any significant difference.
For the individual comparison of the dual groups, post hoc
analysis was done by using Tukey’s test. The level of
significance was selected as 0.05 and the significance levels
lower than this value were considered as statistically different.
RESULTS AND DISCUSSION
Preparation and Characterization of Microparticles
HA microparticles were obtained with a high yield of
about 92 ± 0.8% using spray-drying technique at optimized
conditions (air flow: 600 L/h, feed flow: 50–55 mL/min, inlet
temperature: 180°C, aspirator setting: 100%) with small
particle size (33.97 ± 0.3 μm) and a narrow size distribution
(span: 1.768). The measurement results were recorded as in a
good accordance with the SEM microscopy images (Fig. 1).
Drug loading and drug encapsulation efficiency capacities
were determined using validated UPLC method (r2 = 0.9934)
(LOD: 0.043 ± 0.060 μg/mL and LOQ: 0.144 ± 0.200 μg/mL) and
found as 0.1 mg and 88.56 ± 0.42%, respectively.
DSC results indicated that pure drug exhibited a
sharp endothermic peak at 238°C displaying the crystalline
nature of the drug. In the physical mixture, the melting
endothermic peaks in the DSC curves based on caffeine
and HA were observed. Further, the physical mixture of
caffeine and HA showed an endothermic peak tempera-
ture lower than pure caffeine, which may be due to the
effect of the melted HA because caffeine molecules were
gradually detaching from solid form. On the other hand,
in the thermograms of the caffeine-loaded microparticles,
there was no peak indicating the melting, which proves
the drug molecules are successfully encapsulated in the
microspheres as shown in Fig. 2 (29,30).
Cytotoxicity of microparticles was evaluated by cell
culture studies after being sterilized under UV light. The
MTT test was performed on human dermal fibroblast cells
incubated with the unloaded microparticles and the drug
embedded in microparticles. Cell growth results are
represented in terms of percentage as shown in Fig. 3,
which are expressed with respect to the control group’s
result.
As seen in Fig. 3, caffeine-loaded microparticles showed
non-toxic effect on cells as well as being proliferative at the
concentration of 0.02 mM concentration.
The results of the cell culture toxicity study show the
safety profile of the therapy chosen. This safety profile may
be improved by both increased bioavailability of the drug and
the change of the drug delivery system. The important factor

Fig. 6. Release profiles of caffeine from formulations

Organogels Containing Hyaluronan Microparticles Loaded with Caffeine
Table IV. The release rate (k) and determination coefficient (r2) of
release kinetics for O–MC
Zero order First order Hixon Crowell Higuchi Weibull
k 2.2564 − 0.0549 0.0622 15.1648 0.6600
r2 0.7908 0.9267 0.8883 0.9125 0.9763
here is the choice of excipients in the drug delivery system
where the importance of this choice directly affects the
toxicological profile of the drug molecule (31).
Preparation and Characterization of Organogels
In the literature, it was previously proved that the
phosphatidyl content of lecithin has a critical role on the
initiation of the gellification process of the apolar solvent. In
case this content is smaller than 95%, lecithin cannot show its
initiation effect (25). The major characteristics of the
organogels, which are lecithin based, have been previously
listed as thermos-reversible, transparent, biocompatible and
non-irritant, viscoelastic and thermodynamically stable by
different researchers, which makes them suitable drug
delivery systems in terms of controlled delivery (24,32).
Basically, although lecithin has capability to form organogels
with the addition of slight amount of water, the type and
molar ratio of organic solvent is the rate limiting step where
maximum viscosity is observed. The formation of a viscoelas-
tic organogel is depended on the formation of flexible
cylindrical reverse micelles by lecithin structure resulting in
the entanglement of the micellar network (33,34). The
organogel formulations found a popular trend for various
purposes in a wide set of skin applications. The major reason
for that is the availability of entanglement to form three-
dimensional network structures, limiting fluid flow and
immobilizing solvent molecules (26,35). Depending on the
amphiphilic character of organogels, a number of different
drugs may be incorporated within the structure regardless of
Fig. 7. Accumulated amount of caffeine from formulations
their lipophilic or hydrophilic character. Therefore,
organogels provide suitable platforms especially for the skin
delivery of active ingredients. In this study, homogenous and
yellow colour organogels containing caffeine-loaded HA
microparticles were prepared with lecithin having phosphati-
dyl content 95% (Fig. 4).
The pH value results of organogels including either
caffeine or microparticles were measured as acceptable for
topical use, which may be classified as similar to that of
skin values (average of 5.75 ± 0.2). The viscosity of blank,
caffeine and microparticle-loaded organogel formulations
was found as 4450 ± 12, 4675 ± 18 and 4615 ± 15 cps,
respectively. The values may be classified as suitable for
topical application.
In order to characterize these organogels from the
rheological point of view, oscillation analysis was performed
in the linear viscoelastic region. It is of sure that any possible
interaction existing between the organogel and microparticles
should directly have effect on the viscoelastic properties of
the final formulation. Within the light of this fact, rheological
properties of the final organogel formulations was measured
using a constant strain in the linear viscoelastic regions as a
response to the amount of microparticles added in. The
storage (elastic) modulus (G′) and loss (viscous) modulus (G
″) were measured for microparticle-loaded organogel formu-
lation (36,37). A large value of G′ in comparison of G″
indicated pronounced gel properties of the formulation in 0–
4 Hz (Fig. 5). The elevation of G′ value was depended on the
existence of more polymer chains, which are forming
entanglement of the junctions. This formulation also exhib-
ited shear-thinning behaviour, which we have expected to
have been due to the easiness for application to the skin
surface (38).
The mechanical properties such as adhesiveness, cohesive-
ness, compressibility, hardness and elasticity are defined by the
texture analysis (39). The resulting mechanical properties of
formulations are briefly summarized with average values and
standard deviation in Table I. Microparticle containing organogel
had an acceptable elasticity value indicating that formulations are

rapidly spreadable and non-dripping in nature. All gel formulations
showed high cohesiveness ( 0.811) and acceptable elasticity values
( 0.836).
Stability of Formulations
As a result of stability studies, no significant change was
observed for the physicochemical properties of both micro-
particle and organogel formulations at the end of the 6 months
(p < 0.05) (Tables II and III).
The blank organogels obtained were transparent, al-
though presence of caffeine gave to the preparation non-
transparent appearance. Formulations observed any change
in their homogeneity at the studied temperatures in ICH
guidelines during 6 months.
In Vitro Release Experiments
Release profiles of caffeine powder, caffeine from HA
microparticles (MC), caffeine from organogels (O–C) and
caffeine from microparticle-loaded organogels (O-MC) are
shown in Fig. 6.
The in vitro release profiles clearly indicate that
different release characteristics and trends are significantly
observed for the varying formulation type and character-
istics. It can be seen that for the case of microparticles, ≈
70% of the caffeine has been released within the first 6-h
time. In the case of organogel, the same percentage of
release is reached in 2 h. All caffeine powder release was
completed within 2 h. It is to be noted that caffeine was
released from both microparticle (92.55 ± 1.59%) and
microparticle containing organogel (78.86 ± 1.86) formula-
tions in a controlled fashion at the end of the 24 h. For
the microparticle and microparticles in organogel formu-
lations, at the first sampling time point, a rapid release,
which can also be named as Bburst release,^ was observed.
This term corresponds to the release over 30% in our
case, spectacularly especially for organogel only formula-
tion, this amount was recorded as 51.37 ± 1.24% of the
released amount for caffeine. These results are similar to
the study results that were previously published by
Esposito et al. (11). Concerning HA microspheres which
do not dissolve rapidly in water, but are able to form a
gellified network from controlling the drug release. In a
similar work previously conducted by our research group
previously, controlled release of resveratrol was obtained
with HA microparticles in 24 h (10).
The evaluation of the kinetic results indicated that
caffeine release from microparticle-loaded organogel for-
mulation showed a better fit to the Weibull kinetic model
compared to the other models. The determination coeffi-
cient obtained from the analysis of the dissolution data
was found the highest value (0.9763) for Weibull kinetic
model (Table IV). This means a steeper release at the
beginning and it is followed by controlled drug release
(40).
Skin Permeation Experiments
Ex vivo diffusion studies of the formulations were done
on rat abdominal skin, and the results indicate that the
Simsolo et al.
accumulation of caffeine was extended significantly from the
organogel containing microparticle formulations (p < 0.05).
The caffeine penetration into rat skin from solution,
from organogel and from microparticle-loaded organogel
formulations, was evaluated through Franz cell diffusion
measurements. The results are represented on Fig. 7 as an
amount of release of the applied dose recovered in the
receptor compartment versus time. The amount of caffeine
release from microparticles increased continuously until
6 h, and then presented a plateau between 6 and 24 h.
The amount of caffeine was twice higher from the
aqueous solution than from the microparticle-loaded
organogels at 24 h (8262,673 μg/cm2versus 4676,691 μg/
cm2). It was related to the sustained behaviour of caffeine
release from the microparticles. In this study, for
preparation of organogels, isopropyl myristate was used
as gelator, which is hydrophobic non-ionic molecule
having surface active properties. In addition to that, it
also has the ability to immobilize various solvents. It was
reported that in a previous study, existing organization in
lipid structure of human-derived stratum corneum was
disrupted by the interaction between the isopropyl palmi-
tate or isopropyl myristate (present in lecithin organogels)
and the stratum corneum (41).
These results are found to be in accordance with the ones
that Rodrigues et al. (6) have found. In that study, the
penetration of caffeine from nanostructured lipid carriers
(NLCs) has been investigated in Franz diffusion cells with a
model barrier composed of pig ear skin. According to this
study, caffeine release was nearly completed within 8-h time,
possibly depending on the hydrophilic character of itself. The
authors have also stated that enhanced penetration was
observed for caffeine in case of NLCs with respect to the
hydro-alcoholic solution of caffeine. In another study, the
in vitro transdermal delivery of caffeine has been determined
using Franz-type diffusion cells containing full thickness
porcine ear skin. Linear relationships between the amount
of caffeine liberated from the model patches per unit time
were observed, indicating zero-order release kinetics (42).
CONCLUSION
An innovative therapy was developed with optimized
topical formulation containing the natural components of
the skin which is not only more effective in repairing the
tissue damage caused by cellulite but also has a moistur-
izing effect at the same time. Overall, the results of the
present work confirmed the potential of HA microparti-
cles as carriers for caffeine since they demonstrated
sustained release behaviour and their feasibility for topical
delivery. Based on the above results, it could be con-
cluded that caffeine-loaded HA microparticle containing
lecithin organogel formulation might be used as a
promising formulation in long-term treatment of cellulite.
ACKNOWLEDGEMENTS
This study was supported by the Research Foundation of
Ege University (Grant Number 12/ECZ/029). The authors
are grateful to Prof. Hande Gürer Orhan from Ege
Organogels Containing Hyaluronan Microparticles Loaded with Caffeine
University, Faculty of Pharmacy, Department of Toxicology
for conducting cell culture studies and Ege University, Faculty
of Pharmacy, Pharmaceutical Sciences Research Centre
(FABAL) for equipmental support in UPLC analysis. The
authors would also like to thank TUBITAK for purchasing of
the spray dryer under the project number 111S183.
COMPLIANCE WITH ETHICAL STANDARDS
Wistar Albino rats used for the study were approved by
the Local Animal Ethical Committee.
Conflict of Interest The authors declare that they have no
conflict of interest.
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