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Preparation of transfersomes encapsulating sildenafil aimed for transdermal

drug delivery: Plackett–Burman design and characterization

Article  in  Journal of Liposome Research · August 2014

DOI: 10.3109/08982104.2014.950276 · Source: PubMed

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ISSN: 0898-2104 (print), 1532-2394 (electronic)

J Liposome Res, 2015; 25(1): 1–10

! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/08982104.2014.950276

RESEARCH ARTICLE

Preparation of transfersomes encapsulating sildenafil aimed for

transdermal drug delivery: Plackett–Burman design and
characterization
Tarek A. Ahmed
Journal of Liposome Research Downloaded from [Link] by Dr. Tarek A. Ahmed on 03/03/15

Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Al-Azhar University, Nasr City, Cairo, Egypt

Abstract Keywords
The aim of this work was to study the effect of different processing and formulation Entrapment efficiency, Plackett–Burman
parameters on the preparation of sildenafil (SD) transfersomes utilizing the Plackett–Burman design, sildenafil citrate, transfersomes
design. The drug to phospholipid molar ratio (X1), phospholipid to surfactant ratio (X2),
hydrophilic–lipophilic balance of the surfactant (X3), hydration medium pH (X4), hydration History
time (X5) and the temperature of hydration (X6) were investigated to study their effect on
the vesicle size (Y1) and entrapment efficiency (EE) of the drug (Y2). The preparation Received 1 April 2014
conditions were optimized to minimize the vesicle size and maximize the EE. The prepared Revised 23 June 2014
transfersomes were also subjected to zeta potential measurements, morphological and Accepted 27 July 2014
physicochemical characterization. The combinations of factors that achieve the optimum Published online 22 August 2014
desirability were identified. An optimized formulation was prepared and characterized once
For personal use only.

more for its vesicle size, EE, in vitro permeation and deformability index. The results revealed
that both X3 and X6 had a pronounced effect on Y1, while X1 and X4 showed a significant
effect on Y2. Morphological and physicochemical study confirmed the transfersomes spherical
shape and compatibility of the formulation ingredients. The formulation with optimum
desirability showed EE and vesicle size of 97.21% and 610 nm, respectively. In vitro
permeation of the drug-loaded transfersome showed more than 5-fold higher permeation
rate compared with drug suspension. Deformability index verified elasticity of the
preparation. The significant variables could be optimized again to produce smaller vesicle
size that could increase SD permeation from transdermal delivery systems loaded drug
optimized transfersomes.

Introduction (Sharma et al., 2010). The flexibility of this vesicle minim-

izes the possibility of its rupture especially when applied
Vesicular structures such as liposomes, niosomes, transfer-
onto the skin (Malakar et al., 2012). Transfersomes have
somes and ethosomes that encapsulate pharmacologically
gained a lot of research interest due to their usefulness in
active drugs provide several advantages over the other
delivery of peptides (e.g. insulin and albumin; Cevc, 2003;
non-vesicular ones. These structures are biocompatible,
Cevc et al., 1995; Paul & Cevc, 1995; Yang et al., 2002) and
biodegradable and are able to protect the drug from the
therapeutic drugs (e.g. analgesics, anesthetics, corticosteroids
process of metabolic degradation. Other benefits of these
and sex hormones; Cevc & Blume, 2001; Cevc et al., 1997;
structures include enhanced drug bioavailability, prolongation
Essa et al., 2004; Planas et al., 1992).
of drug action and reduced administered drug toxicity
Men’s sexual arousal is controlled by the nitric oxide/
(Kumar et al., 2012; Mahale et al., 2012). In 1992, Cevc
cyclic guanosine monophosphate pathway that relax the
et al. introduced a novel drug delivery vesicle comprised of
penile corporal smooth muscle, an effect that leads to
phospholipid and single chain surfactant and have assigned
enhancing the corporal blood flow and erection (McMahon
the name ‘‘transfersomes’’. The most important feature of
et al., 2005). Sildenafil (SD) citrate, a worldwide drug, has
this vesicle is the elasticity caused by the presence of the
emerged as an efficient therapy in the treatment of men’s
edge activator, single chain surfactant, which weaken the
erectile dysfunction (Carson, 2003). The possible drug
phospholipid bilayer and render the vesicle ultradeformable
mechanism of action is related to inhibit of phosphodiester-
ase-5 enzyme that hydrolyze cGMP, which reflected in
increasing the effect of NO. Several studies have proposed the
Address for correspondence: Tarek A. Ahmed, Department of usefulness of SD in the treatment of premature ejaculation
Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy,
Al-Azhar University, Nasr City, Cairo, Egypt. Tel: 00201220610496. (McMahon et al., 2005), pulmonary hypertension in preg-
E-mail: dr_tarek_nour@[Link] nancy (Lynch & Laffey, 2006; Warnes, 2004), premature
 2 T. A. Ahmed J Liposome Res, 2015; 25(1): 1–10

labor and intra-uterine growth retardation (Villanueva-Garcia (El-Malah & Nazzal, 2006; Nutan et al., 2007). The model is
et al., 2007) and its role in pulmonary vasodilatation usually described by the following equation:
(Antoniu, 2006; Galie et al., 2005; Sastry et al., 2004;
Y ¼ M0 þ M1 X1 þ M2 X2 þ M3 X3 þ M4 X4 þ    þ Mn Xn
Singh et al., 2006). SD citrate is a slightly water soluble
drug characterized by its rapid oral absorption, but only where Y is the response, M0 is a constant and M1–Mn are the
40% of the administered dose is bioavailable due to the coefficients of the response values, X1–Xn are the factors
hepatic first-pass metabolism (Shin et al., 2006). Its oral under investigation. Analysis of the data is achieved after the
absorption and hence the onset of action is affected by results have been entered. The design enables identifying
food, especially fat-rich meals (Rajagopalan et al., 2003). the effect of the studied variables and present the data in the
Some researchers have developed SD formulations that form of a rank order that depend on the magnitude and sign
overcome the limited drug bioavailability. A sublingual SD of the effects.
formulation characterized by fast onset of action with small The design was developed using StatgraphicsÕ plus,
dose (Deveci et al., 2004), SD intra nasal microemulsion version 4 (Manugistic Inc., Rockville, MD). The possible
(Elshafeey et al., 2009) and transdermal SD citrate-loaded formulation and processing factors used to develop SD
nanostructure lipid carrier and solid lipid nanoparticles transfersomes were investigated. Drug to phospholipid
Journal of Liposome Research Downloaded from [Link] by Dr. Tarek A. Ahmed on 03/03/15

(Elnaggar et al., 2011) have been reported. molar ratio (X1), phospholipid to surfactant ratio (X2),
A well-organized and systematic tool in the design of surfactant HLB (X3), hydration medium pH (X4), hydration
pharmaceutical formulations is the statistical experimental time (X5) and the temperature of hydration (X6) were
designs, also called design of experiments. They permit a selected as independent variables, while the vesicle size
rational study of the effect of formulation and/or process- (Y1) and entrapment efficiency (EE) (Y2) of the prepared SD
ing variables on the selected responses. This allows transfersomes were studied as dependent responses. Table 1
shortening the time of the experiment and an improvement illustrates the independent variables and their level used in
in the development of the research work (Lewis et al., this study.
1999). The possible formulation and processing variables
are first ‘‘screened’’ to verify which are important to the Preparation of SD transfersomes
aim, followed by optimizing the recognized significant
Transfersomes-loaded SD were prepared using lipid film
variables. A polynomial model equation is created to
hydration technique previously described by Cevc et al.
For personal use only.

correlate the relationship between the variables and

(1997), Jain et al. (2003) and El Zaafarany et al. (2010) with
response. Plackett–Burman (PB) design is the most
slight modifications. Based on the composition of each
commonly used tool that rapidly identifies the factors
formulation depicted in Table 2, SD (50 mg), equivalent
most important in the process with the least number of
weight in mg of phospholipid and surfactant was dissolved
experiments (El-Malah & Nazzal, 2006; Ibrahim et al.,
in methanol. The mixture was subjected to sonication
2010; Khan et al., 2000).
using water bath sonicator (QS3, ultrasonic water bath
In this study, the effect of the possible processing and
model F 0023902, Ultrawave Ltd, Cardiff, UK) for five
formulation parameters affecting the preparation of SD
minutes to ensure dissolving of all the components.
transfersomes was studied using PB design. Characterization
Evaporation of the organic solvent was achieved by rotary
of the prepared formulations was achieved to optimize the
evaporation under reduced pressure at a temperature of 45  C
process. This work is the first step in the development of
using (Buchi Rotavapor R-200 Buchi labortechink AG,
optimized SD transfersome preparation suitable for transder-
CH-9230, Flawi, Switzerland). A thin lipid film was
mal drug delivery.
produced inside the flask wall while rotation. This lipid
film was kept overnight under vacuum in a vacuum
Materials and methods oven (Thermo Fisher Scientific, model 6505, Oakwood
Materials Village, OH) for complete solvent evaporation. The dried
film was then hydrated with phosphate buffer of specified
SD citrate, Tween 80 (hydrophilic–lipophilic balance
pH under gentle shaking in the rotavapor for the specified
[HLB] ¼ 15), Span 80 (HLB ¼ 4.3), HCL and methanol
time. The temperature of the rotavapor water bath
were obtained from Sigma-Aldrich (St. Louis, MO).
was adjusted to the corresponding hydration temperature
L-a-phosphatidylcholine (soya 95%) was purchased from
(2–20  C). Ice was added to the water bath to achieve the
Avanti Polar Lipids (Birmingham, AL). Sodium hydroxide
lower temperature of hydration. The obtained transfersome
and potassium dihydrogen phosphate were procured from
suspensions were then subjected to sonication using water
BDH Chemicals Ltd. (Poole, England). All chemicals and
bath sonicator for 30 min.
reagents used were of analytical grade.
Characterization of the formulations
Methods
Measurements of vesicle size and zeta potential. The vesicle
PB design
size and zeta potential of the obtained transfersome disper-
PB design is a preliminary useful tool used to recognize sions were determined by dynamic light scattering using a
the effect of different factors affecting the formulation Zetatrac (Microtrac Inc., York, PA). Other useful properties
process. The processing and formulation factors could such as polarity, dielectric constant (DEC) and viscosity were
be optimized to eliminate or fix the variables under study identified by means of the same apparatus.
 DOI: 10.3109/08982104.2014.950276 Plackett–Burman design and characterization 3
Table 1. Sildenafil transfersomes independent and dependent variables Spectrum One, Model 16 PC, Rheinstetten, Germany).
used in the Plackett–Burman design.
Transparent discs were prepared by mixing each sample
Level
with KBr. The mixture was compressed and tested in the
range 4000–400 cm1.
Low Medium High
Independent variables Placket–Burman design statistical analysis
Drug:lipid molar ratio (X1) 1:2 1:6 1:10
Lipid:surfactant ratio (X2) 95:5 85:15 75:25 The results obtained for both Y1 and Y2 were introduced
Surfactant HLB (X3) 4.3 9.65 15 into the response column of the software and the model was
Hydration medium pH (X4) 5.5 6.5 7.5
Hydration time (X5) 30 75 120
run. Statistical analysis of the data was considered to be
Temperature of hydration (X6) 2 11 20 significant at p value50.05.
Dependent variables Goal
Vesicle size (Y1) Minimize Optimum desirability
Entrapment efficiency (Y2) Maximize
After identifying the significant factors, an optimized SD
transfersome formulation was proposed. This optimized
Journal of Liposome Research Downloaded from [Link] by Dr. Tarek A. Ahmed on 03/03/15

Table 2. Composition of PB sildenafil transfersome formulations. formulation was prepared and characterized to compare the
observed (actual) values of Y1 and Y2 with the predicted
X1 X2 X3 X4 X5 X6 responses. Preparation and determination of the vesicle size
Run (M  R) (%) (HLB) (pH) (min) ( C) and EE were done as previously described.
1 1:10 75:25 15 5.5 120 20
2 1:2 95:5 4.3 7.5 120 20 In vitro permeation study. The optimized SD transfersome
3 1:2 95:5 4.3 5.5 30 2 formulation was also subjected to in vitro permeation study to
4 1:2 75:25 15 5.5 120 2
5 1:10 95:5 15 7.5 30 20 identify the profile of permeation for both the drug and the
6 1:2 95:5 15 7.5 120 2 drug-loaded transfersome. The study was performed accord-
7 1:10 95:5 15 5.5 30 2 ing to the method previously described by Elnaggar et al.
8 1:2 75:25 15 7.5 30 20 (2011) except for slight modification. The experiment was
9 1:10 95:5 4.3 5.5 120 20
10 1:2 75:25 4.3 5.5 30 20 conducted for 24 h at 37  C using Franz diffusion cell
For personal use only.

11 1:10 75:25 4.3 7.5 120 2 apparatus (Hanson Research, MicroettePluss, Chatsworth,
12 1:10 75:25 4.3 7.5 30 2 CA). The donor cells were filled with either 0.3 ml of the
13 1:6 85:15 9.65 6.5 75 11
prepared SD transferosomal suspension or drug suspension,
M  R indicates molar ratio. all at a SD concentration of 0.24%. This concentration was
calculated to achieve the sink condition. The used sample
volume of drug suspension and transfersomes have a constant
drug amount of 0.72 mg. Synthetic nylon membrane of
Determination of EE. Free SD was separated from transfer-
0.45 mm pore size (Whatman Int., Maidstone, Kent, UK)
some-entrapped drug by centrifugation at 15 000 rpm for 1 h
was used as artificial diffusion membrane. The volume of the
at 4  C using (Sigma Laboratory centrifuge, 3K30, Ostrode,
receptor compartment was 7 ml of phosphate buffer, pH 7.4.
Germany). One milliliter of the supernatant was diluted
Samples of 2 ml with replacement were withdrawn at 0.5, 1, 2,
with acidic buffer of pH 1.2 (0.1 N HCL), and the concen-
3, 4, 5, 6, 12 and 24 h. The concentration of the drug was
tration of the drug was determined spectrophotometrically
determined spectrophotometrically at 291 nm. All experi-
at 291 nm. HCL (0.1 N) was selected as the drug exhibits
ments were performed in triplicate, and the obtained results
higher solubility in this solvent as previously reported by
are presented as mean % permeated ± SD.
Elshafeey et al. (2009). Sparsha et al. (2012) demonstrated
UV spectrophotometric determination of SD in pharmaceut-
Morphological study. The morphology of the optimized SD
ical products utilizing HCL. The %EE of SD was calculated
transfersome was identified using scanning electron micro-
using the following equation:
scope (Philips XL30, Eindhoven, the Netherlands). After

 centrifugation, the precipitated transfersome was separated
%EE ¼ Ctotal  Cfree drug =Ct  100
from the supernatant, diluted with distilled water, vortex
where Ctotal represent the total concentration of drug, and mixed and finally subjected to freeze drying in the presence
Cfree drug is the free unentraped drug. and absence of mannitol using (alpha 1-2 LD plus, Christ
lyophilizer, Germany). The lyophilized sample was sputter
Physicochemical characterization. Differential scanning cal- coated with gold before investigation.
orimetry (DSC) was utilized to investigate the thermal
behavior of the drug and the freeze-dried drug-loaded Deformability index. The elasticity of the optimized SD
transfersome formulations namely, F2, F8 and F13 that contain transfersome formulation was evaluated and compared to the
Tween 80, Span 80 and mixture of both, respectively, using formulation with the highest and smallest vesicle size
(Shimadzu DSC TA-50 ESI DSC apparatus, Tokyo, Japan). obtained namely F7 and F2, respectively. Evaluation was
Fourier transforms infrared (FT-IR) spectra of the drug done according to the method previously described by Mahor
alone and selected SD transfersome formulations studied in et al. (2007) except for slight modification. Briefly, each
the FT-IR section were obtained using (Perkin Elmer formulation was extruded from nylon membrane filter with a
 4 T. A. Ahmed J Liposome Res, 2015; 25(1): 1–10

pore size of 0.1 micron under reduced pressure less than Table 3. Characterization results of the prepared sildenafil
1.2 Mpa for a period of 15 min. The deformability index was transfersomes.
calculated using the following equation:
Vesicle Zeta potential Viscosity

2 Formulation size (nm) EE (%) (mv) (cp)
D ¼ J  rv =rp
F1 1317 ± 66 80.14 ± 4.01 1.66 ± 0.08 1.011 ± 0.05
where J is the amount of suspension extruded in 15 min, F2 602 ± 36 92.16 ± 3.15 2.30 ± 0.11 0.983 ± 0.04
rv ¼ vesicles size and rp ¼ pore size of the membrane. F3 1014 ± 41 16.36 ± 0.82 1.52 ± 0.06 0.992 ± 0.03
F4 1644 ± 49 18.06 ± 0.85 1.74 ± 0.05 1.001 ± 0.04
F5 1278 ± 51 87.88 ± 2.64 2.11 ± 0.11 0.977 ± 0.03
Results and discussion F6 1616 ± 80 80.10 ± 3.89 2.60 ± 0.12 1.001 ± 0.05
F7 1880 ± 75 71.22 ± 2.49 3.63 ± 0.18 1.019 ± 0.04
The slightly aqueous solubility and limited oral bioavailability F8 1680 ± 84 90.58 ± 3.98 3.05 ± 0.06 1.011 ± 0.02
(only 40%) of SD render the drug good candidate for F9 689 ± 27 78.26 ± 2.27 2.78 ± 0.09 0.988 ± 0.03
encapsulation into a nano-vesicular structure that is predicted F10 1005 ± 20 34.88 ± 1.72 3.26 ± 0.16 0.997 ± 0.04
to enhance its bioavailability after selecting the suitable F11 965 ± 39 84.24 ± 3.79 2.24 ± 0.11 1.015 ± 0.02
F12 1061 ± 53 86.38 ± 3.02 2.52 ± 0.08 1.022 ± 0.05
delivery system such as transdermal route. As far as we know, F13 859 ± 42 85.18 ± 3.47 2.65 ± 0.12 0.985 ± 0.04
no previous studies have reported the preparation of SD
Journal of Liposome Research Downloaded from [Link] by Dr. Tarek A. Ahmed on 03/03/15

transfersomes. In this study, the influence of various Each reading is an average of three measures.
formulation and processing parameters that affect the vesicle
size and EE of the prepared SD transfersomes was
investigated utilizing PB screening design using a suitable
tool (StatgraphicsÕ plus software) to identify the parameters exhibited lower zeta potential values, which could affect the
that are useful for the aim of this work. stability of the prepared vesicles, a charge-inducing agent
could be added in the next step of optimization to enhance
Selection of the variables and preparation of SD this character. The EE and the vesicle size will be discussed
transfersomes in detail in the next section.

Table 1 illustrates the lower and higher level for the studied Effect of the studied parameters on the vesicle size
independent variables. Wide range of drug to lipid molar and EE
For personal use only.

ratios, lipid to surfactant ratios, different surfactant HLB

Figure 1 illustrates the effect of the studied independent
values, temperature of hydration between 2 and 20  C, which
variables on the size of the prepared SD transfersomes.
was selected to be above the studied lipid transition
Both surfactant HLB (X3) and hydration medium temperature
temperature, phosphate buffer of pH between 5.5–7.5 that
(X6) showed a significant effect on the vesicle size of the
enhance the drug encapsulation in the hydration medium as
prepared transfersomes at p values of 0.0008 and 0.0491,
will described later and hydration time between 30 and
respectively, which is also demonstrated in the Pareto
120 min were screened to illustrate the effect of these
chart and main effects plot portrayed in Figure 1A and B.
parameters on the studied variables.
As the surfactant HLB decreased, the vesicle size increased,
Table 2 lists the composition of the formulation developed
which could be related to the affinity of the studied
by the software. Lipid film hydration technique was utilized
phospholipid toward surfactant. The affinity was highest
in the preparation of SD transfersome formulations. After
with surfactant of HLB 4.3 followed by 9.65 and finally 15.
dissolving the components in methanol, the organic solvent
This finding was in agreement with previous studies
was evaporated to produce a thin lipid film, which was then
demonstrated the effect of surfactant HLB on the entrapment
hydrated to obtain the desired drug lipid-based nano-vesicular
of drug-loaded transfersomes (El Zaafarany et al., 2010;
structure. The prepared transfersome suspensions were sub-
Scognamiglio et al., 2013). They concluded that the lipid
jected to water bath sonication for 30 min to reduce the size of
affinity to the studied surfactants was in the order; Span 85
the prepared vesicles.
(HLB ¼ 1.8)4Span 80 (HLB ¼ 4.3)4Tween 80 (HLB ¼ 15).
Similar finding was reported for the effect of different
Characterization of the formulations
surfactant on vinpocetine niosomes vesicle size prepared
After preparing the 13 PB transfersome-proposed formula- using different non-ionic surfactants (El-Laithy et al., 2011).
tions, each formulation was characterized for vesicle size, EE, They illustrated that niosomes prepared using Span showed
zeta potential, polarity, DEC and viscosity. The results for smaller vesicle size than Tween based and attributed this to
these parameters are depicted in Table 3. All the studied the hydrophobicity of the surfactant. The lipid transition
formulations exhibited a DEC of 79, which is approximately temperature of the used phospholipid (L-a-phosphatidylcho-
the DEC of water; solvent used in preparing the hydration line, soya 95%) is low, less than zero  C (Cevc, 1993; Walde
medium. The viscosity of the formulations was in the range & Ichikawa., 2001). As the temperature of the hydration
0.977–1.022 cp, which is not significantly different from that medium increased from 2 to 20  C, it leads to the formation
of water (1.0020 cp). All the prepared SD transfersomes of more stable smaller vesicles. Temperature of the hydra-
exhibited positive polarity and a zeta potential value in the tion medium has a great effect in the formation of these
range of 1.52–3.63 mv. The positive polarity and small values types of vesicles. It affects their characteristics, especially
for the measured zeta potential are mainly due to the surface their shape and size. The temperature of the hydration
charge on the drug itself. Although the prepared formulations medium should always be above the lipid gel to liquid-phase
 DOI: 10.3109/08982104.2014.950276 Plackett–Burman design and characterization 5
Journal of Liposome Research Downloaded from [Link] by Dr. Tarek A. Ahmed on 03/03/15

Figure 1. Standardized Pareto chart (A), main effects plot (B), estimated response surface (C and D) and contours of estimated response surface
(E and F) for the effect of the studied variables on the vesicle size.

transition temperature. It is previously stated that, this hydration time for transfersomal preparation could vary
temperature affects the assembly of surfactant molecules from minutes to hours. A hydration time of one hour has
into vesicles and also induces changes in vesicle shape during been used in the preparation of sodium diclofenac and
the formation of niosomes (Handjani-Vila, 1990). curcumin transfersomes (Patel et al., 2009; Taghizadeh &
For personal use only.

To illustrate the effect of the studied variables on the Bajgholi, 2011). Mitkari et al. (2010), prepared fluconazole
vesicle size when two variables are changing while the liposomal preparation and allowed the obtained dried
other four variables were kept at their intermediate levels, lipid film to hydrate for 2–3 h. The results for the effect of
estimated response surface (Figure 1C and D) and contours X5 on Y1 demonstrated that F2, F9 and F11, which were
of estimated response surface (Figure 1E and F) were prepared utilizing the maximum level of X5 (120 min),
constructed. The equation of the fitted model is: showed smaller Y1 and F1, F4 and F6 that have the same
level of X5 exhibited higher Y1 value. This could be
Y1 ¼ 1078:3  6:64583  X1 þ 5:375  X2 þ 64:3458 explained as; the prepared vesicles are hydrated rapidly at
 X3  33:25  X4  2:10556  X5  15:3796  X6 higher temperature of hydration (X6), which has the signifi-
cant effect as previously illustrated, and under a minimum
Analysis of variance according to the data of the vesicle level of X5 (30 min). Both X2 and X4 are mainly affecting
size revealed also that X1, X2, X4 and X5 were not Y2 as will be illustrated in the next section.
significantly affecting Y1 at p values of 0.5910, 0.3994, Both the drug to lipid molar ratio (X1) at p value ¼ 0.0307
0.6147 and 0.1481, respectively. This finding indicates that and hydration medium pH (X4) at p value ¼ 0.0071 have a
these factors are not extensively affecting the vesicle size but pronounced effect on the EE as could be concluded from
may be important in another aspect. Trotta et al. (2004) the Pareto chart (Figure 2A) and main effect plot (Figure 2B).
studied the effect of different drug to lipid molar ratios (1:2 As the amount of lipid increased, the EE increased, but
and 1:4) on the preparation of methotrexate deformable further increase in the amount of lipid resulted in
transfersomes and reported that the size was increased as the decreasing the EE. The decrease in the EE as the amount of
drug to lipid ratio increased. They attributed this finding to phospholipid increased is attributed to the competition
formation of multilamellar vesicles as the ratio of the lipid between the drug and the phospholipid in the formed bilayer,
increased, which results in vesicles of large size. Other the effect that may result in excluding the drug and
previous studies also supported this finding (Trotta et al., interrupting the structure of the vesicle membrane as
2002). Our results does not completely support this previously illustrated by El-Laithy et al. (2011). As previously
finding. Although lower level of X1 showed smaller vesicle explained in selection of the variables, SD citrate is slightly
size as in F2 (1:2) and F13 (1:6), yet formulations F3, F4, F6, water soluble, and its solubility is pH dependent (Elnaggar
F8 and F10, which were prepared also with lower level of et al., 2011). The drug solubility decreases with increase of
X1 (1:2), revealed bigger vesicular size. This could be pH. As the pH of the hydration medium increased (5.5–7.5),
attributed to the interaction effect of the other studied the solubility of the drug in the hydration medium decreased
variables and also to the nature of the PC and surfactant the effect that leads to migration of the drug into the
used. Sufficient hydration time (X5) is usually required to transfersomes cavity and lamellar layers. To illustrate the
ensure complete swelling of the dried lipid film and also effect of the studied variables on EE when two variables are
to prepare the desired vesicular dispersion. The usual changing while the other four variables were kept at their
 6 T. A. Ahmed J Liposome Res, 2015; 25(1): 1–10
Journal of Liposome Research Downloaded from [Link] by Dr. Tarek A. Ahmed on 03/03/15

Figure 2. Standardized Pareto chart (A), main effects plot (B), estimated response surface (C and D) and contours of estimated response surface (E and
F) for the effect of the studied variables on entrapment efficiency.

intermediate levels, estimated response surface (Figure 2C The composition of the multiple responses including
and D) and contours of estimated response surface (Figure 2E the optimized desirability was identified. The optimized
and F) were constructed. The equation of the fitted model is: formulation was proposed to contain 1:4.08, 95:5, 4.3, 7.5,
118.6 and 19.8 of X1, X2, X3, X4, X5 and X6, respectively.
Y2 ¼ 89:0088 þ 3:24958  X1  0:264167  X2 This optimized formulation was prepared and characterized
For personal use only.

þ 0:556075  X3 þ 18:535  X4 þ 0:0845556  X5 for its size and EE. The predicted values were compared to
þ 0:995741  X6 the observed ones in which the residual was calculated and
presented in Table 4.
Analysis of variance for the data of the EE also illustrated
that p values for X2, X3, X5 and X6 were 0.5886, 0.5438,
Physicochemical characterization
0.4421 and 0.1007, respectively, and so it was concluded
that these factors were not significantly affecting this The thermal characteristic of the drug and some selected
parameter. Previous reports illustrated the effect of X2 on formulations was studied using DSC. A characteristic SD
EE. El Zaafarany et al. (2010) prepared diclofenac sodium endothermic peak was obtained at 198  C indicating the
transfersomes using PC:EA (w/w) of 98:2, 95:5, 85:15 and melting point of the pure drug as illustrated in Figure 3.
75:25. They concluded that transfersomes prepared with Encapsulation of the drug in the transfersome preparations
95:5% (w/w) (PC:EA) ratio showed the highest drug EE%. containing the phospholipid and studied surfactants, Tween,
Patel et al. (2009) studied the effect of phospholipid to Span or both, does not greatly affect the drug characteristic
surfactant (edge activator) ratio on the preparation and peak except for slight shift in the drug melting endotherm
characterization of curcumin transfersome and illustrated with F13, which could be attributed to the presence of
that the EE of the lipophilic drug, curcumin, was decreased as equal amounts of the two surfactants, Span and Tween, that
the surfactant concentration increased, and the EE was led to slight change in the drug crystalline state or to
increased as the concentration of the phospholipid increased molecular dispersion of the pure drug in the lipid and
(Patel et al., 2009). Our finding revealed that X2 was not surfactant matrix as previously mentioned with the same drug
significantly affecting the prepared SD transfersomes; the during the preparation of SD-loaded PLGA nanoparticles
results of the EE for those formulations prepared with the (Ghasemian et al., 2013). This finding needed further
maximum level of surfactant (25%) were not consistent. investigation to ensure compatibility of the formulation
F1, F8, F11 and F12 showed EE values of 80.14, 90.58, 84.24 ingredients, which was achieved by the IR characterization.
and 86.38, respectively, whereas F4 and F10 demonstrated The IR spectra of the drug showed two characteristic
EE values of 18.06 and 34.88, respectively. This could be peaks at 3614 and 3299 corresponding to the hydroxy (–OH)
attributed to the nature of the surfactant used and to the and amido (–CONH–) groups, respectively. Another two
interaction effect of the other variables. The results for the bands 1800–1695 and 1600–1500 cm1 analogous to the
effect of X5 on Y2 illustrated that all the formulations carbonyl (C¼O) of the pyrazole ring and amino (N–H)
prepared with maximum level of X5 showed higher EE except groups, respectively, as illustrated in Figure 4. This finding is
F4, which may be due to the predominant effect of X1 and in agreement with previous studies for the IR spectra of SD
X3. As previously mentioned in the above section, X6 is (Liu et al., 2011; Patel et al., 2012; Sivakranth et al., 2011).
mainly affecting the formation, size and shape of these types It is previously described that Span and Tween 80, which are
of vesicles and so was insignificantly influencing Y2. non-ionic surfactant of ester and ether–ester linkage,
 DOI: 10.3109/08982104.2014.950276 Plackett–Burman design and characterization 7

respectively, are characterized by a broad band at the transfersome preparation was obviously biphasic where
3422.1 cm1, and a sharp spectrum at 1739.7 cm1 (Shan an initial rapid release phase was observed in the first 4 h
et al., 2010). These characteristic bands corresponding to the followed by a sustained release phase that last for the rest 24 h
surfactants hydroxyl and the ester groups, respectively. The of the study. The fast initial release phase may be attributed
spectrum of the phospholipid usually shows a band approxi-
mately at 3270 cm1 assigned to OH stretching vibration
(Pohle et al., 2001). Another characteristic band for the
phospholipid is between 2700 and 2400 cm1, which is due to
hydride vibration of the phosphine (P-H) group (John, 2000).
After encapsulation of the drug in the transfersome, the
characteristic drug carbonyl of the pyrazole ring was detected
in all the studied formulations, while the hydroxyl, amido and
amino were overlapped with the surfactant hydroxyl group at
3422.1 cm1 and ester group at 1739.7 cm1. The character-
istic phospholipid hydride phosphine group was detected in
Journal of Liposome Research Downloaded from [Link] by Dr. Tarek A. Ahmed on 03/03/15

all the studied transfersomes formulation. The above finding

confirms compatibility of the formulation ingredients.

In vitro permeation study

The in vitro permeation study of the optimized SD
transfersome formulation showed a considerable difference,
more than 5-folds, when compared with the drug suspension
as revealed in Figure 5. The release profile of SD from

Table 4. Multiple response optimization of sildenafil transfersomes.

Optimum
For personal use only.

Factor Low level High level level

X1 1:2 1:10 1:4.08
X2 95:5 75:25 95:5
X3 4.3 15 4.3
X4 5.5 7.5 7.5
X5 30 120 118.6
X6 2 20 19.8
Response Predicted Observed Residual
Figure 3. DSC of sildenafil and drug-loaded transfersome formulations.
Vesicle size (nm) 580 610 30
EE (%) 93.8 97.21 3.41

SD

F2

Egy

F8

F13

4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 400.0
-1
Cm

Figure 4. IR spectra of sildenafil and drug-loaded transfersome formulations.

 8 T. A. Ahmed J Liposome Res, 2015; 25(1): 1–10

to the drug that was adsorbed on the surface of the in Figure 6 that depicts the morphological characteristics
prepared transfersome and to the drug that permeated of the optimized SD transfersome formulation. Micrograph
from the transfersome. This result is in agreement with captured in presence of mannitol for the prepared transfer-
previous study that also described the biphasic release some vesicles partially reduced aggregation but does not
profile of diclofenac sodium from ultradeformable vesicles prevent fusion. Previous reports have illustrated the applic-
(El Zaafarany et al., 2010). In general, the mechanism of drug ability of SEM in characterization the surface morphology
release from the prepared transfersome could be attributed of these kinds of lipid-based vesicles (Chandra & Sharma,
to a diffusion control mechanism as previously illustrated by 2008; El-Laithy et al., 2011). Partial aggregation and
many researchers (Arica et al., 1995; Hathout et al., 2007; fusion during freeze drying did not allow perfect morpho-
Nasr et al., 2008). logical description and so vesicular nanoparticles based on
phospholipid such as liposomes, niosomes and transfersomes
Morphological study are better to characterize using the transmission electron
microscopy that characterize the vesicles suspended in the
It is previously mentioned that aggregation, some time hydration medium without need for freeze drying.
irreversible fusion and destabilization of the prepared
nanosize vesicles, might occur during the freeze drying
Journal of Liposome Research Downloaded from [Link] by Dr. Tarek A. Ahmed on 03/03/15

Deformability index
process (Abdelwahed et al., 2006). One possible mechanism
for this destabilization might be due to the mechanical stress Elasticity of the optimized transfersome was evaluated by
of the ice crystals formed during the freeze drying cycles measuring its ability to pass through pores of a definite
(Abdelwahed et al., 2006). For these aforementioned reasons, size under a suitable gradient pressure, which is mainly
some inert excipients, such as sugars, glycerol, poly vinyl the function of lipid to surfactant ratio (Hofer et al., 2000)
alcohol, glycine or gelatin, are added to prevent this and also the vesicle size. It is previously mentioned that
phenomenon. These inert excipients are called cryoprotectant the transfersome elasticity or transcutaneous efficiency is not
and lyoprotectant (Abdelwahed et al., 2006). The vesicular highly affected after certain level of the membrane component
nature of the prepared formulation was verified as illustrated (Mahor et al., 2007). The deformability index (D-value)
was in the following order; optimized formulation followed
by F2 and finally F7. The optimized formulation, that contain
For personal use only.

35 the optimum phospholipid to surfactant ratio and character-

ized by its lower vesicle size, showed the highest elasticity
30
as indicated by higher D-value of 14.88 ± 1.6, while F7
25 exhibited D-value of 8.52 ± 1.1.
% Drug Permeated

20 Transferosomes Conclusion
15 Drug In this study, different formulation and processing variables
were screened to identify the significant variables that could
10
be optimized to prepare SD transfersome formulation with
5 small vesicle size and high EE. The recognized significant
variable were able to formulate SD transfersome with
0 approximately 610 nm vesicle size, 97.21% EE and character-
0 5 10 15 20 25
Time (h)
ized by more than 5-fold higher in vitro permeation when
compared with the drug suspension. The significant variables
Figure 5. In vitro permeation profiles of sildenafil citrate from drug- need to be optimized again to prepare transfersome formu-
loaded transfersome compared with drug suspension. lation with much smaller vesicle size that will exhibit much

Figure 6. SEM of sildenafil transfersome prepared without (A) and with (B) mannitol.
 DOI: 10.3109/08982104.2014.950276 Plackett–Burman design and characterization 9

higher permeation and will be suitable for preparing well- Hathout RM, Mansour S, Mortada ND, Guinedi AS. (2007). Liposomes
as an ocular delivery system for acetazolamide: in vitro and in vivo
formulated SD transdermal delivery system that is promising studies. AAPS PharmSciTech 8:1.
over the oral route. Hofer C, Hartung R, Gobel R, et al. (2000). New ultradeformable drug
carriers for potential transdermal application of interleukin-2 and
Acknowledgements interferon-alpha: theoretic and practical aspects. World J Surg 24:
1187–9.
The author would like to express his deep thanks to Ibrahim HM, Ahmed TA, Lila AE, et al. (2010). Mucoadhesive
Dr. Osama A. A. Ahmed and Dr. Shaimaa M. Badr-Eldin controlled release microcapsules of indomethacin: optimization and
stability study. J Microencapsul 27:377–86.
for their guidance. Jain S, Jain P, Umamaheshwari RB, Jain NK. (2003). Transfersomes – a
novel vesicular carrier for enhanced transdermal delivery: develop-
Declaration of interest ment, characterization, and performance evaluation. Drug Dev Ind
Pharm 29:1013–26.
The author declares no conflict of interest. John C. (2000). Interpretation of infrared spectra: a practical approach.
In: Meyers RA, ed. Encyclopedia of analytical chemistry. Chichester:
John Wiley & Sons Ltd., 10815–37.
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