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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.

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

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

Simsolo et al.

having narrow particle size distribution, satisfactory level of The purpose of this study is to formulate and optimize
encapsulation efficiency, high product yiled and better stability of microparticles in organogel system for the synergistic effect,
the microparticles (9). Hyaluronan (HA), which is an endoge- which is intended for a long-term treatment of cellulite. For
nous and naturally occurring non-sulphated glycosaminoglycan, this purpose, caffeine-loaded biodegradable microparticle
is also one of the glycosaminoglycan components of skin’s extra formulations were prepared by spray-drying method using
cellular matrix. Its degradation stimulates new cell formation and HA, which is a natural constituent of skin that generates
proliferates skin hydration. During wound healing process, HA is formation and proliferation of new cells having a remarkable
produced in large amounts; therefore, it has many tissue moisterizing ability. The physicochemical properties of the
engineering and viscosupplementation applications (10–12). HA microparticle formulations were characterized and
microparticles have been used recently to improve the physical cytotoxicity/proliferation characteristics were investigated.
properties of powders for inhaled or ocular therapies. High Prepared microparticle formulations were distributed in
lecithin organogels to facilitate drug delivery across the
molecular weight HA was previously proven to prevent
epidermis after topical application and also to give the proper
bronchoconstriction induced in asthmatics because of its high
viscosity for topical application. Characterization (pH, viscos-
molecular weight results in anti-inflammatory effects in mammals ity, rheological, mechanical, stability), in vitro release and
(13–16). Spray-dried HA microparticles have also been evaluated ex vivo permeation studies were conducted to evaluate
as possible materials for bone supplementation (17–19). On the organogels potential for the topical treatment of cellulite
other hand, its usage on topical applications to modulate the drug with caffeine.
release properties has not been extensively studied. In one of the
studies, HA microparticles were investigated for the maintenance MATERIALS AND METHODS
of HA for a longer time in dermis and particle texturing feel. The
authors have suspended HA microparticles in hydrogels and Materials
resulted that these systems may be suitable candidates for dermal
biphasic fillers (20). In another study by Babo et al., HA HA sodium salt 95% (MW: 1000 kDa) was obtained
from Acros Organics (Belgium). Caffeine was obtained from
microparticles have been prepared by spraying the solution of
Boehringer Ingelheim (Germany). Soybean lecithin (Lipoid
HA into a dehydration/crosslinking bath containing isopropyl
S100) was obtained from Lipoid AG (Germany) as a gift. All
alcohol. The resulting microparticles were well characterised with reagents, which are used in the experimental parts, were
respect to the formulation parameters. It was concluded that purchased from Sigma-Aldrich (France). The other chemicals
these systems might be classifies as suitable candidates for were from Merck (Germany), and they were used without
regenerative medicine applications depending on providing further purification.
biochemical cues for endogenous cell migration and proliferation
as well as maintaining a controlled release at the site of action Preparation of Microparticles
(21). Therefore, it is a clear fact that a lacking area is existing in
the field of HA microparticle administration for dermal delivery Microparticle formulations were prepared by using
of chemical agents. BUCHI B-290 mini spray dryer (BUCHI Labortechnik AG,
Particulate formulations have been formulated in a Flawil, Switzerland), in which the solution was sprayed
matrix, such as creams, gels, etc. to facilitate drug delivery through a 0.7-mm diameter two-fluid nozzle (10,14,27). The
across the epidermis after topical application, to enhance spray-drying parameters were set as 600 L/h air flow rate; 50–
drug absorption and to obtain the suitable viscosity for topical 55 mL/min feed-flow rate; 180°C inlet temperature 100%
application (22). Organogel may be classified as a member of aspiration power; and these parameters were kept constant
semi-solid formulations, having viscoelastic properties. The throughout the experiments. HA, the polymeric material for
amphiphilic nature of organogels is the major factor enhanc- microparticles, was firstly dissolved in water (0.5% w/v).
ing dissolution characteristics of of various drug classes. Afterwards, the solution was introduced in the pump of the
Apolar liquid structures of organolgels exist in three- spray dryer under continuous stirring over a magnetic stirrer.
dimensional structure. Lecithin or phosphatidylcholine is a For the loaded formulations, after complete dissolution of
cell component isolated from soya beans or eggs and purified HA, caffeine was added at the ratio of 0.1% (w/v). The
to show excellent gelation in non-polar solvents when resulting microparticles were collected from the vessel and
combined with water. Lecithin results in isotropic reverse stored in amber vials at room temperature in a vacuum
micelle solutions upon mixing with the organic solvents, and desiccator. The preparation efficiency was calculated by the
after adding small quantities of polar solvents, these ratio of resulting microparticles to the ratio of solid materials
cyclindrical micelles constantly get larger they form a gel used for preparation.
structure network (23). Lecithin organogels (LO) can form a
heat-stable, visco-elastic, optically transparent and non- CHARACTERIZATION OF MICROPARTICLES
birefringent micellar system. The moisture-insensitive nature
of the gels makes them resistant to microbial contamination. Particle Size Measurement and Morphology
It can dissolve both hydrophilic and lipophilic drugs and
hence acts as an effective vehicle to deliver wide variety of Particle size distribution of the microparticle formula-
drugs across the skin. It serves as an organic medium to tions was measured by Mastersizer 2000 (Malvern Instru-
enhance dermal permeation of poorly permeable drugs by ments, France), in which the basic principle depends on light
effectively partitioning into the skin (24–26). diffraction. Each sample was measured six times. Morphology
Organogels Containing Hyaluronan Microparticles Loaded with Caffeine

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
Fig. 1. SEM images of caffeine-loaded microparticle (× 1.60 K, (DSC) (Perkin Elmer DSC-8000). Measurements were
3.00 kV, 3.2 mm) 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.

of the microparticles was investigated by scanning electron Cytotoxicity Assay

microscopy (SEM) equipped with a LEO1530 microscope
(LEO Electron Microscopy Inc., Thornwood, USA). Indirect cytotoxicity method has been used for the
investigation of any possible effect of the microparticles
on cell viability. Human dermal fibroblast cells were
Drug Loading and Encapsulation Efficiency seeded onto 96-well plates with a density of 5 × 103 cells/
cm2. These cells were incubated at 37°C in a humid
For this purpose, firstly 10 mg of microparticles was atmosphere containing 5% CO2 for 24 h for the adhesion
accurately weighed and added into falcon tubes containing of the cells. After 24 h, the cells were incubated for 24 h
25 mL of phosphate buffer saline (PBS) solution. Afterwards, with 5.55, 16.66, 50 and 150 μM of the formulations or
falcon tubes were vortexed for a duration time of 3 min and caffeine solution calculated by their caffeine
finally centrifuged for 15 min at 5000 rpm. The supernatant concentration. Vehicle only, negative (medium only) and
was withdrawn, and for quantification of caffeine, this positive (15 mM of TritonX-100) controls were also
solution was injected to ultra-performance liquid included in every experiment. After removing the

Fig. 2. DSC thermograms of a caffeine; b HA; c physical mixture of caffeine and unloaded microparticles; d unloaded microparticles; e caffeine-
loaded microparticles
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,


Measurement of the pH Value

Fig. 3. Cytotoxicity evaluation from the MTT assay: concentrations
are the final concentration of API in the incubation media. Bars The pH value of the organogel formulations was done by
represent Bmean ± SD^ from four separate studies (n = 4). The using a digital pH meter by inserting the probe into the
numbers present on top of the bars are % cell viability compared to
organogel formulations (Beckman Coulter, USA).
the control values. *p < 0.05, ** p < 0.005 compared to the control
Viscosity and Rheological Properties

Brookfield viscometer (Model DV-III) was used to

medium, cells were washed with PBS. Medium of 100 μL measure the viscosity of the organogel formulations at 25 ±
and 20 μL of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5- 1°C. The spindle number was 29 and it was rotated at the rate
diphenyltetrazolium bromide) solution (5 mg/mL in PBS) of 25 rpm. For the quantification of rheological properties,
were added to each as well. After incubation for another HAAKE rheometer (Thermo Fisher Scientific, Waltham,
4 h, the medium was removed, and 100 μL of dimethyl MA, USA) was used. Following the determination of linear
sulfoxide (DMSO) was added to each well in order to viscoeleastic region, oscillatory analysis of the formulations
dissolve the formazan crystals. The absorbance of the has been investigated, which was also followed by frequency
purple solution was measured at 490 nm in microplate sweep analysis within the range of 0.1–10.0 Hz via application
reader. The cell viability results are expressed in terms of of a constant stress. The measurement was expressed over the
percentage with respect to the untreated cells existing in results of three replicate analyses, and then, the storage
the pure medium. Acceptance criteria were set as 90% of modulus (G′) and loss modulus (G′) were determined using
greater for the absence of cytotoxic effects. HAAKE RheoWin software (Thermo Fisher Scientific,
Preparation of Organogel Formulations
Mechanical Properties
Organogels were prepared by dissolving lecithin (25.6 g)
in isopropylmyristate (52.8 mL) at 40°C with constant stirring For the determination of mechanical properties, we
at 200 rpm to obtain a homogeneous mixture. Distilled water have investigated hardness, adhesiveness cohesiveness,
of 1.6 mL and glycerol mixture (1:1) were added to this elasticity and compressibility of the formulations with
mixture, through a micropipette, and stirring was continued texture analyser. (TA.XTplus; Stable Micro Systems, UK).
The analyser was equipped with 5 kg of load cell. The

Fig. 4. Macroscopic appearance of organogel formulations (1,

organogel; 2, caffeine-loaded organogel; 3, caffeine microparticle- Fig. 5. The modulus plotted versus frequency of microparticle-loaded
loaded organogel) 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 encapsulated in microparticles) in 1 mL PBS or caffeine
immersed into the beakers for a 15-mm deepness with a rate in 1 mL organogel or MC (that amount corresponds to
of 2 mm/s. There was a lag time of 15 s between two 10 mg caffeine encapsulated in microparticles) in 1 mL
consecutive immersions. A minimum number of five repli- organogel were placed in the dialysis bag. Afterwards, they
cate was done for the analyses with a fresh sample at room were placed in a water bath at 37 ± 0.5°C over constant
temperature. Texture Exponent software (version magnetic stirring (200 rpm) and tightly covered for
was used for the data collection and necessary calculations, preventing evaporation during release studies (n = 6).
which is included in the device configuration by the Certain amount of samples was withdrawn and immedi-
manufacturer. The mechanical parameters for each formu- ately replaced with fresh medium in order to maintain sink
lation were defined by the force versus time plotting. conditions. Fore mentioned analytical method has been
used for the quantification of caffeine amount in the
Stability of Formulations samples.
The release profiles were evaluated kinetically by zero-
Stability of microparticle and organogel formulations order, first-order, Higuchi (square root of time), Hixson-
were evaluated according to the International Conference Crowell (cube-root) and Weibull (RRSBW) models. The
on Harmonisation (ICH) Q1A stability conditions at 25 ± determination coefficients (r 2) and the residuals were
2°C and 60 ± 5% relative humidity and 40 ± 2°C and 75 ± analysed by GraphPad software. The best model was chosen
5% relative humidity (TK 252 Test Cabinet; Nüve) in to analyse the release profiles of caffeine from microparticle-
tightly closed glass vials for 6 months (28). Microparticles loaded organogel (O–MC) formulation.
were evaluated periodically in terms of particle size
distribution and encapsulation efficiency parameters; on
the other hand, organogels were evaluated for visual Experiments for Determination of Skin Permeation
inspection (phase separation and transparency), pH and
viscosity during 6 months. The ex vivo permeation experiments of caffeine solution,
caffeine-loaded organogel (O–C) and microparticle loaded
In Vitro Drug Release from Formulations organogel (O–MC) were performed using Franz-type diffu-
sion cells across rat abdominal skin. Wistar Albino rats used
Caffeine, caffeine-loaded microparticles (MC), for the study were approved by the Local Animal Ethical
caffeine-loaded organogel (O–C) and microparticle-loaded Committee. Receptor compartment (5 mL) was phosphate
organogel (O–MC) formulations were used for in vitro buffer saline (PBS, pH:7.4) and the solution was maintained
drug release studies. Caffeine release was determined by to keep its temperature at 37 C ± 0.5°C during the experi-
the dialysis bag method (cutoff 12 kDa Spectra/Por®, ments. At predetermined time points, samples of 300 μL were
Canada). A total of 200 mL pH 7.4 phosphate-buffered collected up to to 24 h. Infinite dose regimen was applied in
saline (PBS) was used as medium. Caffeine in 1 mL PBS all experiments. The amount of caffeine in the permeation
or MC (that amount corresponds to 10 mg caffeine 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


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 nature of the drug. In the physical mixture, the melting
endothermic peaks in the DSC curves based on caffeine
The statistical comparison of the results was investigated and HA were observed. Further, the physical mixture of
by ANOVA for the existence of any significant difference. caffeine and HA showed an endothermic peak tempera-
For the individual comparison of the dual groups, post hoc ture lower than pure caffeine, which may be due to the
analysis was done by using Tukey’s test. The level of effect of the melted HA because caffeine molecules were
significance was selected as 0.05 and the significance levels gradually detaching from solid form. On the other hand,
lower than this value were considered as statistically different. in the thermograms of the caffeine-loaded microparticles,
there was no peak indicating the melting, which proves
RESULTS AND DISCUSSION the drug molecules are successfully encapsulated in the
microspheres as shown in Fig. 2 (29,30).
Preparation and Characterization of Microparticles Cytotoxicity of microparticles was evaluated by cell
culture studies after being sterilized under UV light. The
HA microparticles were obtained with a high yield of MTT test was performed on human dermal fibroblast cells
about 92 ± 0.8% using spray-drying technique at optimized incubated with the unloaded microparticles and the drug
conditions (air flow: 600 L/h, feed flow: 50–55 mL/min, inlet embedded in microparticles. Cell growth results are
temperature: 180°C, aspirator setting: 100%) with small represented in terms of percentage as shown in Fig. 3,
particle size (33.97 ± 0.3 μm) and a narrow size distribution which are expressed with respect to the control group’s
(span: 1.768). The measurement results were recorded as in a result.
good accordance with the SEM microscopy images (Fig. 1). As seen in Fig. 3, caffeine-loaded microparticles showed
Drug loading and drug encapsulation efficiency capacities non-toxic effect on cells as well as being proliferative at the
were determined using validated UPLC method (r2 = 0.9934) concentration of 0.02 mM concentration.
(LOD: 0.043 ± 0.060 μg/mL and LOQ: 0.144 ± 0.200 μg/mL) and The results of the cell culture toxicity study show the
found as 0.1 mg and 88.56 ± 0.42%, respectively. safety profile of the therapy chosen. This safety profile may
DSC results indicated that pure drug exhibited a be improved by both increased bioavailability of the drug and
sharp endothermic peak at 238°C displaying the crystalline 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 their lipophilic or hydrophilic character. Therefore,
release kinetics for O–MC organogels provide suitable platforms especially for the skin
delivery of active ingredients. In this study, homogenous and
yellow colour organogels containing caffeine-loaded HA
Zero order First order Hixon Crowell Higuchi Weibull microparticles were prepared with lecithin having phosphati-
dyl content 95% (Fig. 4).
k 2.2564 − 0.0549 0.0622 15.1648 0.6600
The pH value results of organogels including either
r2 0.7908 0.9267 0.8883 0.9125 0.9763
caffeine or microparticles were measured as acceptable for
topical use, which may be classified as similar to that of
here is the choice of excipients in the drug delivery system skin values (average of 5.75 ± 0.2). The viscosity of blank,
where the importance of this choice directly affects the caffeine and microparticle-loaded organogel formulations
toxicological profile of the drug molecule (31). was found as 4450 ± 12, 4675 ± 18 and 4615 ± 15 cps,
respectively. The values may be classified as suitable for
Preparation and Characterization of Organogels topical application.
In order to characterize these organogels from the
In the literature, it was previously proved that the rheological point of view, oscillation analysis was performed
phosphatidyl content of lecithin has a critical role on the in the linear viscoelastic region. It is of sure that any possible
initiation of the gellification process of the apolar solvent. In interaction existing between the organogel and microparticles
case this content is smaller than 95%, lecithin cannot show its should directly have effect on the viscoelastic properties of
initiation effect (25). The major characteristics of the the final formulation. Within the light of this fact, rheological
organogels, which are lecithin based, have been previously properties of the final organogel formulations was measured
listed as thermos-reversible, transparent, biocompatible and using a constant strain in the linear viscoelastic regions as a
non-irritant, viscoelastic and thermodynamically stable by response to the amount of microparticles added in. The
different researchers, which makes them suitable drug storage (elastic) modulus (G′) and loss (viscous) modulus (G
delivery systems in terms of controlled delivery (24,32). ″) were measured for microparticle-loaded organogel formu-
Basically, although lecithin has capability to form organogels lation (36,37). A large value of G′ in comparison of G″
with the addition of slight amount of water, the type and indicated pronounced gel properties of the formulation in 0–
molar ratio of organic solvent is the rate limiting step where 4 Hz (Fig. 5). The elevation of G′ value was depended on the
maximum viscosity is observed. The formation of a viscoelas- existence of more polymer chains, which are forming
tic organogel is depended on the formation of flexible entanglement of the junctions. This formulation also exhib-
cylindrical reverse micelles by lecithin structure resulting in ited shear-thinning behaviour, which we have expected to
the entanglement of the micellar network (33,34). The have been due to the easiness for application to the skin
organogel formulations found a popular trend for various surface (38).
purposes in a wide set of skin applications. The major reason The mechanical properties such as adhesiveness, cohesive-
for that is the availability of entanglement to form three- ness, compressibility, hardness and elasticity are defined by the
dimensional network structures, limiting fluid flow and texture analysis (39). The resulting mechanical properties of
immobilizing solvent molecules (26,35). Depending on the formulations are briefly summarized with average values and
amphiphilic character of organogels, a number of different standard deviation in Table I. Microparticle containing organogel
drugs may be incorporated within the structure regardless of had an acceptable elasticity value indicating that formulations are

Fig. 7. Accumulated amount of caffeine from formulations

Simsolo et al.

rapidly spreadable and non-dripping in nature. All gel formulations accumulation of caffeine was extended significantly from the
showed high cohesiveness ( 0.811) and acceptable elasticity values organogel containing microparticle formulations (p < 0.05).
( 0.836). The caffeine penetration into rat skin from solution,
from organogel and from microparticle-loaded organogel
Stability of Formulations formulations, was evaluated through Franz cell diffusion
measurements. The results are represented on Fig. 7 as an
As a result of stability studies, no significant change was amount of release of the applied dose recovered in the
observed for the physicochemical properties of both micro- receptor compartment versus time. The amount of caffeine
particle and organogel formulations at the end of the 6 months release from microparticles increased continuously until
(p < 0.05) (Tables II and III). 6 h, and then presented a plateau between 6 and 24 h.
The blank organogels obtained were transparent, al- The amount of caffeine was twice higher from the
though presence of caffeine gave to the preparation non- aqueous solution than from the microparticle-loaded
transparent appearance. Formulations observed any change organogels at 24 h (8262,673 μg/cm2versus 4676,691 μg/
in their homogeneity at the studied temperatures in ICH cm2). It was related to the sustained behaviour of caffeine
guidelines during 6 months. release from the microparticles. In this study, for
preparation of organogels, isopropyl myristate was used
as gelator, which is hydrophobic non-ionic molecule
In Vitro Release Experiments
having surface active properties. In addition to that, it
also has the ability to immobilize various solvents. It was
Release profiles of caffeine powder, caffeine from HA
reported that in a previous study, existing organization in
microparticles (MC), caffeine from organogels (O–C) and
lipid structure of human-derived stratum corneum was
caffeine from microparticle-loaded organogels (O-MC) are
disrupted by the interaction between the isopropyl palmi-
shown in Fig. 6.
tate or isopropyl myristate (present in lecithin organogels)
The in vitro release profiles clearly indicate that
and the stratum corneum (41).
different release characteristics and trends are significantly
These results are found to be in accordance with the ones
observed for the varying formulation type and character-
istics. It can be seen that for the case of microparticles, ≈ that Rodrigues et al. (6) have found. In that study, the
70% of the caffeine has been released within the first 6-h penetration of caffeine from nanostructured lipid carriers
time. In the case of organogel, the same percentage of (NLCs) has been investigated in Franz diffusion cells with a
release is reached in 2 h. All caffeine powder release was model barrier composed of pig ear skin. According to this
completed within 2 h. It is to be noted that caffeine was study, caffeine release was nearly completed within 8-h time,
released from both microparticle (92.55 ± 1.59%) and possibly depending on the hydrophilic character of itself. The
microparticle containing organogel (78.86 ± 1.86) formula- authors have also stated that enhanced penetration was
tions in a controlled fashion at the end of the 24 h. For
observed for caffeine in case of NLCs with respect to the
the microparticle and microparticles in organogel formu-
lations, at the first sampling time point, a rapid release, hydro-alcoholic solution of caffeine. In another study, the
which can also be named as Bburst release,^ was observed. in vitro transdermal delivery of caffeine has been determined
This term corresponds to the release over 30% in our using Franz-type diffusion cells containing full thickness
case, spectacularly especially for organogel only formula- porcine ear skin. Linear relationships between the amount
tion, this amount was recorded as 51.37 ± 1.24% of the of caffeine liberated from the model patches per unit time
released amount for caffeine. These results are similar to were observed, indicating zero-order release kinetics (42).
the study results that were previously published by
Esposito et al. (11). Concerning HA microspheres which CONCLUSION
do not dissolve rapidly in water, but are able to form a
gellified network from controlling the drug release. In a An innovative therapy was developed with optimized
similar work previously conducted by our research group topical formulation containing the natural components of
previously, controlled release of resveratrol was obtained the skin which is not only more effective in repairing the
with HA microparticles in 24 h (10). tissue damage caused by cellulite but also has a moistur-
The evaluation of the kinetic results indicated that izing effect at the same time. Overall, the results of the
caffeine release from microparticle-loaded organogel for- present work confirmed the potential of HA microparti-
mulation showed a better fit to the Weibull kinetic model cles as carriers for caffeine since they demonstrated
compared to the other models. The determination coeffi- sustained release behaviour and their feasibility for topical
cient obtained from the analysis of the dissolution data delivery. Based on the above results, it could be con-
was found the highest value (0.9763) for Weibull kinetic cluded that caffeine-loaded HA microparticle containing
model (Table IV). This means a steeper release at the lecithin organogel formulation might be used as a
beginning and it is followed by controlled drug release promising formulation in long-term treatment of cellulite.
Skin Permeation Experiments
This study was supported by the Research Foundation of
Ex vivo diffusion studies of the formulations were done Ege University (Grant Number 12/ECZ/029). The authors
on rat abdominal skin, and the results indicate that the are grateful to Prof. Hande Gürer Orhan from Ege
Organogels Containing Hyaluronan Microparticles Loaded with Caffeine

University, Faculty of Pharmacy, Department of Toxicology acid-DPPC microparticles in diabetic rats. Eur J Pharm
for conducting cell culture studies and Ege University, Faculty B i o p h a r m . 2 0 1 7 ; 11 9 : 1 7 – 2 7 . h t t p s : / / d o i . o rg / 1 0 . 1 0 1 6 /
of Pharmacy, Pharmaceutical Sciences Research Centre 13. Gomez-Gaete C, Tsapis N, Silva L, Bourgaux C, Besnard M,
(FABAL) for equipmental support in UPLC analysis. The Bochot A, et al. Supramolecular organization and release
authors would also like to thank TUBITAK for purchasing of properties of phospholipid-hyaluronan microparticles encapsu-
the spray dryer under the project number 111S183. lating dexamethasone. Eur J Pharm Biopharm. 2008;70(1):116–
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